Phytopathogenic fungi are known to produce several types of enzymes usually involved in plant cell wall degradation and pathogenesis. The increasing of global temperature may induce fungi, such as Lasiodiplodia theobromae (L. theobromae), to alter its behavior. Nonetheless, there is only limited information regarding the effect of temperature on L. theobromae production of enzymes. The need for new, thermostable enzymes, that are biotechnologically relevant, led us to investigate the effect of temperature on the production of several extracellular enzymatic activities by different L. theobromae strains. Fungi were grown at 25 °C, 30 °C and 37 °C and the enzymatic activities were detected by plate assays, quantified by spectrophotometric methods and characterized by zymography. The thermostability (25–80 °C) of the enzymes produced was also tested. Strains CAA019, CBS339.90, LA-SOL3, LA-SV1 and LA-MA-1 produced amylases, gelatinases, caseinases, cellulases, lipases, laccases, xylanases, pectinases and pectin liases. Temperature modulated the expression of the enzymes, and this effect was more visible when fungi were grown at 37 °C than at lower temperatures. Contrary to proteolytic and endoglucanolytic activities, whose highest activities were detected when fungi were grown at 30 °C, lipolytic activity was not detected at this growth temperature. Profiles of proteases and endoglucanases of fungi grown at different temperatures were characterized by zymography. Enzymes were shown to be more thermostable when fungi were grown at 30 °C. Proteases were active up to 50 °C and endoglucanases up to 70 °C. Lipases were the least stable, with activities detected up to 45 °C. The enzymatic profiles detected for L. theobromae strains tested showed to be temperature and strain-dependent, making this species a good target for biotechnological applications. Full article

Hepatocellular carcinoma (HCC) frequently develops from hepatitis C virus (HCV) and hepatitis B virus (HBV) infection. We previously reported that peretinoin, an acyclic retinoid, inhibits HCV replication. This study aimed to examine the influence of peretinoin on the HBV lifecycle. HBV-DNA and covalently closed circular DNA (cccDNA) were evaluated by a qPCR method in HepG2.2.15 cells. Peretinoin significantly reduced the levels of intracellular HBV-DNA, nuclear cccDNA, and HBV transcript at a concentration that did not induce cytotoxicity. Conversely, other retinoids, such as 9-cis, 13-cis retinoic acid (RA), and all-trans-retinoic acid (ATRA), had no effect or rather increased HBV replication. Mechanistically, although peretinoin increased the expression of HBV-related transcription factors, as observed for other retinoids, peretinoin enhanced the binding of histone deacetylase 1 (HDAC1) to cccDNA in the nucleus and negatively regulated HBV transcription. Moreover, peretinoin significantly inhibited the expression of SPHK1, a potential inhibitor of HDAC activity, and might be involved in hepatic inflammation, fibrosis, and HCC. SPHK1 overexpression in cells cancelled the inhibition of HBV replication induced by peretinoin. This indicates that peretinoin activates HDAC1 and thereby suppresses HBV replication by inhibiting the sphingosine metabolic pathway. Therefore, peretinoin may be a novel therapeutic agent for HBV replication and chemoprevention against HCC. Full article

Paleo-colour scientists have recently made the transition from describing melanin-based colouration in fossil specimens to inferring life-history traits of the species involved. Two such cases correspond to counter-shaded dinosaurs: dark-coloured due to melanins dorsally, and light-coloured ventrally. We believe that colour reconstruction of fossils based on the shape of preserved microstructures—the majority of paleo-colour studies involve melanin granules—is not without risks. In addition, animals with contrasting dorso-ventral colouration may be under different selection pressures beyond the need for camouflage, including, for instance, visual communication or ultraviolet (UV) protection. Melanin production is costly, and animals may invest less in areas of the integument where pigments are less needed. In addition, melanocytes exposed to UV radiation produce more melanin than unexposed melanocytes. Pigment economization may thus explain the colour pattern of some counter-shaded animals, including extinct species. Even in well-studied extant species, their diversity of hues and patterns is far from being understood; inferring colours and their functions in species only known from one or few specimens from the fossil record should be exerted with special prudence. Full article

Wheat (Triticum aestivum L.) is an important food crop, and biotic and abiotic stresses significantly impact grain yield. Wheat leaf and stem surface waxes are associated with traits of biological importance, including stress resistance. Past studies have characterized the composition of wheat cuticular waxes, however protocols can be relatively low-throughput and narrow in the range of metabolites detected. Here, gas chromatography-mass spectrometry (GC-MS) metabolomics methods were utilized to provide a comprehensive characterization of the chemical composition of cuticular waxes in wheat leaves and stems. Further, waxes from four wheat cultivars were assayed to evaluate the potential for GC-MS metabolomics to describe wax composition attributed to differences in wheat genotype. A total of 263 putative compounds were detected and included 58 wax compounds that can be classified (e.g., alkanes and fatty acids). Many of the detected wax metabolites have known associations to important biological functions. Principal component analysis and ANOVA were used to evaluate metabolite distribution, which was attributed to both tissue type (leaf, stem) and cultivar differences. Leaves contained more primary alcohols than stems such as 6-methylheptacosan-1-ol and octacosan-1-ol. The metabolite data were validated using scanning electron microscopy of epicuticular wax crystals which detected wax tubules and platelets. Conan was the only cultivar to display alcohol-associated platelet-shaped crystals on its abaxial leaf surface. Taken together, application of GC-MS metabolomics enabled the characterization of cuticular wax content in wheat tissues and provided relative quantitative comparisons among sample types, thus contributing to the understanding of wax composition associated with important phenotypic traits in a major crop. Full article

Aim: To develop an innovative delivery system for temozolomide (TMZ) in solid lipid nanoparticles (SLN), which has been preliminarily investigated for the treatment of melanoma. Materials and Methods: SLN-TMZ was obtained through fatty acid coacervation. Its pharmacological effects were assessed and compared with free TMZ in in vitro and in vivo models of melanoma and glioblastoma. Results: Compared to the standard free TMZ, SLN-TMZ exerted larger effects, when cell proliferation of melanoma cells, and neoangiogeneis were evaluated. SLN-TMZ also inhibited growth and vascularization of B16-F10 melanoma in C57/BL6 mice, without apparent toxic effects. Conclusion: SLN could be a promising strategy for the delivery of TMZ, allowing an increased stability of the drug and thereby its employment in the treatment of aggressive malignacies. Full article

Tigerinin-1R (Arg–Val–Cys–Ser–Ala–Ile–Pro–Leu–Pro–Ile–Cys–His–NH₂), a cationic 12-mer peptide containing a disulfide bond extracted from frog skin secretions, lacks antibacterial activity, but has the ability to stimulate insulin release both in vitro and in vivo. To study the structure–function relationships of tigerinin-1R, we designed and synthesized five analogs, including tigerinin-cyclic, tigerinin-1R-L4, tigerinin-linear, [C3K]tigerinin-1R, and [C11K]tigerinin-1R. Tigerinin-1R promoted insulin secretion in a concentration-dependent manner in INS-1 cells without obvious cytotoxicity. At a concentration of 10⁻⁵ M, [C11K]tigerinin-1R exhibited the highest stimulation ability, suggesting that the positive charge at the C-terminus may contribute to the in vitro insulin-releasing activity of tigerinin-1R. Tigerinin-1R peptides stimulated insulin release in INS-1 cells through a universal mechanism that involves mobilization of intracellular calcium without disrupting the cell membrane. In vivo experiments showed that both tigerinin-1R and [C11K]tigerinin-1R improved glucose tolerance in overnight-fasted mice. Due to its structural stability, tigerinin-1R showed superior hypoglycemic activity to [C11K]tigerinin-1R, which suggested a critical role of the disulfide bonds. In addition, we also identified a protective effect of tigerinin-1R peptides in apoptosis induced by oxidative stress. These results further confirm the potential for the development of tigerinin-1R as an anti-diabetic therapeutic agent in clinical practice. Full article

Initial studies showed that ligand-activated hormone receptors act by binding to the proximal promoters of individual target genes. Genome-wide studies have now revealed that regulation of transcription by steroid hormones mainly depends on binding of the receptors to distal regulatory elements. Those distal elements, either enhancers or silencers, act on the regulation of target genes by chromatin looping to the gene promoters. In the nucleus, this level of chromatin folding is integrated within dynamic higher orders of genome structures, which are organized in a non-random fashion. Terminally differentiated cells exhibit a tissue-specific three-dimensional (3D) organization of the genome that favors or restrains the activity of transcription factors and modulates the function of steroid hormone receptors, which are transiently activated upon hormone exposure. Conversely, integration of the hormones signal may require modifications of the 3D organization to allow appropriate transcriptional outcomes. In this review, we summarize the main levels of organization of the genome, review how they can modulate the response to steroids in a cell specific manner and discuss the role of receptors in shaping and rewiring the structure in response to hormone. Taking into account the dynamics of 3D genome organization will contribute to a better understanding of the pleiotropic effects of steroid hormones in normal and cancer cells. Full article

2-Methyl-6-(phenylethynyl)pyridine (MPEP), a negative allosteric modulator of the metabotropic glutamate receptor (mGluR) 5, protects hepatocytes from ischemic injury. In astrocytes and microglia, MPEP depletes ATP. These findings seem to be self-contradictory, since ATP depletion is a fundamental stressor in ischemia. This study attempted to reconstruct the mechanism of MPEP-mediated ATP depletion and the consequences of ATP depletion on protection against ischemic injury. We compared the effects of MPEP and other mGluR5 negative modulators on ATP concentration when measured in rat hepatocytes and acellular solutions. We also evaluated the effects of mGluR5 blockade on viability in rat hepatocytes exposed to hypoxia. Furthermore, we studied the effects of MPEP treatment on mouse livers subjected to cold ischemia and warm ischemia reperfusion. We found that MPEP and 3-[(2-methyl-1,3-thiazol-4-yl)ethynyl]pyridine (MTEP) deplete ATP in hepatocytes and acellular solutions, unlike fenobam. This finding suggests that mGluR5s may not be involved, contrary to previous reports. MPEP, as well as MTEP and fenobam, improved hypoxic hepatocyte viability, suggesting that protection against ischemic injury is independent of ATP depletion. Significantly, MPEP protected mouse livers in two different ex vivo models of ischemia reperfusion injury, suggesting its possible protective deployment in the treatment of hepatic inflammatory conditions. Full article

Cytoplasmic male sterility (CMS) where no functional pollen is produced has important roles in wheat breeding. The anther is a unique organ for male gametogenesis and its abnormal development can cause male sterility. However, the mechanisms and regulatory networks related to plant male sterility are poorly understood. In this study, we conducted comparative analyses using isobaric tags for relative and absolute quantification (iTRAQ) of the pollen proteins in a CMS line and its wheat maintainer. Differentially abundant proteins (DAPs) were analyzed based on Gene Ontology classifications, metabolic pathways and transcriptional regulation networks using Blast2GO. We identified 5570 proteins based on 23,277 peptides, which matched with 73,688 spectra, including proteins in key pathways such as glyceraldehyde-3-phosphate dehydrogenase, pyruvate kinase and 6-phosphofructokinase 1 in the glycolysis pathway, isocitrate dehydrogenase and citrate synthase in the tricarboxylic acid cycle and nicotinamide adenine dinucleotide (NADH)-dehydrogenase and adenosine-triphosphate (ATP) synthases in the oxidative phosphorylation pathway. These proteins may comprise a network that regulates male sterility in wheat. Quantitative real time polymerase chain reaction (qRT-PCR) analysis, ATP assays and total sugar assays validated the iTRAQ results. These DAPs could be associated with abnormal pollen grain formation and male sterility. Our findings provide insights into the molecular mechanism related to male sterility in wheat. Full article

The maintenance of ordinal cell cycle phases is a critical biological process in cancer genesis, which is a crucial target for anti-cancer drugs. As an important natural isoquinoline alkaloid from Chinese herbal medicine, Berberine (BBR) has been reported to possess anti-cancer potentiality to induce cell cycle arrest in hepatocellular carcinoma cells (HCC). However, the underlying mechanism remains to be elucidated. In our present study, G0/G1 phase cell cycle arrest was observed in berberine-treated Huh-7 and HepG2 cells. Mechanically, we observed that BBR could deactivate the Akt pathway, which consequently suppressed the S-phase kinase-associated protein 2 (Skp2) expression and enhanced the expression and translocation of Forkhead box O3a (FoxO3a) into nucleus. The translocated FoxO3a on one hand could directly promote the transcription of cyclin-dependent kinase inhibitors (CDKIs) p21^Cip1 and p27^Kip1, on the other hand, it could repress Skp2 expression, both of which lead to up-regulation of p21^Cip1 and p27^Kip1, causing G0/G1 phase cell cycle arrest in HCC. In conclusion, BBR promotes the expression of CDKIs p21^Cip1 and p27^Kip1 via regulating the Akt/FoxO3a/Skp2 axis and further induces HCC G0/G1 phase cell cycle arrest. This research uncovered a new mechanism of an anti-cancer effect of BBR. Full article

Recent studies have demonstrated that flavonoid glucuronides can be deconjugated to the active form aglycone by β-glucuronidase-expressing macrophages. Keigairengyoto (KRT) is a flavonoid-rich traditional Japanese medicine reported to enhance bacterial clearance through immune modulation. Our aims are to examine the pharmacokinetics of KRT flavonoids and to identify active flavonoids contributing to the adjuvant effects of KRT. KRT was evaluated at pharmacokinetic analysis to quantify absorbed flavonoids, and cutaneous infection assay induced in mice by inoculation of Staphylococcus aureus. Preventive or therapeutic KRT administration reduced the number of bacteria in the infection site as well as macroscopic and microscopic lesion scores with efficacies similar to antibiotics. Pharmacokinetic study revealed low plasma levels of flavonoid aglycones after KRT administration; however, plasma concentrations were enhanced markedly by β-glucuronidase treatment, with baicalein the most abundant (C_max, 1.32 µg/mL). In random screening assays, flavonoids such as bacalein, genistein, and apigenin enhanced bacteria phagocytosis by macrophages. Glucuronide bacalin was converted to aglycone baicalein by incubation with living macrophages, macrophage lysate, or skin homogenate. Taken together, the adjuvant effect of KRT may be due to some blood-absorbed flavonoids which enhance macrophage functions in host defense. Flavonoid-rich KRT may be a beneficial treatment for infectious skin inflammation. Full article

Bone tissue engineering is one of the main branches of regenerative medicine. In this field, the use of a scaffold, which supported bone development, in combination with mesenchymal stem cells (MSCs), has promised better outcomes for bone regeneration. In particular, human gingival mesenchymal stem cells (hGMSCs) may present advantages compared to other MSCs, including the easier isolation. However, MSCs’ secretome has attracted much attention for its potential use in tissue regeneration, such as conditioned medium (CM) that contains different soluble factors proved to be useful for the regenerative purposes. In this study, we evaluated the osteogenic capacity of a poly-(lactide) (3D-PLA) scaffold enriched with hGMSCs and hGMSCs derived CM and its ability to regenerate bone defects in rat calvarias. 3D-PLA alone, 3D-PLA + CM or 3D-PLA + hGMSCs with/without CM were implanted in Wistar male rats subjected to calvarial defects. We observed that 3D-PLA scaffold enriched with hGMSCs and CM showed a better osteogenic capacity, being able to repair the calvarial defect as revealed in vivo by morphological evaluation. Moreover, transcriptomic analysis in vitro revealed the upregulation of genes involved in ossification and regulation of ossification in the 3D-PLA + CM + hGMSCs group. All of these results indicate the great osteogenic ability of 3D-PLA + CM + hGMSCs supporting its use in bone regenerative medicine, in particular in the repair of cranial bone defects. Especially, hGMSCs derived CM played a key role in the induction of the osteogenic process and in bone regeneration. Full article

The identification of components of the kallikrein–kinin system in the vitreous from patients with microvascular retinal diseases suggests that bradykinin (BK) signaling may contribute to pathogenesis of retinal vascular complications. BK receptor 2 (B2R) signaling has been implicated in both pro-inflammatory and pro-angiogenic effects promoted by BK. Here, we investigated the role of BK/B2R signaling in the retinal neovascularization in the oxygen-induced retinopathy (OIR) model. Blockade of B2R signaling by the antagonist fasitibant delayed retinal vascularization in mouse pups, indicating that the retinal endothelium is a target of the BK/B2R system. In the rabbit cornea assay, a model of pathological neoangiogenesis, the B2 agonist kallidin induced vessel sprouting and promoted cornea opacity, a sign of edema and tissue inflammation. In agreement with these results, in the OIR model, a blockade of B2R signaling significantly reduced retinal neovascularization, as determined by the area of retinal tufts, and, in the retinal vessel, it also reduced vascular endothelial growth factor and fibroblast growth factor-2 expression. All together, these findings show that B2R blockade reduces retinal neovascularization and inhibits the expression of proangiogenic and pro-inflammatory cytokines, suggesting that targeting B2R signaling may be an effective strategy for treating ischemic retinopathy. Full article

Oligodendrocyte progenitor cells (OPCs) constitute one of the main populations of dividing cells in the central nervous system (CNS). Physiologically, OPCs give rise to mature, myelinating oligodendrocytes and confer trophic support to their neighboring cells within the nervous tissue. OPCs are known to be extremely sensitive to the influence of exogenous clues which might affect their crucial biological processes, like survival, proliferation, differentiation, and the ability to generate a myelin membrane. Alterations in their differentiation influencing their final potential for myelinogenesis are usually the leading cause of CNS dys- and demyelination, contributing to the development of leukodystrophic disorders. The evaluation of the mechanisms that cause oligodendrocytes to malfunction requires detailed studies based on designed in vitro models. Since OPCs readily respond to changes in local homeostasis, it is crucial to establish restricted culture conditions to eliminate the potential stimuli that might influence oligodendrocyte biology. Additionally, the in vitro settings should mimic the physiological conditions to enable the obtained results to be translated to future preclinical studies. Therefore, the aim of our study was to investigate OPC differentiation in physiological normoxia (5% O₂) and a restricted in vitro microenvironment. To evaluate the impact of the combined microenvironmental clues derived from other components of the nervous tissue, which are also influenced by the local oxygen concentration, the process of generating OPCs was additionally analyzed in organotypic hippocampal slices. The obtained results show that OPC differentiation, although significantly slowed down, proceeded correctly through its typical stages in the physiologically relevant conditions created in vitro. The established settings were also conducive to efficient cell proliferation, exerting also a neuroprotective effect by promoting the proliferation of neurons. In conclusion, the performed studies show how oxygen tension influences OPC proliferation, differentiation, and their ability to express myelin components, and should be taken into consideration while planning preclinical studies, e.g., to examine neurotoxic compounds or to test neuroprotective strategies. Full article

A tumor vasculature network undergoes intense growth and rebuilding during tumor growth. Traditionally, vascular networks are histologically examined using parameters such as vessel density determined from two-dimensional slices of the tumor. Two-dimensional probing of a complicated three-dimensional (3D) structure only provides partial information. Therefore, we propose the use of microcomputed tomography (micro-CT) imaging to analyze the evolution of a tumor vasculature in an experimental ocular tumor model. A Bomirski Hamster Melanoma was implanted in the anterior chamber of a hamster eye. Ultrasound (US) imaging of the same tumor was performed in vivo, and the vascular results obtained using the two methods were compared. Normal ocular tissues, a tumor, and a tumor vascular structure were revealed with high accuracy using micro-CT. The vessels that grew within the tumor were chaotic, leaky, and contained many convoluted micro-vessels and embolizations. They comprised 20–38% of the tumor mass. The blood flow in the larger functional vessels was in the range from 10 to 25 mm/s, as determined by in vivo Doppler US. The micro-CT imaging of the hamster eyeball enabled both qualitative and quantitative 3D analyses of the globe at a histological level. Although the presented images were obtained ex vivo, micro-CT noninvasive imaging is being developed intensively, and high-resolution in vivo imaging is feasible. Full article

Replicative capacity of a cell is strongly correlated with telomere length regulation. Aberrant lengthening or reduction in the length of telomeres can lead to health anomalies, such as cancer or premature aging. Telomerase is a master regulator for maintaining replicative potential in most eukaryotic cells. It does so by controlling telomere length at chromosome ends. Akin to cancer cells, most single-cell eukaryotic pathogens are highly proliferative and require persistent telomerase activity to maintain constant length of telomere and propagation within their host. Although telomerase is key to unlimited cellular proliferation in both cases, not much was known about the role of telomerase in human parasites (malaria, Trypanosoma, etc.) until recently. Since telomerase regulation is mediated via its own structural components, interactions with catalytic reverse transcriptase and several factors that can recruit and assemble telomerase to telomeres in a cell cycle-dependent manner, we compare and discuss here recent findings in telomerase biology in cancer, aging and parasitic diseases to give a broader perspective of telomerase function in human diseases. Full article

α-Synuclein (αS) is an intrinsically disordered protein that is associated with Parkinson’s disease (PD) through its ability to self-assemble into oligomers and fibrils. Inhibition of this oligomerization cascade is an interesting approach to developing therapeutical strategies and β-synuclein (βS) has been described as a natural negative regulator of this process. However, the biological background and molecular mechanisms by which this inhibition occurs is unclear. Herein, we focused on assessing the effect of βS on the aggregation of five αS pathological mutants linked to early-onset PD (A30P, E46K, H50Q, G51D and A53T). By coupling single molecule fluorescence spectroscopy to a cell-free protein expression system, we validated the ability of βS to act as a chaperone of αS, effectively inhibiting its aggregation. Interestingly, we found that βS does so in a selective manner, i.e., is a more effective inhibitor for certain αS pathological mutants—A30P and G51D—as compared to E46K, H50Q and A53T. Moreover, two-color coincidence experiments proved that this discrepancy is due to a preferential incorporation of βS into smaller oligomers of αS. This was validated by showing that the chaperoning effect was lost when proteins were mixed after being expressed individually. This study highlights the potential of fluorescence spectroscopy to deconstruct αS aggregation cascade and its interplay with βS. Full article

Lignin is one of the main components of plant cell wall and it is a natural phenolic polymer with high molecular weight, complex composition and structure. Lignin biosynthesis extensively contributes to plant growth, tissue/organ development, lodging resistance and the responses to a variety of biotic and abiotic stresses. In the present review, we systematically introduce the biosynthesis of lignin and its regulation by genetic modification and summarize the main biological functions of lignin in plants and their applications. We hope this review will give an in-depth understanding of the important roles of lignin biosynthesis in various plants’ biological processes and provide a theoretical basis for the genetic improvement of lignin content and composition in energy plants and crops. Full article

Amyloid-β and α-synuclein are intrinsically disordered proteins (IDPs), which are at the center of Alzheimer’s and Parkinson’s disease pathologies, respectively. These IDPs are extremely flexible and do not adopt stable structures. Furthermore, both amyloid-β and α-synuclein can form toxic oligomers, amyloid fibrils and other type of aggregates in Alzheimer’s and Parkinson’s diseases. Experimentalists face challenges in investigating the structures and thermodynamic properties of these IDPs in their monomeric and oligomeric forms due to the rapid conformational changes, fast aggregation processes and strong solvent effects. Classical molecular dynamics simulations complement experiments and provide structural information at the atomic level with dynamics without facing the same experimental limitations. Artificial missense mutations are employed experimentally and computationally for providing insights into the structure-function relationships of amyloid-β and α-synuclein in relation to the pathologies of Alzheimer’s and Parkinson’s diseases. Furthermore, there are several natural genetic variations that play a role in the pathogenesis of familial cases of Alzheimer’s and Parkinson’s diseases, which are related to specific genetic defects inherited in dominant or recessive patterns. The present review summarizes the current understanding of monomeric and oligomeric forms of amyloid-β and α-synuclein, as well as the impacts of artificial and pathological missense mutations on the structural ensembles of these IDPs using molecular dynamics simulations. We also emphasize the recent investigations on residual secondary structure formation in dynamic conformational ensembles of amyloid-β and α-synuclein, such as β-structure linked to the oligomerization and fibrillation mechanisms related to the pathologies of Alzheimer’s and Parkinson’s diseases. This information represents an important foundation for the successful and efficient drug design studies. Full article

Candida albicans is the most commonly encountered human fungal pathogen, and it is traditionally treated with antimicrobial chemical agents. The antimicrobial effect of these agents is largely weakened by drug resistance and biofilm-associated virulence. Enhancement of the antimicrobial activity of existing agents is needed for effective candidiasis treatment. Our aim was to develop a therapy that combined biofilm disruption with existing antimicrobial agents. Photodynamic therapy (PDT) utilizing curcumin and blue light was tested as an independent therapy and in combination with fluconazole treatment. Viability assays and morphology analysis were used to assess the effectiveness of C. albicans treatment. Results showed that fluconazole treatment decreased the viability of planktonic C. albicans, but the decrease was not as pronounced in adherent C. albicans because its biofilm form was markedly more resistant to the antimicrobiotic. PDT effectively eradicated C. albicans biofilms, and when combined with fluconazole, PDT significantly inhibited C. albicans to a greater extent. This study suggests that the addition of PDT to fluconazole to treat C. albicans infection enhances its effectiveness and can potentially be used clinically. Full article

Helicobacter pylori (Hp) is one of the most important human pathogens that can cause duodenal and gastric ulcers, gastritis and stomach cancer. Hp infection is considered to be a cause of limiting access to bariatric surgery. The aim of this study was to determine the prevalence of Hp in patients with obesity going into bariatric surgery and to reveal the relationship between Hp and clinical data. The study group was formed of 68 preoperative bariatric surgery patients (body mass index (BMI) 44.7 ± 4.8). Gastric biopsies (antrum and corpus) were used for histological and molecular (caqA and glmM genes) examinations. The PCR method revealed Hp infection in 64.7% of obese patients that is higher in comparison with histological analysis (55.9%). The prevalence of cagA and glmM genes in antrum mucosa was 45.6% and 47.0% while in the corpus it was 41.2% and 38.3%, respectively. The coincidence of both cagA and glmM virulence genes in the antrum and corpus mucosa was 33.8% and 22.1%, respectively. Either of the genes was found in 58.8% of antrum and 57.3% of corpus mucosa. Presence of caqA and glmM genes was in association with active and atrophic chronic gastritis. In conclusion, our study demonstrated that two thirds of morbidly obese patients undergoing bariatric surgery are infected with Hp and have a high prevalence of cagA and glmM virulence genes that points out the necessity for diagnostics and treatment of this infection before surgery. Full article

Modulation of the immune system by cancer protective food bioactives has preventive and therapeutic importance in prostate cancer, but the mechanisms remain largely unclear. The current study tests the hypothesis that the diet-derived cancer protective compounds, indole-3-carbinol (I3C) and 3,3′-diindolylmethane (DIM), affect the tumor microenvironment by regulation of inflammatory responses in monocytes and macrophages. We also ask whether I3C and DIM act through the aryl hydrocarbon (AHR)-dependent pathway or the signaling lymphocyte activation molecule (SLAM) family protein CD84-mediated pathway. The effect of I3C and DIM was examined using the human THP-1 monocytic cell in its un-differentiated (monocyte) and differentiated (macrophage) state. We observed that I3C and DIM inhibited lipopolysaccharide (LPS) induction of IL-1β mRNA and protein in the monocyte form but not the macrophage form of THP-1. Interestingly, CD84 mRNA but not protein was inhibited by I3C and DIM. AHR siRNA knockdown experiments confirmed that the inhibitory effects of I3C and DIM on IL-1β as well as CD84 mRNA are regulated through AHR-mediated pathways. Additionally, the AHR ligand appeared to differentially regulate other LPS-induced cytokines expression. Hence, cross-talk between AHR and inflammation-mediated pathways, but not CD84-mediated pathways, in monocytes but not macrophages may contribute to the modulation of tumor environments by I3C and DIM in prostate cancer. Full article

Chimeric antigen receptor (CAR) T cell therapy represents the first U.S. Food and Drug Administration approved gene therapy and these engineered cells function with unprecedented efficacy in the treatment of refractory CD19 positive hematologic malignancies. CAR translation to solid tumors is also being actively investigated; however, efficacy to date has been variable due to tumor-evolved mechanisms that inhibit local immune cell activity. To bolster the potency of CAR-T cells, modulation of the immunosuppressive tumor microenvironment with immune-checkpoint blockade is a promising strategy. The impact of this approach on hematological malignancies is in its infancy, and in this review we discuss CAR-T cells and their synergy with immune-checkpoint blockade. Full article

Antidepressant-like effects of ethanolic extract of Hericium erinaceus (HE) mycelium enriched in erinacine A on depressive mice challenged by repeated restraint stress (RS) were examined. HE at 100, 200 or 400 mg/kg body weight/day was orally given to mice for four weeks. After two weeks of HE administration, all mice except the control group went through with 14 days of RS protocol. Stressed mice exhibited various behavioral alterations, such as extending immobility time in the tail suspension test (TST) and forced swimming test (FST), and increasing the number of entries in open arm (POAE) and the time spent in the open arm (PTOA). Moreover, the levels of norepinephrine (NE), dopamine (DA) and serotonin (5-HT) were decreased in the stressed mice, while the levels of interleukin (IL)-6 and tumor necrosis factor (TNF)-α were increased. These changes were significantly inverted by the administration of HE, especially at the dose of 200 or 400 mg/kg body weight/day. Additionally, HE was shown to activate the BDNF/TrkB/PI3K/Akt/GSK-3β pathways and block the NF-κB signals in mice. Taken together, erinacine A-enriched HE mycelium could reverse the depressive-like behavior caused by RS and was accompanied by the modulation of monoamine neurotransmitters as well as pro-inflammatory cytokines, and regulation of BDNF pathways. Therefore, erinacine A-enriched HE mycelium could be an attractive agent for the treatment of depressive disorders. Full article

Osteoarthritis (OA) is a joint disease in which cartilage degenerates as a result of mechanical and biochemical changes. The main OA symptom is chronic pain involving both peripheral and central mechanisms of nociceptive processing. Our previous studies have implicated the benefits of dual- over single-acting compounds interacting with the endocannabinoid system (ECS) in OA treatment. In the present study, we focused on the specific molecular alterations associated with pharmacological treatment. OA was induced in Wistar rats by intra-articular injection of 3 mg of monoiodoacetate (MIA). Single target compounds (URB597, an FAAH inhibitor, and SB366791, a TRPV1 antagonist) and a dual-acting compound OMDM198 (FAAH inhibitor/TRPV1 antagonist) were used in the present study. At day 21 post-MIA injection, rats were sacrificed 1 h after i.p. treatment, and changes in mRNA expression were evaluated in the lumbar spinal cord by RT-qPCR. Following MIA administration, we observed 2-4-fold increase in mRNA expression of targeted receptors (Cnr1, Cnr2, and Trpv1), endocannabinoid degradation enzymes (Faah, Ptgs2, and Alox12), and TRPV1 sensitizing kinases (Mapk3, Mapk14, Prkcg, and Prkaca). OMDM198 treatment reversed some of the MIA effects on the spinal cord towards intact levels (Alox12, Mapk14, and Prkcg). Apparent regulation of ECS and TRPV1 in response to pharmacological intervention is a strong justification for novel ECS-based multi-target drug treatment in OA. Full article

Chronic obstructive pulmonary disease (COPD) is associated with severe chronic inflammation that promotes irreversible tissue destruction. Moreover, the most broadly accepted cause of COPD is exposure to cigarette smoke. There is no effective cure and significantly, the mechanism behind the development and progression of this disease remains unknown. Our laboratory has demonstrated that Bruton’s tyrosine kinase (Btk) is a critical regulator of pro-inflammatory processes in the lungs and that Btk controls expression of matrix metalloproteinase-9 (MMP-9) in the alveolar compartment. For this study apolipoprotein E null (ApoE^−/−) mice were exposed to SHS to facilitate study in a COPD/atherosclerosis comorbidity model. We applied two types of treatments, animals received either a pharmacological inhibitor of Btk or MMP-9 specific siRNA to minimize MMP-9 expression in endothelial cells or neutrophils. We have shown that these treatments had a protective effect in the lung. We have noted a decrease in alveolar changes related to SHS induced inflammation in treated animals. In summary, we are presenting a novel concept in the field of COPD, i.e., that Btk may be a new drug target for this disease. Moreover, cell specific targeting of MMP-9 may also benefit patients affected by this disease. Full article

Recent experiments showed a potential cardiotoxic effect of the macrolide antibiotic (tulathromycin). This study was performed to investigate whether diclofenac sodium (DFS) potentiates the cardiotoxicity of tulathromycin and increases the cardioprotective effects of lycopene against DFS and tulathromycin. Seven groups (eight per group) of adult Swiss albino mice received saline (control), tulathromycin (a single subcutaneous dose of 28 mg/kg/bw on day 14), DFS (a single oral dose of 100 mg/kg/bw on day 14), tulathromycin plus DFS, or lycopene (oral, 10 mg/kg/bw daily for 15 d) combined with tulathromycin, DFS, or both. Compared to the control group, the administration of tulathromycin or DFS (individually or in combination) caused significantly elevated (p < 0.05) serum levels of Creatine kinase-myocardial B fraction (CK-MB), lactate dehydrogenase, and cardiac-specific troponin-T and tissue levels of nitric oxide and malondialdehyde that were accompanied by significantly decreased tissue reduced glutathione content and glutathione peroxidase, superoxide dismutase, and catalase antioxidant enzyme activity. Upon histopathological and immunohistochemical examination, the mean pathology scores and the percentages of caspase-3-, Bax-, and CK-positive regions were significantly higher in the tulathromycin- and/or DFS-treated groups than in control mice. For all these parameters, the pathological changes were more significant in the tulathromycin–DFS combination group than in mice treated with either drug individually. Interestingly, co-administration of lycopene with tulathromycin and/or DFS significantly ameliorated the changes described above. In conclusion, DFS could potentiate the cardiotoxic effects of tulathromycin, whereas lycopene can serve as a cardioprotective agent against DFS and tulathromycin. Full article

N-acetylglutamate synthase deficiency (NAGSD) is an extremely rare urea cycle disorder (UCD) with few adult cases so far described. Diagnosis of late-onset presentations is difficult and delayed treatment may increase the risk of severe hyperammonemia. We describe a 52-year-old woman with recurrent headaches who experienced an acute onset of NAGSD. As very few papers focus on headaches in UCDs, we also report a literature review of types and pathophysiologic mechanisms of UCD-related headaches. In our case, headaches had been present since puberty (3–4 days a week) and were often accompanied by nausea, vomiting, or behavioural changes. Despite three previous episodes of altered consciousness, ammonia was measured for the first time at 52 years and levels were increased. Identification of the new homozygous c.344C>T (p.Ala115Val) NAGS variant allowed the definite diagnosis of NAGSD. Bioinformatic analysis suggested that an order/disorder alteration of the mutated form could affect the arginine-binding site, resulting in poor enzyme activation and late-onset presentation. After optimized treatment for NAGSD, ammonia and amino acid levels were constantly normal and prevented other headache bouts. The manuscript underlies that headache may be the presenting symptom of UCDs and provides clues for the rapid diagnosis and treatment of late-onset NAGSD. Full article

Cationic liposomes are frequently used as carrier particles for nucleic acid delivery. The most popular formulation is the equimolar mixture of two components, a cationic lipid and a neutral phosphoethanolamine. Its uptake pathway has been described as endocytosis. The presence of an aromatic molecule as a third component strongly influences the cellular uptake process and results in complete membrane fusion instead of endocytosis. Here, we systematically varied all three components of this lipid mixture and determined how efficiently the resulting particles fused with the plasma membrane of living mammalian cells. Our results show that an aromatic molecule and a cationic lipid component with conical molecular shape are essential for efficient fusion induction. While a neutral lipid is not mandatory, it can be used to control fusion efficiency and, in the most extreme case, to revert the uptake mechanism back to endocytosis. Full article

Tuberculosis remains one of the deadliest diseases. Emergence of drug-resistant and multidrug-resistant M. tuberculosis strains makes treating tuberculosis increasingly challenging. In order to develop novel intervention strategies, detailed understanding of the molecular mechanisms behind the success of this pathogen is required. Here, we review recent literature to provide a systems level overview of the molecular and cellular components involved in divalent metal homeostasis and their role in regulating the three main virulence strategies of M. tuberculosis: immune modulation, dormancy and phagosomal rupture. We provide a visual and modular overview of these components and their regulation. Our analysis identified a single regulatory cascade for these three virulence strategies that respond to limited availability of divalent metals in the phagosome. Full article

Institute Goeorges Lopez 1 (IGL-1) and Histidine-Tryptophan-Ketoglutarate (HTK) preservation solutions are regularly used in clinical for liver transplantation besides University of Wisconsin (UW) solution and Celsior. Several clinical trials and experimental works have been carried out comparing all the solutions, however the comparative IGL-1 and HTK appraisals are poor; especially when they deal with the underlying protection mechanisms of the fatty liver graft during cold storage. Fatty livers from male obese Zücker rats were conserved for 24 h at 4 °C in IGL-1 or HTK preservation solutions. After organ recovery and rinsing of fatty liver grafts with Ringer Lactate solution, we measured the changes in mechanistic target of rapamycin (mTOR) signaling activation, liver autophagy markers (Beclin-1, Beclin-2, LC3B and ATG7) and apoptotic markers (caspase 3, caspase 9 and TUNEL). These determinations were correlated with the prevention of liver injury (aspartate and alanine aminostransferase (AST/ALT), histology) and mitochondrial damage (glutamate dehydrogenase (GLDH) and confocal microscopy findings). Liver grafts preserved in IGL-1 solution showed a marked reduction on p-TOR/mTOR ratio when compared to HTK. This was concomitant with significant increased cyto-protective autophagy and prevention of liver apoptosis, including inflammatory cytokines such as HMGB1. Together, our results revealed that IGL-1 preservation solution better protected fatty liver grafts against cold ischemia damage than HTK solution. IGL-1 protection was associated with a reduced liver damage, higher induced autophagy and decreased apoptosis. All these effects would contribute to limit the subsequent extension of reperfusion injury after graft revascularization in liver transplantation procedures. Full article

With the increasing interest in the potential benefits of nanotechnologies, concern is still growing that they may present emerging risks for workers. Various strategies have been developed to assess the exposure to nano-objects and their agglomerates and aggregates (NOAA) in the workplace, integrating different aerosol measurement instruments and taking into account multiple parameters that may influence NOAA toxicity. The present study proposes a multi-metric approach for measuring and sampling NOAA in the workplace, applied to three case studies in laboratories each dedicated to materials with different shapes and dimensionalities: graphene, nanowires, and nanoparticles. The study is part of a larger project with the aim of improving risk management tools in nanomaterials research laboratories. The harmonized methodology proposed by the Organization for Economic Cooperation and Development (OECD) has been applied, including information gathering about materials and processes, measurements with easy-to-use and hand-held real-time devices, air sampling with personal samplers, and off-line analysis using scanning electron microscopy. Significant values beyond which an emission can be attributed to the NOAA production process were identified by comparison of the particle number concentration (PNC) time series and the corresponding background levels in the three laboratories. We explored the relations between background PNC and microclimatic parameters. Morphological and elemental analysis of sampled filters was done to identify possible emission sources of NOAA during the production processes: rare particles, spherical, with average diameter similar to the produced NOAA were identified in the nanoparticles laboratory, so further investigation is recommended to confirm the potential for worker exposure. In conclusion, the information obtained should provide a valuable basis for improving risk management strategies in the laboratory at work. Full article

Banana Fusarium wilt caused by Fusarium oxysporum f. sp. cubense (Foc) is one of the most destructive soil-borne diseases. In this study, young tissue-cultured plantlets of banana (Musa spp. AAA) cultivars differing in Foc susceptibility were used to reveal their differential responses to this pathogen using digital gene expression (DGE). Data were evaluated by various bioinformatic tools (Venn diagrams, gene ontology (GO) annotation and Kyoto encyclopedia of genes and genomes (KEGG) pathway analyses) and immunofluorescence labelling method to support the identification of gene candidates determining the resistance of banana against Foc. Interestingly, we have identified MaWRKY50 as an important gene involved in both constitutive and induced resistance. We also identified new genes involved in the resistance of banana to Foc, including several other transcription factors (TFs), pathogenesis-related (PR) genes and some genes related to the plant cell wall biosynthesis or degradation (e.g., pectinesterases, β-glucosidases, xyloglucan endotransglucosylase/hydrolase and endoglucanase). The resistant banana cultivar shows activation of PR-3 and PR-4 genes as well as formation of different constitutive cell barriers to restrict spreading of the pathogen. These data suggest new mechanisms of banana resistance to Foc. Full article

Pressure-driven membrane-based technologies represent a valid approach to reduce the environmental pollution of several agro-food by-products. Recently, in relation to the major interest for natural compounds with biological activities, their use has been also addressed to the recovery, separation and fractionation of phenolic compounds from such by-products. In particular, tight ultrafiltration (UF) and nanolfiltration (NF) membranes have been recognized for their capability to recover phenolic compounds from several types of agro-food by-products. The separation capability of these membranes, as well as their productivity, depends on multiple factors such as membrane material, molecular weight cut-off (MWCO) and operating conditions (e.g., pressure, temperature, feed flow rate, volume reduction factor, etc.). This paper aims at providing a critical overview of the influence of these parameters on the recovery of phenolic compounds from agro-food by-products by using tight UF and NF membranes. The literature data are analyzed and discussed in relation to separation processes, molecule properties, membrane characteristics and other phenomena occurring in the process. Current extraction methodologies of phenolic compounds from raw materials are also introduced in order to drive the implementation of integrated systems for the production of actractive phenolic formulations of potential interest as food antioxidants. Full article

Mesenchymal stem cells (MSCs) could be a promising solution in the treatment of various diseases including chronic kidney disease (CKD). However, endoplasmic reticulum (ER) stress induced by ischemia in the area of application limits the integration and survival of MSCs in patients. In our study, we generated ER stress-induced conditions in MSCs using P-cresol. As P-cresol is a toxic compound accumulated in the body of CKD patients and induces apoptosis and inflammation through reactive oxygen species (ROS), we observed ER stress-induced MSC apoptosis activated by oxidative stress, which in turn resulted from ROS generation. To overcome stress-induced apoptosis, we investigated the protective effects of tauroursodeoxycholic acid (TUDCA), a bile acid, on ER stress in MSCs. In ER stress, TUDCA treatment of MSCs reduced ER stress-associated protein activation, including GRP78, PERK, eIF2α, ATF4, IRE1α, and CHOP. Next, to explore the protective mechanism adopted by TUDCA, TUDCA-mediated cellular prion protein (PrP^C) activation was assessed. We confirmed that PrP^C expression significantly increased ROS, which was eliminated by superoxide dismutase and catalase in MSCs. These findings suggest that TUDCA protects from inflammation and apoptosis in ER stress via PrP^C expression. Our study demonstrates that TUDCA protects MSCs against inflammation and apoptosis in ER stress by PrP^C expression in response to P-cresol exposure. Full article

Potassium channel openers (KCOs) have been shown to play a role in cytoprotection through the activation of mitochondrial potassium channels. Recently, in several reports, a number of data has been described as off-target actions for KCOs. In the present study, we investigated the effects of BK_Ca channel openers CGS7181, CGS7184, NS1619, and NS004 in neuronal cells. For the purpose of this research, we used a rat brain, the mouse hippocampal HT22 cells, and the human astrocytoma U-87 MG cell line. We showed that CGS7184 activated the mitochondrial BK_Ca (mitoBK_Ca) channel in single-channel recordings performed on astrocytoma mitoplasts. Moreover, when applied to the rat brain homogenate or isolated rat brain mitochondria, CGS7184 increased the oxygen consumption rate, and can thus be considered a potentially cytoprotective agent. However, experiments on intact neuronal HT22 cells revealed that both CGS7181 and CGS7184 induced HT22 cell death in a concentration- and time-dependent manner. By contrast, we did not observe cell death when NS1619 or NS004 was applied. CGS7184 toxicity was not abolished by BK_Ca channel inhibitors, suggesting that the observed effects were independent of a BK_Ca-type channel activity. CGS7184 treatment resulted in an increase of cytoplasmic Ca²⁺ concentration that likely involved efflux from internal calcium stores and the activation of calpains (calcium-dependent proteases). The cytotoxic effect of the channel opener was partially reversed by a calpain inhibitor. Our data show that KCOs under study not only activate mitoBK_Ca channels from brain tissue, but also induce cell death when used in cellular models. Full article

Functionalization has been shown to alter toxicity of multi-walled carbon nanotube (MWCNT) in several studies. This study varied the degree of functionalization (viz., amount of MWCNT surface carboxylation) to define the relationship between the extent of carboxylation and effects in a variety of in vitro cell models and short-term ex vivo/in vivo particle exposures. Studies with vitamin D₃ plus phorbol ester transformed THP-1 macrophages demonstrated that functionalization, regardless of amount, corresponded with profoundly decreased NLRP3 inflammasome activation. However, all MWCNT variants were slightly toxic in this model. Alternatively, studies with A549 epithelial cells showed some varied effects. For example, IL-33 and TNF-α release were related to varying amounts of functionalization. For in vivo particle exposures, autophagy of alveolar macrophages, measured using green fluorescent protein (GFP)- fused-LC3 transgenic mice, increased for all MWCNT tested three days after exposure, but, by Day 7, autophagy was clearly dependent on the amount of carboxylation. The instilled source MWCNT continued to produce cellular injury in alveolar macrophages over seven days. In contrast, the more functionalized MWCNT initially showed similar effects, but reduced over time. Dark-field imaging showed the more functionalized MWCNTs were distributed more uniformly throughout the lung and not isolated to macrophages. Taken together, the results indicated that in vitro and in vivo bioactivity of MWCNT decreased with increased carboxylation. Functionalization by carboxylation eliminated the bioactive potential of the MWCNT in the exposure models tested. The observation that maximally functionalized MWCNT distribute more freely throughout the lung with the absence of cellular damage, and extended deposition, may establish a practical use for these particles as a safer alternative for unmodified MWCNT. Full article

Stress can lead to inflammation, accelerated aging, and some chronic diseases condition. Mentha arvensis (MA) is a traditional medicine having antioxidant and anti-inflammatory activities. The present study investigated the anti-stress role of MA and fermented MA (FMA) extract in immobilized rats. We studied the lipopolysaccharide (LPS)-induced inflammation in RAW 264.7 cells and rats were immobilized for 2 h per day for 14 days using a restraining cage. MA (100 mg/kg) and FMA (100 mg/kg) were orally administered to rats 1 h prior to immobilization. Using high-performance liquid chromatography (HPLC) analysis, we determined the rosmarinic acid content of MA and FMA. The generation of malondialdehyde (MDA) and nitric oxide (NO) in RAW 246.7 cells were suppressed by both MA and FMA. In rats, MA and FMA notably improved the body weight, daily food intake, and duodenum histology. MDA and NO level were gradually decreased by MA and FMA treatment. MA and FMA significantly controlled the stress-related hormones by decreasing corticosterone and β-endorphin and increasing serotonin level. Moreover, protein expression levels of mitogen activated protein kinases (MAPK) and cyclooxygenase-2 (COX-2) were markedly downregulated by MA and FMA. Taken together, MA and FMA could ameliorate immobilized-stress by reducing oxidative stress, regulating stress-related hormones, and MAPK/COX-2 signaling pathways in rats. Particularly, FMA has shown greater anti-stress activities than MA. Full article

The aging process, comorbidities, and age-associated diseases are closely dependent on each other. Cerebral ischemia impacts a wide range of systems in an age-dependent manner. However, the aging process has many facets which are influenced by the genetic background and epigenetic or environmental factors, which can explain why some people age differently than others. Therefore, there is an urgent need to identify age-related changes in body functions or structures that increase the risk for stroke and which are associated with a poor outcome. Multimodal imaging, electrophysiology, cell biology, proteomics, and transcriptomics, offer a useful approach to link structural and functional changes in the aging brain, with or without comorbidities, to post-stroke rehabilitation. This can help us to improve our knowledge about senescence firstly, and in this context, aids in elucidating the pathophysiology of age-related diseases that allows us to develop therapeutic strategies or prevent diseases. These processes, including potential therapeutical interventions, need to be studied first in relevant preclinical models using aged animals, with and without comorbidities. Therefore, preclinical research on ischemic stroke should consider age as the most important risk factor for cerebral ischemia. Furthermore, the identification of effective therapeutic strategies, corroborated with successful translational studies, will have a dramatic impact on the lives of millions of people with cerebrovascular diseases. Full article

Eggplant cultivation is limited by numerous diseases, including the devastating bacterial wilt (BW) caused by the Ralstonia solanacearum species complex (RSSC). Within the RSSC, Ralstonia pseudosolanacearum (including phylotypes I and III) causes severe damage to all solanaceous crops, including eggplant. Therefore, the creation of cultivars resistant to R. pseudosolanacearum strains is a major goal for breeders. An intraspecific eggplant population, segregating for resistance, was created from the cross between the susceptible MM738 and the resistant EG203 lines. The population of 123 doubled haploid lines was challenged with two strains belonging to phylotypes I (PSS4) and III (R3598), which both bypass the published EBWR9 BW-resistance quantitative trait locus (QTL). Ten and three QTLs of resistance to PSS4 and to R3598, respectively, were detected and mapped. All were strongly influenced by environmental conditions. The most stable QTLs were found on chromosomes 3 and 6. Given their estimated physical position, these newly detected QTLs are putatively syntenic with BW-resistance QTLs in tomato. In particular, the QTLs’ position on chromosome 6 overlaps with that of the major broad-spectrum tomato resistance QTL Bwr-6. The present study is a first step towards understanding the complex polygenic system, which underlies the high level of BW resistance of the EG203 line. Full article

Cytokine-induced killer (CIK) cells are T lymphocytes that have acquired, in vitro, following extensive manipulation by Interferon gamma (IFN-γ), OKT3 and Interleukin 2 (IL-2) addition, the expression of several Natural Killer (NK) cell-surface markers. CIK cells have a dual “nature”, due to the presence of functional TCR as well as NK molecules, even if the antitumoral activity can be traced back only to the NK-like structures (DNAM-1, NKG2D, NKp30 and CD56). In addition to antineoplastic activity in vitro and in several in-vivo models, CIK cells show very limited, if any, GvHD toxicity as well as a strong intratumoral homing. For all such reasons, CIK cells have been proposed and tested in many clinical trials in cancer patients both in autologous and allogeneic combinations, up to haploidentical mismatching. Indeed, genetic modification of CIK cells as well as the possibility of combining them with specific monoclonal antibodies will further expand the possibility of their clinical utilization. Full article

Human mesenchymal stem cells (hMSCs) have been widely studied for therapeutic development in tissue engineering and regenerative medicine. They can be harvested from human donors via tissue biopsies, such as bone marrow aspiration, and cultured to reach clinically relevant cell numbers. However, an unmet issue lies in the fact that the hMSC donors for regenerative therapies are more likely to be of advanced age. Their stem cells are not as potent compared to those of young donors, and continue to lose healthy, stemness-related activities when the hMSCs are serially passaged in tissue culture plates. Here, we have developed a cheap, scalable, and effective copolymer film to culture hMSCs obtained from aged human donors over several passages without loss of reactive oxygen species (ROS) handling or differentiation capacity. Assays of cell morphology, reactive oxygen species load, and differentiation potential demonstrate the effectiveness of copolymer culture on reduction in senescence-related activities of aging donor-derived hMSCs that could hinder the therapeutic potential of autologous stem cell therapies. Full article

Osteoporosis is a progressive skeletal disease characterized by decreased bone mass and degraded bone microstructure, which leads to increased bone fragility and risks of bone fracture. Osteoporosis is generally age related and has become a major disease of the world. Uncovering the molecular mechanisms underlying osteoporosis and developing effective prevention and therapy methods has great significance for human health. Mesenchymal stem cells (MSCs) are multipotent cells capable of differentiating into osteoblasts, adipocytes, or chondrocytes, and have become the favorite source of cell-based therapy. Evidence shows that during osteoporosis, a shift of the cell differentiation of MSCs to adipocytes rather than osteoblasts partly contributes to osteoporosis. Thus, uncovering the molecular mechanisms of the osteoblast or adipocyte differentiation of MSCs will provide more understanding of MSCs and perhaps new methods of osteoporosis treatment. The MSCs have been applied to both preclinical and clinical studies in osteoporosis treatment. Here, we review the recent advances in understanding the molecular mechanisms regulating osteoblast differentiation and adipocyte differentiation of MSCs and highlight the therapeutic application studies of MSCs in osteoporosis treatment. This will provide researchers with new insights into the development and treatment of osteoporosis. Full article

Empirically validated interventions addressing childhood psychological problems are now readily available, but success likely depends in part on accurately identifying which children will benefit from which intervention. This pilot study examined the stress activation and response system, first as a way to differentiate high versus low-risk children, and second to explore indicators of the stress system associated with favorable intervention response. Method. Participants (N = 43, 58% male) were school-aged children who qualified for inclusion in the Early Risers “Skills for Success” Prevention Program based on their elevated levels of aggressive and/or socially withdrawn behavior and a normally developing comparison group. Compared to the normally developing group, children who were participants in the intervention exhibited a more blunted cortisol response to the stress paradigm. However, for the children in the intervention group, elevated cortisol levels at the start of the stress paradigm were concurrently associated with internalizing problems and predictive of improvement in internalizing problems over time. These findings provide preliminary evidence that hypothalamic pituitary adrenal (HPA) axis biological variables may be helpful tools for identifying children who would benefit from intervention and personalizing interventions. Full article

Acidic chitinase (Chia) has been implicated in asthma, allergic inflammations, and food processing. We have purified Chia enzymes with striking acid stability and protease resistance from chicken and pig stomach tissues using a chitin column and 8 M urea (urea-Chia). Here, we report that acetic acid is a suitable agent for native Chia purification from the stomach tissues using a chitin column (acetic acid-Chia). Chia protein can be eluted from a chitin column using 0.1 M acetic acid (pH 2.8), but not by using Gly-HCl (pH 2.5) or sodium acetate (pH 4.0 or 5.5). The melting temperatures of Chia are not affected substantially in the elution buffers, as assessed by differential scanning fluorimetry. Interestingly, acetic acid appears to be more effective for Chia-chitin dissociation than do other organic acids with similar structures. We propose a novel concept of this dissociation based on competitive interaction between chitin and acetic acid rather than on acid denaturation. Acetic acid-Chia also showed similar chitinolytic activity to urea-Chia, indicating that Chia is extremely stable against acid, proteases, and denaturing agents. Both acetic acid- and urea-Chia seem to have good potential for supplementation or compensatory purposes in agriculture or even biomedicine. Full article

Though Oldenlandia diffusa Herba (ODH) has been known to exhibit anti-cancer and anti-inflammatory effects, its anti-amnestic effect has never been reported so far. The aim of this present study was to elucidate the anti-amnestic effect of ODH. ODH pretreatment significantly reduced escape latency of scopolamine treated Institute of Cancer Research (ICR) mice compared to untreated control groups in a Morris water maze test. Similarly, the passive avoidance test showed that ODH treatment recovered the scopolamine induced amnesia in the ICR mouse model. Concentration of Ach in brains of ODH treated mice was increased compared to that of scopolamine treated mice. In addition, activity of acetylcholinesterase (AChE) was notably decreased by ODH. The protein expression of brain-derived neurotrophic factor (BDNF) and phospho-cAMP response element-binding protein (p-CREB) (Ser133) was increased in ODH pretreated group compared to control group. Consistently, immunohistochemistry (IHC) revealed the elevated expression of brain-derived neurotrophic factor (BDNF) and p-CREB in brains of ODH treated mice compared to the control group. Overall, these findings suggest that ODH has anti-amnestic potential via activation of BDNF and p-CREB and inhibition of AChE in mice with scopolamine induced amnesia. Full article

Phosphorus (P) is an essential mineral for the development and maintenance of the vertebrate skeletal system. Modulation of P levels is believed to influence metabolism and the physiological responses of gene expression. In this study, we investigated the influence of dietary P on skeletal deformities and osteocalcin gene expression in zebrafish (Danio rerio), and sought to determine appropriate levels in a diet. We analyzed a total of 450 zebrafish within 31 days of hatching. Animals were distributed in a completely randomized experimental design that consisted of five replications. After an eight-week experiment, fish were diaphanized to evaluate cranial and spinal bone deformities. Increases in dietary phosphorus were inversely proportional to the occurrence of partial spine fusions, the absence of spine fusions, absence of parallelism between spines, intervertebral spacing, vertebral compression, scoliosis, lordosis, ankylosis, fin caudal insertion, and craniofacial deformities. Additionally, osteocalcin expression was inversely correlated to P levels, suggesting a physiological recovery response for bone mineralization deficiency. Our data showed that dietary P concentration was a critical factor in the occurrence of zebrafish skeletal abnormalities. We concluded that 1.55% P in the diet significantly reduces the appearance of skeletal deformities and favors adequate bone mineralization through the adjustment of osteocalcin expression. Full article

The interaction strength of progressively longer oligomers of glycine, (Gly), di-Gly, tri-Gly, and penta-Gly, with a natural pyrite surface was directly measured using the force mode of an atomic force microscope (AFM). In recent years, selective activation of abiotically formed amino acids on mineral surfaces, especially that of pyrite, has been proposed as an important step in many origins of life scenarios. To investigate such notions, we used AFM-based force measurements to probe possible non-covalent interactions between pyrite and amino acids, starting from the simplest amino acid, Gly. Although Gly itself interacted with the pyrite surface only weakly, progressively larger unbinding forces and binding frequencies were obtained using oligomers from di-Gly to penta-Gly. In addition to an expected increase of the configurational entropy and size-dependent van der Waals force, the increasing number of polar peptide bonds, among others, may be responsible for this observation. The effect of chain length was also investigated by performing similar experiments using l-lysine vs. poly-l-lysine (PLL), and l-glutamic acid vs. poly-l-glutamic acid. The results suggest that longer oligomers/polymers of amino acids can be preferentially adsorbed on pyrite surfaces. Full article

More than 140 million people live and works (in a chronic or intermittent form) above 2500 m worldwide and 35 million live in the Andean Mountains. Furthermore, in Chile, it is estimated that 55,000 persons work in high altitude shifts, where stays at lowlands and interspersed with working stays at highlands. Acute exposure to high altitude has been shown to induce oxidative stress in healthy human lowlanders, due to an increase in free radical formation and a decrease in antioxidant capacity. However, in animal models, intermittent hypoxia (IH) induce preconditioning, like responses and cardioprotection. Here, we aimed to describe in a rat model the responses on cardiac and vascular function to 4 cycles of intermittent hypobaric hypoxia (IHH). Twelve adult Wistar rats were randomly divided into two equal groups, a four-cycle of IHH, and a normobaric hypoxic control. Intermittent hypoxia was induced in a hypobaric chamber in four continuous cycles (1 cycle = 4 days hypoxia + 4 days normoxia), reaching a barometric pressure equivalent to 4600 m of altitude (428 Torr). At the end of the first and fourth cycle, cardiac structural, and functional variables were determined by echocardiography. Thereafter, ex vivo vascular function and biomechanical properties were determined in femoral arteries by wire myography. We further measured cardiac oxidative stress biomarkers (4-Hydroxy-nonenal, HNE; nytrotirosine, NT), reactive oxygen species (ROS) sources (NADPH and mitochondrial), and antioxidant enzymes activity (catalase, CAT; glutathione peroxidase, GPx, and superoxide dismutase, SOD). Our results show a higher ejection and shortening fraction of the left ventricle function by the end of the 4th cycle. Further, femoral vessels showed an improvement of vasodilator capacity and diminished stiffening. Cardiac tissue presented a higher expression of antioxidant enzymes and mitochondrial ROS formation in IHH, as compared with normobaric hypoxic controls. IHH exposure determines a preconditioning effect on the heart and femoral artery, both at structural and functional levels, associated with the induction of antioxidant defence mechanisms. However, mitochondrial ROS generation was increased in cardiac tissue. These findings suggest that initial states of IHH are beneficial for cardiovascular function and protection. Full article

Joint tissues like synovium, articular cartilage, meniscus and subchondral bone, are targets for neuropeptides. Resident cells of these tissues express receptors for various neuroendocrine-derived peptides including proopiomelanocortin (POMC)-derived peptides, i.e., α-melanocyte-stimulating hormone (α-MSH), adrenocorticotropin (ACTH) and β-endorphin (β-ED), and sympathetic neuropeptides like vasoactive intestinal peptide (VIP) and neuropeptide y (NPY). Melanocortins attained particular attention due to their immunomodulatory and anti-inflammatory effects in several tissues and organs. In particular, α-MSH, ACTH and specific melanocortin-receptor (MCR) agonists appear to have promising anti-inflammatory actions demonstrated in animal models of experimentally induced arthritis and osteoarthritis (OA). Sympathetic neuropeptides have obtained increasing attention as they have crucial trophic effects that are critical for joint tissue and bone homeostasis. VIP and NPY are implicated in direct and indirect activation of several anabolic signaling pathways in bone and synovial cells. Additionally, pituitary adenylate cyclase-activating polypeptide (PACAP) proved to be chondroprotective and, thus, might be a novel target in OA. Taken together, it appears more and more likely that the anabolic effects of these neuroendocrine peptides or their respective receptor agonists/antagonists may be exploited for the treatment of patients with inflammatory and degenerative joint diseases in the future. Full article

Plakins are a family of seven cytoskeletal cross-linker proteins (microtubule-actin crosslinking factor 1 (MACF), bullous pemphigoid antigen (BPAG1) desmoplakin, envoplakin, periplakin, plectin, epiplakin) that network the three major filaments that comprise the cytoskeleton. Plakins have been found to be involved in disorders and diseases of the skin, heart, nervous system, and cancer that are attributed to autoimmune responses and genetic alterations of these macromolecules. Despite their role and involvement across a spectrum of several diseases, there are no current drugs or pharmacological agents that specifically target the members of this protein family. On the contrary, microtubules have traditionally been targeted by microtubule inhibiting agents, used for the treatment of diseases such as cancer, in spite of the deleterious toxicities associated with their clinical utility. The Research Collaboratory for Structural Bioinformatics (RCSB) was used here to identify therapeutic drugs targeting the plakin proteins, particularly the spectraplakins MACF1 and BPAG1, which contain microtubule-binding domains. RCSB analysis revealed that plakin proteins had 329 ligands, of which more than 50% were MACF1 and BPAG1 ligands and 10 were documented, clinically or experimentally, to have several therapeutic applications as anticancer, anti-inflammatory, and antibiotic agents. Full article

Using renewable and biocompatible natural-based resources to construct functional biomaterials has attracted great attention in recent years. In this work, we successfully prepared a series of steroid-based cationic lipids by integrating various steroid skeletons/hydrophobes with (l-)-arginine headgroups via facile and efficient synthetic approach. The plasmid DNA (pDNA) binding affinity of the steroid-based cationic lipids, average particle sizes, surface potentials, morphologies and stability of the steroid-based cationic lipids/pDNA lipoplexes were disclosed to depend largely on the steroid skeletons. Cellular evaluation results revealed that cytotoxicity and gene transfection efficiency of the steroid-based cationic lipids in H1299 and HeLa cells strongly relied on the steroid hydrophobes. Interestingly, the steroid lipids/pDNA lipoplexes inclined to enter H1299 cells mainly through caveolae and lipid-raft mediated endocytosis pathways, and an intracellular trafficking route of “lipid-raft-mediated endocytosis→lysosome→cell nucleic localization” was accordingly proposed. The study provided possible approach for developing high-performance steroid-based lipid gene carriers, in which the cytotoxicity, gene transfection capability, endocytosis pathways, and intracellular trafficking/localization manners could be tuned/controlled by introducing proper steroid skeletons/hydrophobes. Noteworthy, among the lipids, Cho-Arg showed remarkably high gene transfection efficacy, even under high serum concentration (50% fetal bovine serum), making it an efficient gene transfection agent for practical application. Full article

In this study, we first used gelatin/chondroitin-6-sulfate/hyaluronan/chitosan highly elastic cryogels, which showed total recovery from large strains during repeated compression cycles, as 3D scaffolds to study the effects of cyclic dynamic compressive loading on chondrocyte gene expression and extracellular matrix (ECM) production. Dynamic culture of porcine chondrocytes was studied at 1 Hz, 10% to 40% strain and 1 to 9 h/day stimulation duration, in a mechanical-driven multi-chamber bioreactor for 14 days. From the experimental results, we could identify the optimum dynamic culture condition (20% and 3 h/day) to enhance the chondrocytic phenotype of chondrocytes from the expression of marker (Col I, Col II, Col X, TNF-α, TGF-β1 and IGF-1) genes by quantitative real-time polymerase chain reactions (qRT-PCR) and production of ECM (GAGs and Col II) by biochemical analysis and immunofluorescence staining. With up-regulated growth factor (TGF-β1 and IGF-1) genes, co-culture of chondrocytes with porcine adipose-derived stem cells (ASCs) was employed to facilitate chondrogenic differentiation of ASCs during dynamic culture in cryogel scaffolds. By replacing half of the chondrocytes with ASCs during co-culture, we could obtain similar production of ECM (GAGs and Col II) and expression of Col II, but reduced expression of Col I, Col X and TNF-α. Subcutaneous implantation of cells/scaffold constructs in nude mice after mono-culture (chondrocytes or ASCs) or co-culture (chondrocytes + ASCs) and subject to static or dynamic culture condition in vitro for 14 days was tested for tissue-engineering applications. The constructs were retrieved 8 weeks post-implantation for histological analysis by Alcian blue, Safranin O and Col II immunohistochemical staining. The most abundant ectopic cartilage tissue was found for the chondrocytes and chondrocytes + ASCs groups using dynamic culture, which showed similar neo-cartilage formation capability with half of the chondrocytes replaced by ASCs for co-culture. This combined co-culture/dynamic culture strategy is expected to cut down the amount of donor chondrocytes needed for cartilage-tissue engineering. Full article

Expression cloning from cDNA is an important technique for acquiring genes encoding novel fluorescent proteins. However, the probability of in-frame cDNA insertion following the first start codon of the vector is normally only 1/3, which is a cause of low cloning efficiency. To overcome this issue, we developed a new expression plasmid vector, pRSET-TriEX, in which transcriptional slippage was induced by introducing a DNA sequence of (dT)₁₄ next to the first start codon of pRSET. The effectiveness of frame-insensitive cloning was validated by inserting the gene encoding eGFP with all three possible frames to the vector. After transformation with one of these plasmids, E. coli cells expressed eGFP with no significant difference in the expression level. The pRSET-TriEX vector was then used for expression cloning of a novel fluorescent protein from Scolionema suvaense. We screened 3658 E. coli colonies transformed with pRSET-TriEX containing Scolionema suvaense cDNA, and found one colony expressing a novel green fluorescent protein, ScSuFP. The highest score in protein sequence similarity was 42% with the chain c of multi-domain green fluorescent protein like protein “ember” from Anthoathecata sp. Variations in the N- and/or C-terminal sequence of ScSuFP compared to other fluorescent proteins indicate that the expression cloning, rather than the sequence similarity-based methods, was crucial for acquiring the gene encoding ScSuFP. The absorption maximum was at 498 nm, with an extinction efficiency of 1.17 × 10⁵ M⁻¹·cm⁻¹. The emission maximum was at 511 nm and the fluorescence quantum yield was determined to be 0.6. Pseudo-native gel electrophoresis showed that the protein forms obligatory homodimers. Full article

Galectin-3 (Gal-3) is recognized as a prognostic marker in several cancer types. Its involvement in tumor development and proliferation makes this lectin a promising target for early cancer diagnosis and anti-cancer therapies. Gal-3 recognizes poly-N-acetyllactosamine (LacNAc)-based carbohydrate motifs of glycoproteins and glycolipids with a high specificity for internal LacNAc epitopes. This study analyzes the mode and kinetics of binding of Gal-3 to a series of multivalent neo-glycoproteins presenting complex poly-LacNAc-based oligosaccharide ligands on a scaffold of bovine serum albumin. These neo-glycoproteins rank among the strongest Gal-3 ligands reported, with K_d reaching sub-nanomolar values as determined by surface plasmon resonance. Significant differences in the binding kinetics were observed within the ligand series, showing the tetrasaccharide capped with N,N′-diacetyllactosamine (LacdiNAc) as the strongest ligand of Gal-3 in this study. A molecular model of the Gal-3 carbohydrate recognition domain with docked oligosaccharide ligands is presented that shows the relations in the binding site at the molecular level. The neo-glycoproteins presented herein may be applied for selective recognition of Gal-3 both on the cell surface and in blood serum. Full article

Kiwifruit bacterial canker caused by Pseudomonas syringae pv. actinidiae (Psa) has brought about a severe threat to the kiwifruit industry worldwide since its first outbreak in 2008. Studies on other pathovars of P. syringae are revealing the pathogenesis of these pathogens, but little about the mechanism of kiwifruit bacterial canker is known. In order to explore the species-specific interaction between Psa and kiwifruit, we analyzed the transcriptomic profile of kiwifruit infected by Psa. After 48 h, 8255 differentially expressed genes were identified, including those involved in metabolic process, secondary metabolites metabolism and plant response to stress. Genes related to biosynthesis of terpens were obviously regulated, indicating terpens may play roles in suppressing the growth of Psa. We identified 283 differentially expressed resistant genes, of which most U-box domain containing genes were obviously up regulated. Expression of genes involved in plant immunity was detected and some key genes showed differential expression. Our results suggest that Psa induced defense response of kiwifruit, including PAMP (pathogen/microbe-associated molecular patterns)-triggered immunity, effector-triggered immunity and hypersensitive response. Metabolic process was adjusted to adapt to these responses and production of secondary metabolites may be altered to suppress the growth of Psa. Full article

Acute ischemia of an extremity occurs in several stages, a lack of oxygen being the primary contributor of the event. Although underlying patho-mechanisms are similar, it is important to determine whether it is an acute or chronic event. Healthy tissue does not contain enlarged collaterals, which are formed in chronically malperfused tissue and can maintain a minimum supply despite occlusion. The underlying processes for enhanced collateral blood flow are sprouting vessels from pre-existing vessels (via angiogenesis) and a lumen extension of arterioles (via arteriogenesis). While disturbed flow patterns with associated local low shear stress upregulate angiogenesis promoting genes, elevated shear stress may trigger arteriogenesis due to increased blood volume. In case of an acute ischemia, especially during the reperfusion phase, fluid transfer occurs into the tissue while the vascular bed is simultaneously reduced and no longer reacts to vaso-relaxing factors such as nitric oxide. This process results in an exacerbative cycle, in which increased peripheral resistance leads to an additional lack of oxygen. This whole process is accompanied by an inundation of inflammatory cells, which amplify the inflammatory response by cytokine release. However, an extremity is an individual-specific composition of different tissues, so these processes may vary dramatically between patients. The image is more uniform when broken down to the single cell stage. Because each cell is dependent on energy produced from aerobic respiration, an event of acute hypoxia can be a life-threatening situation. Aerobic processes responsible for yielding adenosine triphosphate (ATP), such as the electron transport chain and oxidative phosphorylation in the mitochondria, suffer first, thus disrupting the integrity of cellular respiration. One consequence of this is irreparable damage of the cell membrane due to an imbalance of electrolytes. The eventual increase in net fluid influx associated with a decrease in intracellular pH is considered an end-stage event. Due to the lack of ATP, individual cell organelles can no longer sustain their activity, thus initiating the cascade pathways of apoptosis via the release of cytokines such as the BCL2 associated X protein (BAX). As ischemia may lead to direct necrosis, inflammatory processes are further aggravated. In the case of reperfusion, the flow of nascent oxygen will cause additional damage to the cell, further initiating apoptosis in additional surrounding cells. In particular, free oxygen radicals are formed, causing severe damage to cell membranes and desoxyribonucleic acid (DNA). However, the increased tissue stress caused by this process may be transient, as radical scavengers may attenuate the damage. Taking the above into final consideration, it is clearly elucidated that acute ischemia and subsequent reperfusion is a process that leads to acute tissue damage combined with end-organ loss of function, a condition that is difficult to counteract. Full article

This brief review of the neurological effects of growth hormone (GH) and gonadotropin-releasing hormone (GnRH) in the brain, particularly in the cerebral cortex, hypothalamus, hippocampus, cerebellum, spinal cord, neural retina, and brain tumors, summarizes recent information about their therapeutic potential as treatments for different neuropathologies and neurodegenerative processes. The effect of GH and GnRH (by independent administration) has been associated with beneficial impacts in patients with brain trauma and spinal cord injuries. Both GH and GnRH have demonstrated potent neurotrophic, neuroprotective, and neuroregenerative action. Positive behavioral and cognitive effects are also associated with GH and GnRH administration. Increasing evidence suggests the possibility of a multifactorial therapy that includes both GH and GnRH. Full article

Triptolide (TP), a major extract of the herb Tripterygium wilfordii Hook F (TWHF), has been shown to exert potent pharmacological effects, especially an immunosuppressive effect in the treatment of rheumatoid arthritis (RA). However, its multiorgan toxicity prevents it from being widely used in clinical practice. Recently, several attempts are being performed to reduce TP toxicity. In this review, recent progress in the use of TP for RA, including its pharmacological effects and toxicity, is summarized. Meanwhile, strategies relying on chemical structural modifications, innovative delivery systems, and drug combinations to alleviate the disadvantages of TP are also reviewed. Furthermore, we also discuss the challenges and perspectives in their clinical translation. Full article

Alterations in the immune response of patients with autoimmune diseases may predispose to malignancies, and a link between chronic autoimmune gastritis and gastric cancer has been reported in many studies. Intestinal metaplasia with dysplasia of the gastric corpus-fundus mucosa and hyperplasia of chromaffin cells, which are typical features of late-stage autoimmune gastritis, are considered precursor lesions. Autoimmune gastritis has been associated with the development of two types of gastric neoplasms: intestinal type and type I gastric carcinoid. Here, we review the association of autoimmune gastritis with gastric cancer and other autoimmune features present in gastric neoplasms. Full article

Proinsulin-transferrin fusion protein (ProINS-Tf) has been designed and successfully expressed from the mammalian HEK293 cells (HEK-ProINS-Tf). It was found that HEK-ProINS-Tf could be converted into an activated form in the liver. Furthermore, HEK-ProINS-Tf was demonstrated as an extra-long acting insulin analogue with liver-specific insulin action in streptozotocin (STZ)-induced type 1 diabetic mice. However, due to the low production yield from transfected HEK293 cells, there are other interesting features, including the oral bioavailability, which have not been fully explored and characterized. To improve the protein production yield, an alternative protein expression system, ExpressTec using transgenic rice (Oryza sativa L.), was used. The intact and active rice-derived ProINS-Tf (ExpressTec-ProINS-Tf) was successfully expressed from the transgenic rice expression system. Our results suggested that, although the insulin-like bioactivity of ExpressTec-ProINS-Tf was slightly lower in vitro, its potency of in vivo blood glucose control was considerably stronger than that of HEK-ProINS-Tf. The oral delivery studies in type 1 diabetic mice demonstrated a prolonged control of blood glucose to near-normal levels after oral administration of ExpressTec-ProINS-Tf. Results in this report suggest that ExpressTec-ProINS-Tf is a promising insulin analog with advantages including low cost, prolonged and liver targeting effects, and most importantly, oral bioactivity. Full article

Galectin-3 (Gal-3) regulates basic cellular functions such as cell–cell and cell–matrix interactions, growth, proliferation, differentiation, and inflammation. It is not surprising, therefore, that this protein is involved in the pathogenesis of many relevant human diseases, including cancer, fibrosis, chronic inflammation and scarring affecting many different tissues. The papers published in the literature have progressively increased in number during the last decades, testifying the great interest given to this protein by numerous researchers involved in many different clinical contexts. Considering the crucial role exerted by Gal-3 in many different clinical conditions, Gal-3 is emerging as a new diagnostic, prognostic biomarker and as a new promising therapeutic target. The current review aims to extensively examine the studies published so far on the role of Gal-3 in all the clinical conditions and diseases, listed in alphabetical order, where it was analyzed. Full article

Corylin is a flavonoid extracted from the nuts of Psoralea corylifolia L. (Fabaceae), which is a widely used anti-inflammatory and anticancer herb in China. Recent studies revealed antioxidant, anti-inflammatory, and bone differentiation–promoting effects of corylin. However, there are no studies examining the anticancer activity of corylin. In this study, we used cells and animal models to examine the antitumor effects of corylin on hepatocellular carcinoma (HCC) and then studied its downstream regulatory mechanisms. The results showed that corylin significantly inhibited the proliferation, migration, and invasiveness of HCC cells and suppressed epithelial–mesenchymal transition. We found that the anti-HCC mechanism of corylin’s action lies in the upregulation of tumor suppressor long noncoding RNA growth arrest-specific transcript 5 (GAS5) and the activation of its downstream anticancer pathways. In animal experiments, we also found that corylin can significantly inhibit tumor growth without significant physiological toxicity. The above results suggest that corylin has anti-HCC effects and good potential as a clinical treatment. Full article

Intrinsically disordered proteins (IDPs) represent approximately 30% of the human genome and play key roles in cell proliferation and cellular signaling by modulating the function of target proteins via protein–protein interactions. In addition, IDPs are involved in various human disorders, such as cancer, neurodegenerative diseases, and amyloidosis. To understand the underlying molecular mechanism of IDPs, it is important to study their structural features during their interactions with target proteins. However, conventional biochemical and biophysical methods for analyzing proteins, such as X-ray crystallography, have difficulty in characterizing the features of IDPs because they lack an ordered three-dimensional structure. Here, we present biochemical and biophysical studies on nucleolar phosphoprotein 140 (Nopp140), which mostly consists of disordered regions, during its interaction with casein kinase 2 (CK2), which plays a central role in cell growth. Surface plasmon resonance and electron paramagnetic resonance studies were performed to characterize the interaction between Nopp140 and CK2. A single-molecule fluorescence resonance energy transfer study revealed conformational change in Nopp140 during its interaction with CK2. These studies on Nopp140 can provide a good model system for understanding the molecular function of IDPs. Full article

Constitutive signaling through the phosphatidylinositol-3-kinase-Akt-mechanistic target of rapamycin (PI3K-Akt-mTOR) pathway is present in acute myeloid leukemia (AML) cells. However, AML is a heterogeneous disease, and we therefore investigated possible associations between cellular metabolism and sensitivity to PI3K-Akt-mTOR pathway inhibitors. We performed non-targeted metabolite profiling to compare the metabolome differences of primary human AML cells derived from patients susceptible or resistant to the in vitro antiproliferative effects of mTOR and PI3K inhibitors. In addition, the phosphorylation status of 18 proteins involved in PI3K-Akt-mTOR signaling and the effect of the cyclooxygenase inhibitor indomethacin on their phosphorylation status was investigated by flow cytometry. Strong antiproliferative effects by inhibitors were observed only for a subset of patients. We compared the metabolite profiles for responders and non-responders towards PI3K-mTOR inhibitors, and 627 metabolites could be detected. Of these metabolites, 128 were annotated and 15 of the annotated metabolites differed significantly between responders and non-responders, including metabolites involved in energy, amino acid, and lipid metabolism. To conclude, leukemia cells that are susceptible or resistant to PI3K-Akt-mTOR inhibitors differ in energy, amino acid, and arachidonic acid metabolism, and modulation of arachidonic acid metabolism alters the activation of mTOR and its downstream mediators. Full article

The study of the human microbiome has become a very popular topic. Our microbial counterpart, in fact, appears to play an important role in human physiology and health maintenance. Accordingly, microbiome alterations have been reported in an increasing number of human diseases. Despite the huge amount of data produced to date, less is known on how a microbial dysbiosis effectively contributes to a specific pathology. To fill in this gap, other approaches for microbiome study, more comprehensive than 16S rRNA gene sequencing, i.e., shotgun metagenomics and metatranscriptomics, are becoming more widely used. Methods standardization and the development of specific pipelines for data analysis are required to contribute to and increase our understanding of the human microbiome relationship with health and disease status. Full article

A crucial neuronal structure for the development and regeneration of neuronal networks is the axonal growth cone. Affected by different guidance cues, it grows in a predetermined direction to reach its final destination. One of those cues is the vascular endothelial growth factor (VEGF), which was identified as a positive effector for growth cone movement. These positive effects are mainly mediated by a reorganization of the actin network. This study shows that VEGF triggers a tight colocalization of cofilin and the Arp2/3 complex to the actin cytoskeleton within chicken dorsal root ganglia (DRG). Live cell imaging after microinjection of GFP (green fluorescent protein)-cofilin and RFP (red fluorescent protein)-LifeAct revealed that both labeled proteins rapidly redistributed within growth cones, and showed a congruent distribution pattern after VEGF supplementation. Disruption of signaling upstream of cofilin via blocking LIM-kinase (LIMK) activity resulted in growth cones displaying regressive growth behavior. Microinjection of GFP-p16b (a subunit of the Arp2/3 complex) and RFP-LifeAct revealed that both proteins redistributed into lamellipodia of the growth cone within minutes after VEGF stimulation. Disruption of the signaling to the Arp2/3 complex in the presence of VEGF by inhibition of N-WASP (neuronal Wiskott–Aldrich–Scott protein) caused retraction of growth cones. Hence, cofilin and the Arp2/3 complex appear to be downstream effector proteins of VEGF signaling to the actin cytoskeleton of DRG growth cones. Our data suggest that VEGF simultaneously affects different pathways for signaling to the actin cytoskeleton, since activation of cofilin occurs via inhibition of LIMK, whereas activation of Arp2/3 is achieved by stimulation of N-WASP. Full article

Fed-batch animal cell culture is the most common method for commercial production of recombinant proteins. However, higher cell densities in these platforms are still limited due to factors such as excessive ammonium production, lactic acid production, nutrient limitation, and/or hyperosmotic stress related to nutrient feeds and base additions to control pH. To partly overcome these factors, we investigated a simple method to reduce both ammonium and lactic acid production—termed Lactate Supplementation and Adaptation (LSA) technology—through the use of CHO cells adapted to a lactate-supplemented medium. Using this simple method, we achieved a reduction of nearly 100% in lactic acid production with a simultaneous 50% reduction in ammonium production in batch shaker flasks cultures. In subsequent fed-batch bioreactor cultures, lactic acid production and base addition were both reduced eight-fold. Viable cell densities of 35 million cells per mL and integral viable cell days of 273 million cell-days per mL were achieved, both among the highest currently reported for a fed-batch animal cell culture. Investigating the benefits of LSA technology in animal cell culture is worthy of further consideration and may lead to process conditions more favorable for advanced industrial applications. Full article

Presently, many studies have focused on exploring in silico approaches in the identification and development of alternative therapy for the treatment and management of cancer. Solute carrier family-2-member-4-gene (Slc2a4) which encodes glucose transporter 4 protein (GLUT4), has been identified as a promising therapeutic target for cancer. Though Slc2a4 is known to play a major regulatory role in the pathophysiology of type 2 diabetes, emerging evidence suggests that successful pharmacological inhibition of this protein may lead to the development of a novel drug candidate for the treatment of cancer. In this study, Slc2a4 protein sequence was retrieved and analysed using in silico approaches, and we identified seven putative antimicrobial peptides (AMPs; RAB1-RAB7) as anti-cancer. The structures of the protein and AMPs were modelled using I-TASSER server, and the overall quality of the Slc2a4 model was validated using PROCHECK. Subsequently, the probable motifs and active site of the protein were forecasted. Also, the molecular interaction between the AMPs and Slc2a4 was ascertained using PatchDock. The result revealed that, all the AMPs are good Slc2a4 inhibitors with RAB1 having the highest binding affinity of 12,392 and binding energy of −39.13 kcal/mol. Hence, this study reveals that all the generated AMPs can serve as therapeutic drug in treating cancer by inhibiting Slc2a4 which is responsible for the production of energy for cancer cells during angiogenesis. This is the first report on AMPs as inhibitors of Slc2a4 for the treatment of cancer. Full article

Mucoadhesive gelling systems with tannic acid modified silver nanoparticles were developed for effective treatment of herpes virus infections. To increase nanoparticle residence time after local application, semi solid formulations designed from generally regarded as safe (GRAS) excipients were investigated for their rheological and mechanical properties followed with ex vivo mucoadhesive behavior to the porcine vaginal mucosa. Particular effort was made to evaluate the activity of nanoparticle-based hydrogels toward herpes simplex virus (HSV) type 1 and 2 infection in vitro in immortal human keratinocyte cell line and in vivo using murine model of HSV-2 genital infection. The effect of infectivity was determined by real time quantitative polymerase chain reaction, plaque assay, inactivation, attachment, penetration and cell-to-cell assessments. All analyzed nanoparticle-based hydrogels exhibited pseudoplastic and thixotropic properties. Viscosity and mechanical measurements of hydrogels were found to correlate with the mucoadhesive properties. The results confirmed the ability of nanoparticle-based hydrogels to affect viral attachment, impede penetration and cell-to-cell transmission, although profound differences in the activity evoked by tested preparations toward HSV-1 and HSV-2 were noted. In addition, these findings demonstrated the in vivo potential of tannic acid modified silver nanoparticle-based hydrogels for vaginal treatment of HSV-2 genital infection. Full article

A generic research platform with 2-dimensional (2D) cell culture technology, a 3-dimensional (3D) in vitro tissue model, and a scaled-down cell culture and imaging system in between, was utilized to address the problematic issues associated with the use of serum in skin tissue engineering. Human dermal fibroblasts (HDFs) and immortalized keratinocytes (HaCat cells) mono- or co-cultured in serum or serum-free medium were compared and analyzed via the platform. It was demonstrated that serum depletion had significant influence on the attachment of HaCat cells onto tissue culture plastic (TCP), porous substrates and cellulosic scaffolds, which was further enhanced by the pre-seeded HDFs. The complex structures formed by the HDFs colonized within the porous substrates and scaffolds not only prevented the seeded HaCat cells from filtering through the open pores, but also acted as cellular substrates for HaCat cells to attach onto. When mono-cultured on TCP, both HDFs and HaCat cells were less proliferative in medium without serum than with serum. However, both cell types were successfully co-cultured in 2D using serum-free medium if the initial cell seeding density was higher than 80,000 cells/cm² (with 1:1 ratio). Based on the results from 2D cultures, co-culture of both cell types on modular substrates with small open pores (125 μm) and cellulosic scaffolds with open pores of varying sizes (50–300 µm) were then conducted successfully in serum-free medium. This study demonstrated that the generic research platform had great potential for in-depth understanding of HDFs and HaCat cells cultivated in serum-free medium, which could inform the processes for manufacturing skin cells or tissues for clinical applications. Full article

Hypoxia-inducible factor (HIF) plays a crucial role in the response to hypoxia at the cellular, tissue, and organism level. New agents under development to pharmacologically manipulate HIF may provide new and exciting possibilities in the treatment of anemia of chronic kidney disease (CKD) as well as in multiple other disease states involving ischemia–reperfusion injury. This article provides an overview of recent studies describing current standards of care for patients with anemia in CKD and associated clinical issues, and those supporting the clinical potential for targeting HIF stabilization with HIF prolyl-hydroxylase inhibitors (HIF-PHI) in these patients. Additionally, articles reporting the clinical potential for HIF-PHIs in ‘other’ putative therapeutic areas, the tissue and intracellular distribution of HIF- and prolyl-hydroxylase domain (PHD) isoforms, and HIF isoforms targeted by the different PHDs, were identified. There is increasing uncertainty regarding the optimal treatment for anemia of CKD with poorer outcomes associated with treatment to higher hemoglobin targets, and the increasing use of iron and consequent risk of iron imbalance. Attainment and maintenance of more physiologic erythropoietin levels associated with HIF stabilization may improve the management of patients resistant to treatment with erythropoiesis-stimulating agents and improve outcomes at higher hemoglobin targets. Full article

Multiple factors influence immunoglobulin G glycosylation, which in turn affect the glycoproteins’ function on eliciting an anti-inflammatory or pro-inflammatory response. It is prudent to underscore these processes when considering the use of immunoglobulin G N-glycan moieties as an indication of disease presence, progress, or response to therapeutics. It has been demonstrated that the altered expression of genes that encode enzymes involved in the biosynthesis of immunoglobulin G N-glycans, receptors, or complement factors may significantly modify immunoglobulin G effector response, which is important for regulating the immune system. The immunoglobulin G N-glycome is highly heterogenous; however, it is considered an interphenotype of disease (a link between genetic predisposition and environmental exposure) and so has the potential to be used as a dynamic biomarker from the perspective of predictive, preventive, and personalised medicine. Undoubtedly, a deeper understanding of how the multiple factors interact with each other to alter immunoglobulin G glycosylation is crucial. Herein we review the current literature on immunoglobulin G glycoprotein structure, immunoglobulin G Fc glycosylation, associated receptors, and complement factors, the downstream effector functions, and the factors associated with the heterogeneity of immunoglobulin G glycosylation. Full article

Introduction: Sphingosine-1-phosphate (S1P) regulates the migration of follicular B cells (B2 cells) and directs the positioning of Marginal zone B cells (MZ B cells) within the spleen. The function of S1P signalling in the third B cell lineage, B1 B cells, mainly present in the pleural and peritoneal cavity, has not yet been determined. Methods: S1P receptor expression was analysed in peritoneal B cells by real-time polymerase chain reaction (qPCR). The chemotactic response to S1P was studied in vitro. The role of S1P signalling was further explored in a s1p₄^−/− mouse strain. Results: Peritoneal B cells expressed considerable amounts of the S1P receptors 1 and 4 (S1P₁ and S1P₄, respectively). S1P₁ showed differential expression between the distinct peritoneal B cell lineages. While B2 cells showed no chemotactic response to S1P, B1 B cells showed a migration response to S1P. s1p₄^−/− mice displayed significant alterations in the composition of peritoneal B cell populations, as well as a significant reduction of mucosal immunoglobulin A (IgA) in the gut. Discussion: S1P signalling influences peritoneal B1 B cell migration. S1P₄ deficiency alters the composition of peritoneal B cell populations and reduces secretory IgA levels. These findings suggest that S1P signalling may be a target to modulate B cell function in inflammatory intestinal pathologies. Full article

Galectins are β-galactoside-binding proteins. As carbohydrate-binding proteins, they participate in intracellular trafficking, cell adhesion, and cell–cell signaling. Accumulating evidence indicates that they play a pivotal role in numerous physiological and pathological activities, such as the regulation on cancer progression, inflammation, immune response, and bacterial and viral infections. Galectins have drawn much attention as targets for therapeutic interventions. Several molecules have been developed as galectin inhibitors. In particular, TD139, a thiodigalactoside derivative, is currently examined in clinical trials for the treatment of idiopathic pulmonary fibrosis. Herein, we provide an in-depth review on the development of galectin inhibitors, aiming at the dissection of the structure–activity relationship to demonstrate how inhibitors interact with galectin(s). We especially integrate the structural information established by X-ray crystallography with several biophysical methods to offer, not only in-depth understanding at the molecular level, but also insights to tackle the existing challenges. Full article

Acute leukemia is one of the commonly diagnosed neoplasms and causes human death. However, the treatment for acute leukemia is not yet satisfactory. Studies have shown that mushroom-derived polysaccharides display low toxicity and have been used clinically for cancer therapy. Therefore, we set out to evaluate the anti-cancerous efficacy of a water-soluble polysaccharide extract from Inonotus taiwanensis (WSPIS) on human acute monocytic leukemia THP-1 and U937 cell lines in vitro. Under our experimental conditions, WSPIS elicited dose-dependent growth retardation and induced apoptotic cell death. Further analysis showed that WSPIS-induced apoptosis was associated with a mitochondrial apoptotic pathway, such as the disruption of mitochondrial membrane potential (MMP), followed by the activation of caspase-9, caspase-3, and PARP (poly(ADP-ribose) polymerase) cleavage. However, a broad caspase inhibitor, Z-VAD.fmk, could not prevent WSPIS-induced apoptosis. These data imply that mechanism(s) other than caspase might be involved. Thus, the involvement of endonuclease G (endoG), a mediator arbitrating caspase-independent oligonucleosomal DNA fragmentation, was examined. Western blotting demonstrated that WSPIS could elicit nuclear translocation of endoG. MMP disruption after WSPIS treatment was accompanied by intracellular reactive oxygen species (ROS) generation. However, pretreatment with N-acetyl-l-cysteine (NAC) could not attenuate WSPIS-induced apoptosis. In addition, our data also show that WSPIS could inhibit autophagy. Activation of autophagy by rapamycin decreased WSPIS-induced apoptosis and cell death. Taken together, our findings suggest that cell cycle arrest, endonuclease G-mediated apoptosis, and autophagy inhibition contribute to the anti-cancerous effect of WSPIS on human acute monocytic leukemia cells. Full article

Melanoma remains mostly an untreatable fatal disease despite advances in decoding cancer genomics and developing new therapeutic modalities. Progress in patient care would benefit from additional predictive models germane for human disease mechanisms, tumor heterogeneity, and therapeutic responses. Toward this aim, this review documents comparative aspects of human and naturally occurring canine melanomas. Clinical presentation, pathology, therapies, and genetic alterations are highlighted in the context of current basic and translational research in comparative oncology. Somewhat distinct from sun exposure-related human cutaneous melanomas, there is growing evidence that a variety of gene copy number alterations and protein structure/function mutations play roles in canine melanomas, in circumstances more analogous to human mucosal melanomas and to some extent other melanomas with murine sarcoma viral oncogene homolog B (BRAF), Neuroblastoma RAS Viral (V-Ras) Oncogene Homolog (NRAS), and neurofibromin 1 tumor suppressor NF1 triple wild-type genotype. Gaps in canine genome annotation, as well as an insufficient number and depth of sequences covered, remain considerable barriers to progress and should be collectively addressed. Preclinical approaches can be designed to include canine clinical trials addressing immune modulation as well as combined-targeted inhibition of Rat Sarcoma Superfamily/Mitogen-activated protein kinase (RAS/MAPK) and/or Phosphatidylinositol-3-Kinase/Protein Kinase B/Mammalian target of rapamycin (PI3K/AKT/mTOR) signal transduction, pathways frequently activated in both human and canine melanomas. Future investment should be aimed towards improving understanding of canine melanoma as a predictive preclinical surrogate for human melanoma and for mutually benefiting these uniquely co-dependent species. Full article

Glioblastoma multiforme (GBM) is one of the most aggressive and malignant forms of brain tumors. Despite recent advances in operative and postoperative treatments, it is almost impossible to perform complete resection of these tumors owing to their invasive and diffuse nature. Several natural plant-derived products, however, have been demonstrated to have promising therapeutic effects, such that they may serve as resources for anticancer drug discovery. The therapeutic effects of one such plant product, n-butylidenephthalide (BP), are wide-ranging in nature, including impacts on cancer cell apoptosis, cell cycle arrest, and cancer cell senescence. The compound also exhibits a relatively high level of penetration through the blood-brain barrier (BBB). Taken together, its actions have been shown to have anti-proliferative, anti-chemoresistance, anti-invasion, anti-migration, and anti-dissemination effects against GBM. In addition, a local drug delivery system for the subcutaneous and intracranial implantation of BP wafers that significantly reduce tumor size in xenograft models, as well as orthotopic and spontaneous brain tumors in animal models, has been developed. Isochaihulactone (ICL), another kind of plant product, possesses a broad spectrum of pharmacological activities, including impacts on cancer cell apoptosis and cell cycle arrest, as well as anti-proliferative and anti-chemoresistance effects. Furthermore, these actions have been specifically shown to have cancer-fighting effects on GBM. In short, the results of various studies reviewed herein have provided substantial evidence indicating that BP and ICH are promising novel anticancer compounds with good potential for clinical applications. Full article

Flight capacity is important for invasive pests during entry, establishment and spreading. Both Bactrocera dorsalis Hendel and Bactrocera correcta Bezzi are invasive fruit flies but their flight capacities differ. Here, a tethered flight mill test demonstrated that B. dorsalis exhibits a greater flight capacity than B. correcta. RNA-Seq was used to determine the transcriptomic differences associated with the flight capacity of two Bactrocera species. Transcriptome data showed that 6392 unigenes were differentially expressed between the two species in the larval stage, whereas in the adult stage, 4104 differentially expressed genes (DEGs) were identified in females, and 3445 DEGs were observed in males. The flight capacity appeared to be correlated with changes in the transcriptional levels of genes involved in wing formation, flight muscle structure, energy metabolism, and hormonal control. Using RNA interference (RNAi) to verify the function of one DEG, the epidermal growth factor receptor (EGFR), we confirmed the role of this gene in regulating wing development, and thereby flight capacity, in both species. This work reveals the flight mechanism of fruit flies and provides insight into fundamental transcriptomics for further studies on the flight performance of insects. Full article

Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumors of gastrointestinal tract. GISTs have very different clinical phenotypes and underlying molecular characteristics that are not yet completely understood. microRNAs (miRNAs) have been shown to participate in carcinogenesis pathways through post-transcriptional regulation of gene expression in different tumors. Over the last years emerging evidence has highlighted the role of miRNAs in GISTs. This review provides an overview of original research papers that analyze miRNA deregulation patterns, functional role, diagnostic, therapeutic and prognostic implications in GIST as well as provides directions for further research in the field. Full article

The role of sex hormone receptors in human cancer development and progression has been well documented in numerous studies, as has the success of sex hormone antagonists in the biological therapy of many human tumors. In salivary gland tumors (SGTs), little and conflicting information about the role of the estrogen receptor alpha (ERα), progesterone receptor (PgR) and androgen receptor (AR) has been described and in most cases the use of sex hormone antagonists is not contemplated in clinical practice. In this study, we analyzed a panel of sex hormone receptors that have not been widely investigated in SGTs—ERα, PgR, AR, but also ERβ and GPR30—to define their expression pattern and their prognostic and predictive value in a case series of 69 benign and malignant SGTs. We showed the aberrant expression of AR in mucoepidermoid and oncocytic carcinoma, a strong relation between cytoplasmic ERβ expression and tumor grade, and a strong correlation between nuclear GPR30 expression and disease-free survival (DFS) of SGT patients. Full article

The morning surge in blood pressure (BP) coincides with increased cardiovascular (CV) events. This strongly suggests that an altered circadian rhythm of BP plays a crucial role in the development of CV disease (CVD). A disrupted circadian rhythm of BP, such as the non-dipping type of hypertension (i.e., absence of nocturnal BP decline), is frequently observed in metabolic disorders and chronic kidney disease (CKD). The circadian timing system, controlled by the central clock in the suprachiasmatic nucleus of the hypothalamus and/or by peripheral clocks in the heart, vasculature, and kidneys, modulates the 24 h oscillation of BP. However, little information is available regarding the molecular and cellular mechanisms of an altered circadian timing system-mediated disrupted dipping pattern of BP in metabolic disorders and CKD that can lead to the development of CV events. A more thorough understanding of this pathogenesis could provide novel therapeutic strategies for the management of CVD. This short review will address our and others’ recent findings on the molecular mechanisms that may affect the dipping pattern of BP in metabolic dysfunction and kidney disease and its association with CV disorders. Full article

Intrinsically disordered proteins (IDPs) are an emerging phenomenon. They may have a high degree of flexibility in their polypeptide chains, which lack a stable 3D structure. Although several biological functions of IDPs have been proposed, their general function is not known. The only finding related to their function is the genetically conserved YSK₂ motif present in plant dehydrins. These proteins were shown to be IDPs with the YSK₂ motif serving as a core region for the dehydrins’ cryoprotective activity. Here we examined the cryoprotective activity of randomly selected IDPs toward the model enzyme lactate dehydrogenase (LDH). All five IDPs that were examined were in the range of 35–45 amino acid residues in length and were equally potent at a concentration of 50 μg/mL, whereas folded proteins, the PSD-95/Dlg/ZO-1 (PDZ) domain, and lysozymes had no potency. We further examined their cryoprotective activity toward glutathione S-transferase as an example of the other enzyme, and toward enhanced green fluorescent protein as a non-enzyme protein example. We further examined the lyophilization protective activity of the peptides toward LDH, which revealed that some IDPs showed a higher activity than that of bovine serum albumin (BSA). Based on these observations, we propose that cryoprotection is a general feature of IDPs. Our findings may become a clue to various industrial applications of IDPs in the future. Full article

Increased expression of heat shock proteins (HSPs) following heat stress or other stress conditions is a common physiological response in almost all living organisms. Modification of cytosolic proteins including HSPs by O-GlcNAc has been shown to enhance their capabilities for counteracting lethal levels of cellular stress. Since HSPs are key players in stress resistance and protein homeostasis, we aimed to analyze their forms at the cellular and molecular level using camel and human HSPs as models for efficient and moderate thermotolerant mammals, respectively. In this study, we cloned the cDNA encoding two inducible HSP members, HSPA6 and CRYAB from both camel (Camelus dromedarius) and human in a Myc-tagged mammalian expression vector. Expression of these chaperones in COS-1 cells revealed protein bands of approximately 25-kDa for both camel and human CRYAB and 70-kDa for camel HSPA6 and its human homologue. While localization and trafficking of the camel and human HSPs revealed similar cytosolic localization, we could demonstrate altered glycan structure between camel and human HSPA6. Interestingly, the glycoform of camel HSPA6 was rapidly formed and stabilized under normal and stress culture conditions whereas human HSPA6 reacted differently under similar thermal and hypoxic stress conditions. Our data suggest that efficient glycosylation of camel HSPA6 is among the mechanisms that provide camelids with a superior capability for alleviating stressful environmental circumstances. Full article

Recent clinical trials have shown that adoptive chimeric antigen receptor (CAR) T cell therapy is a very potent and possibly curative option in the treatment of B cell leukemias and lymphomas. However, targeting a single antigen may not be sufficient, and relapse due to the emergence of antigen negative leukemic cells may occur. A potential strategy to counter the outgrowth of antigen escape variants is to broaden the specificity of the CAR by incorporation of multiple antigen recognition domains in tandem. As a proof of concept, we here describe a bispecific CAR in which the single chain variable fragment (scFv) is replaced by a tandem of two single-antibody domains or nanobodies (nanoCAR). High membrane nanoCAR expression levels are observed in retrovirally transduced T cells. NanoCARs specific for CD20 and HER2 induce T cell activation, cytokine production and tumor lysis upon incubation with transgenic Jurkat cells expressing either antigen or both antigens simultaneously. The use of nanobody technology allows for the production of compact CARs with dual specificity and predefined affinity. Full article

Reasons for the development of chronic tendon pathologies are still under debate and more basic knowledge is needed about the different diseases. The aim of the present study was therefore to characterize different acute and chronic Achilles tendon disorders. Achilles tendon samples from patients with chronic tendinopathy (n = 7), chronic ruptures (n = 6), acute ruptures (n = 13), and intact tendons (n = 4) were analyzed. The histological score investigating pathological changes was significantly increased in tendinopathy and chronic ruptures compared to acute ruptures. Inflammatory infiltration was detected by immunohistochemistry in all tendon pathology groups, but was significantly lower in tendinopathy compared to chronic ruptures. Quantitative real-time PCR (qRT-PCR) analysis revealed significantly altered expression of genes related to collagens and matrix modeling/remodeling (matrix metalloproteinases, tissue inhibitors of metalloproteinases) in tendinopathy and chronic ruptures compared to intact tendons and/or acute ruptures. In all three tendon pathology groups markers of inflammation (interleukin (IL) 1β, tumor necrosis factor α, IL6, IL10, IL33, soluble ST2, transforming growth factor β1, cyclooxygenase 2), inflammatory cells (cluster of differentaition (CD) 3, CD68, CD80, CD206), fat metabolism (fatty acid binding protein 4, peroxisome proliferator-activated receptor γ, CCAAT/enhancer-binding protein α, adiponectin), and innervation (protein gene product 9.5, growth associated protein 43, macrophage migration inhibitory factor) were detectable, but only in acute ruptures significantly regulated compared to intact tendons. The study gives an insight into structural and molecular changes of pathological processes in tendons and might be used to identify targets for future therapy of tendon pathologies. Full article

Skeletal muscle comprising approximately 40% of body weight is highly important for locomotion and metabolic homeostasis. The growth and regeneration of skeletal muscle are highly organized processes; thus, it is not surprising to reveal certain complexity during these regulatory processes. Recently, a large number of evidence indicate that microRNAs can result in obvious impacts on growth, regeneration and metabolism of skeletal muscle. In this review, recent research achievements of microRNAs in regulating myogenesis, atrophy and aging during exercise intervention are discussed, which will provide the guidance for developing potential applications of microRNAs in health promotion and rehabilitation of sports injuries. Full article

Cancers that exhibit the Warburg effect may elevate expression of glyoxylase 1 (GLO1) to detoxify the toxic glycolytic byproduct methylglyoxal (MG) and inhibit the formation of pro-apoptotic advanced glycation endproducts (AGEs). Inhibition of GLO1 in cancers that up-regulate glycolysis has been proposed as a therapeutic targeting strategy, but this approach has not been evaluated for glioblastoma multiforme (GBM), the most aggressive and difficult to treat malignancy of the brain. Elevated GLO1 expression in GBM was established in patient tumors and cell lines using bioinformatics tools and biochemical approaches. GLO1 inhibition in GBM cell lines and in an orthotopic xenograft GBM mouse model was examined using both small molecule and short hairpin RNA (shRNA) approaches. Inhibition of GLO1 with S-(p-bromobenzyl) glutathione dicyclopentyl ester (p-BrBzGSH(Cp)₂) increased levels of the DNA-AGE N²-1-(carboxyethyl)-2′-deoxyguanosine (CEdG), a surrogate biomarker for nuclear MG exposure; substantially elevated expression of the immunoglobulin-like receptor for AGEs (RAGE); and induced apoptosis in GBM cell lines. Targeting GLO1 with shRNA similarly increased CEdG levels and RAGE expression, and was cytotoxic to glioma cells. Mice bearing orthotopic GBM xenografts treated systemically with p-BrBzGSH(Cp)₂ exhibited tumor regression without significant off-target effects suggesting that GLO1 inhibition may have value in the therapeutic management of these drug-resistant tumors. Full article

Electrospinning has been used for decades to generate nano-fibres via an electrically charged jet of polymer solution. This process is established on a spinning technique, using electrostatic forces to produce fine fibres from polymer solutions. Amongst, the electrospinning of available biopolymers (silk, cellulose, collagen, gelatine and hyaluronic acid), chitosan (CH) has shown a favourable outcome for tissue regeneration applications. The aim of the current review is to assess the current literature about electrospinning chitosan and its composite formulations for creating fibres in combination with other natural polymers to be employed in tissue engineering. In addition, various polymers blended with chitosan for electrospinning have been discussed in terms of their potential biomedical applications. The review shows that evidence exists in support of the favourable properties and biocompatibility of chitosan electrospun composite biomaterials for a range of applications. However, further research and in vivo studies are required to translate these materials from the laboratory to clinical applications. Full article

Some novel anthranilate diamides derivatives 4a–e, 6a–c and 9a–d were designed and synthesized to be evaluated for their in vitro anticancer activity. Structures of all newly synthesized compounds were confirmed by infra-red (IR), high-resolution mass (HR-MS) spectra, ¹H nuclear magnetic resonance (NMR) and ¹³C nuclear magnetic resonance (NMR) analyses. Cytotoxic screening was performed according to (3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide) tetrazolium (MTT) assay method using erlotinib as a reference drug against two different types of breast cancer cells. The molecular docking study was performed for representative compounds against two targets, epidermal growth factor receptor (EGFR) and tubulin in colchicine binding site to assess their binding affinities in order to rationalize their anticancer activity in a qualitative way. The data obtained from the molecular modeling was correlated with that obtained from the biological screening. These data showed considerable anticancer activity for these newly synthesized compounds. Biological data for most of the anthranilate diamide showed excellent activity with nanomolar or sub nanomolar half maximal inhibitory concentration (IC₅₀) values against tumor cells. EGFR tyrosine kinase (TK) inhibition assay, tubulin inhibition assay and apoptosis analysis were performed for selected compounds to get more details about their mechanism of action. Extensive structure activity relationship (SAR) analyses were also carried out. Full article

Phyllostachys nigra var. henosis, a domestic bamboo species, has been attracting much attention; its bioactive compounds (especially in the leaf) show antioxidant, anti-inflammatory, and anti-obesity activities. Little information is available on the antioxidative and anti-melanogenetic activities of the bioactive compounds in bamboo stems. The anti-melanogenic and antioxidative activities of the EtOAc fraction (PN3) of a P. nigra stem extract were investigated in a cell-free system and in B16F10 melanoma cells. PN3 consisted of a mixture of flavonoids, such as catechin, chlorogenic acid, caffeic acid, and p-coumaric acid. The antioxidant activity (2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS)), and hydroxyl radical scavenging) was evaluated, as well as the inhibition of reactive oxygen species (ROS) produced by the Fenton reaction. PN3 showed in vitro tyrosinase inhibition activity with the half maximal inbihitory concentration (IC₅₀) values of 240 μg/mL, and in vivo cytotoxic concentration ranges > 100 μg/mL. The protein expression levels and mRNA transcription levels of TYR, TRP-1, and MITF were decreased in a dose-dependent manner by the treatment with PN3. PN3 interfered with the phosphorylation of intracellular protein kinase A (PKA)/cAMP response element-binding protein (CREB), demonstrating potent anti-melanogenic effects. PN3 could inhibit PKA/CREB and the subsequent degradation of microphthalmia-associated transcription factor (MITF), resulting in the suppression of melanogenic enzymes and melanin production, probably because of the presence of flavonoid compounds. These properties make it a candidate as an additive to whitening cosmetics. Full article

Post-transcriptional regulation of gene expression plays a crucial role in many processes. In cells, it is mediated by diverse RNA-binding proteins. These proteins can influence mRNA stability, translation, and localization. The PUF protein family (Pumilio and FBF) is composed of RNA-binding proteins highly conserved among most eukaryotic organisms. Previous investigations indicated that they could be involved in many processes by binding corresponding motifs in the 3′UTR or by interacting with other proteins. To date, most of the investigations on PUF proteins have been focused on Caenorhabditis elegans, Drosophila melanogaster, and Saccharomyces cerevisiae, while only a few have been conducted on Arabidopsis thaliana. The present article provides an overview of the PUF protein family. It addresses their RNA-binding motifs, biological functions, and post-transcriptional control mechanisms in Caenorhabditis elegans, Drosophila melanogaster, Saccharomyces cerevisiae, and Arabidopsis thaliana. These items of knowledge open onto new investigations into the relevance of PUF proteins in specific plant developmental processes. Full article

The detection of human anti-estrogen receptor α antibodies (ERαABs) inducing estrogenic responses in MCF-7 mammary tumor cells suggests their implication in breast cancer emergence and/or evolution. A recent report revealing a correlation between the titer of such antibodies in sera from patients suffering from this disease and the percentage of proliferative cells in samples taken from their tumors supports this concept. Complementary evidence of the ability of ERαABs to interact with an epitope localized within the estradiol-binding core of ERα also argues in its favor. This epitope is indeed inserted in a regulatory platform implicated in ERα-initiated signal transduction pathways and transcriptions. According to some experimental observations, two auto-immune reactions may already be advocated to explain the emergence of ERαABs: one involving probably the idiotypic network to produce antibodies acting as estrogenic secretions and the other based on antibodies able to abrogate the action of a natural ERα inhibitor or to prevent the competitive inhibitory potency of released receptor degradation products able to entrap circulating estrogens and co-activators. All of this information, the aspect of which is mainly fundamental, may open new ways in the current tendency to combine immunological and endocrine approaches for the management of breast cancer. Full article

Carbohydrate esterases family 4 (CE4 enzymes) includes chitin and peptidoglycan deacetylases, acetylxylan esterases, and poly-N-acetylglucosamine deacetylases that act on structural polysaccharides, altering their physicochemical properties, and participating in diverse biological functions. Chitin and peptidoglycan deacetylases are not only involved in cell wall morphogenesis and remodeling in fungi and bacteria, but they are also used by pathogenic microorganisms to evade host defense mechanisms. Likewise, biofilm formation in bacteria requires partial deacetylation of extracellular polysaccharides mediated by poly-N-acetylglucosamine deacetylases. Such biological functions make these enzymes attractive targets for drug design against pathogenic fungi and bacteria. On the other side, acetylxylan esterases deacetylate plant cell wall complex xylans to make them accessible to hydrolases, making them attractive biocatalysts for biomass utilization. CE4 family members are metal-dependent hydrolases. They are highly specific for their particular substrates, and show diverse modes of action, exhibiting either processive, multiple attack, or patterned deacetylation mechanisms. However, the determinants of substrate specificity remain poorly understood. Here, we review the current knowledge on the structure, activity, and specificity of CE4 enzymes, focusing on chitin deacetylases and related enzymes active on N-acetylglucosamine-containing oligo and polysaccharides. Full article

Osteoarthritis (OA) is a common joint disorder found mostly in elderly people. The role of mechanical behavior in the progression of OA is complex and remains unclear. The stress-relaxation behavior of human articular cartilage in clinically defined osteoarthritic stages may have importance in diagnosis and prognosis of OA. In this study we investigated differences in the biomechanical responses among human cartilage of ICRS grades I, II and III using polymer dynamics theory. We collected 24 explants of human articular cartilage (eight each of ICRS grade I, II and III) and acquired stress-relaxation data applying a continuous load on the articular surface of each cartilage explant for 1180 s. We observed a significant decrease in Young’s modulus, stress-relaxation time, and stretching exponent in advanced stages of OA (ICRS grade III). The stretch exponential model speculated that significant loss in hyaluronic acid polymer might be the reason for the loss of proteoglycan in advanced OA. This work encourages further biomechanical modelling of osteoarthritic cartilage utilizing these data as input parameters to enhance the fidelity of computational models aimed at revealing how mechanical behaviors play a role in pathogenesis of OA. Full article

The present study analysed changes in the distribution pattern of cocaine- and amphetamine-regulated transcript (CART) in the enteric nervous system (ENS) of the human colon challenged by adenocarcinoma invasion, using the double-labelling immunofluorescence technique. In control specimens, CART immunoreactivity was found in neurons of all studied plexuses, representing 30.1 ± 4.1%, 12.9 ± 5.2%, and 4.1 ± 1.3% of all neurons forming the myenteric plexus (MP), outer submucous plexus (OSP), and inner submucous plexus (ISP), respectively. Tumour growth into the colon wall caused an increase in the relative frequency of CART-like immunoreactive (CART-LI) neurons in enteric plexuses located in the vicinity of the infiltrating neoplasm (to 36.1 ± 6.7%, 32.7 ± 7.3% and 12.1 ± 3.8% of all neurons in MP, OSP and ISP, respectively). The density of CART-LI nerves within particular layers of the intestinal wall did not differ between control and adenocarcinoma-affected areas of the human colon. This is the first detailed description of the CART distribution pattern within the ENS during the adenocarcinoma invasion of the human colon wall. The obtained results suggest that CART probably acts as a neuroprotective factor and may be involved in neuronal plasticity evoked by the progression of a neoplastic process. Full article

Urological cancers include a spectrum of malignancies affecting organs of the reproductive and/or urinary systems, such as prostate, kidney, bladder, and testis. Despite improved primary prevention, detection and treatment, urological cancers are still characterized by an increasing incidence and mortality worldwide. While advances have been made towards understanding the molecular bases of these diseases, a complete understanding of the pathological mechanisms remains an unmet research goal that is essential for defining safer pharmacological therapies and prognostic factors, especially for the metastatic stage of these malignancies for which no effective therapies are currently being used. Glyoxalases, consisting of glyoxalase 1 (Glo1) and glyoxalase 2 (Glo2), are enzymes that catalyze the glutathione-dependent metabolism of cytotoxic methylglyoxal (MG), thus protecting against cellular damage and apoptosis. They are generally overexpressed in numerous cancers as a survival strategy by providing a safeguard through enhancement of MG detoxification. Increasing evidence suggests that glyoxalases, especially Glo1, play an important role in the initiation and progression of urological malignancies. In this review, we highlight the critical role of glyoxalases as regulators of tumorigenesis in the prostate through modulation of various critical signaling pathways, and provide an overview of the current knowledge on glyoxalases in bladder, kidney and testis cancers. We also discuss the promise and challenges for Glo1 inhibitors as future anti-prostate cancer (PCa) therapeutics and the potential of glyoxalases as biomarkers for PCa diagnosis. Full article

The frequency and volume of Asian sand dust (ASD) (Kosa) are increasing in Japan, and it has been reported that ASD may cause adverse respiratory effects. The pulmonary toxicity of ASD has been previously analyzed in mice exposed to ASD particles by intratracheal instillation. To study the pulmonary toxicity induced by inhalation of ASD, ICR mice were exposed by inhalation to 50 or 200 mg/m³ Kanto loam powder, which resembles ASD in elemental composition and particle size, for 6 h a day over 1, 3, 6, 9, or 15 consecutive days. Histological examination revealed that Kanto loam powder induced acute inflammation in the whole lung at all the time points examined. The lesions were characterized by infiltration of neutrophils and macrophages. The intensity of the inflammatory changes in the lung and number of neutrophils in both histological lesions and bronchoalveolar lavage fluid (BALF) appeared to increase over time. Immunohistochemical staining showed interleukin (IL)-6- and tumor necrosis factor (TNF)-α-positive macrophages and a decrease in laminin positivity in the inflammatory lesions of the lung tissues. Electron microscopy revealed vacuolar degeneration in the alveolar epithelial cells close to the Kanto loam particles. The nitric oxide level in the BALF increased over time. These results suggest that inhaled Kanto loam powder may induce diffuse and acute pulmonary inflammation, which is associated with increased expression of inflammatory cytokines and oxidative stress. Full article

Ischemia reperfusion (IR), present in myocardial infarction or extremity injuries, is a major clinical issue and leads to substantial tissue damage. Molecular mechanisms underlying IR injury in striated muscles involve the production of reactive oxygen species (ROS). Excessive ROS accumulation results in cellular oxidative stress, mitochondrial dysfunction, and initiation of cell death by activation of the mitochondrial permeability transition pore. Elevated ROS levels can also decrease myofibrillar Ca²⁺ sensitivity, thereby compromising muscle contractile function. Low levels of ROS can act as signaling molecules involved in the protective pathways of ischemic preconditioning (IPC). By scavenging ROS, antioxidant therapies aim to prevent IR injuries with positive treatment outcomes. Novel therapies such as postconditioning and pharmacological interventions that target IPC pathways hold great potential in attenuating IR injuries. Factors such as aging and diabetes could have a significant impact on the severity of IR injuries. The current paper aims to provide a comprehensive review on the multifaceted roles of ROS in IR injuries, with a focus on cardiac and skeletal muscle, as well as recent advancement in ROS-related therapies. Full article

The protease-cleaved osteopontin (OPN) was proposed to enhance the migration of memory T cells to granulomas in tuberculosis. Various forms of OPN were identified in human monocytic THP-1 cells stimulated by phorbol 12-myristate 13-acetate (PMA). Antibodies O-17, 10A16 and 34E3, which recognize N-terminus, the C-half, and thrombin-cleaved site of OPN, respectively, all detected distinct bands on Western blots following PMA stimulation. Bands corresponding to 18 and 30 kD were detected by antibodies 34E3 and 10A16, indicating that OPN cleavage occurred by endogenous proteases in the PMA-stimulated THP-1 cells. In immune-fluorescence (IF) assay, 34E3 positive signals were detected in intracellular space of non-infected and bacillus Calmette-Guérin (BCG)-infected cells; however, 10A16 positive signals were confirmed in extracellular area in PMA-stimulated cells followed by BCG infection. Small amounts of full-length (FL) and thrombin-cleaved (Tr) OPN were detected by ELISA in the supernatants of non-PMA-stimulated cells, and increased levels of all forms, including undefined (Ud) OPN, in PMA-stimulated cells. ELISA showed a decrease in OPN synthesis during BCG infection. To our knowledge, this is the first report of OPN cleavage in THP-1 macrophages after PMA stimulation, and of enhanced cleavage induced by BCG infection. Full article

SERCA2a is the Ca²⁺ ATPase playing the major contribution in cardiomyocyte (CM) calcium removal. Its activity can be regulated by both modulatory proteins and several post-translational modifications. The aim of the present work was to investigate whether the function of SERCA2 can be modulated by treating CMs with the histone deacetylase (HDAC) inhibitor suberanilohydroxamic acid (SAHA). The incubation with SAHA (2.5 µM, 90 min) of CMs isolated from rat adult hearts resulted in an increase of SERCA2 acetylation level and improved ATPase activity. This was associated with a significant improvement of calcium transient recovery time and cell contractility. Previous reports have identified K464 as an acetylation site in human SERCA2. Mutants were generated where K464 was substituted with glutamine (Q) or arginine (R), mimicking constitutive acetylation or deacetylation, respectively. The K464Q mutation ameliorated ATPase activity and calcium transient recovery time, thus indicating that constitutive K464 acetylation has a positive impact on human SERCA2a (hSERCA2a) function. In conclusion, SAHA induced deacetylation inhibition had a positive impact on CM calcium handling, that, at least in part, was due to improved SERCA2 activity. This observation can provide the basis for the development of novel pharmacological approaches to ameliorate SERCA2 efficiency. Full article

Sphingolipids, sphingolipid metabolizing enzymes, and their receptors network are being recognized as part of the signaling mechanisms, which govern breast cancer cell growth, migration, and survival during chemotherapy treatment. Approximately 70% of breast cancers are estrogen receptor (ER) positive and, thus, rely on estrogen signaling. Estrogen activates an intracellular network composed of many cytoplasmic and nuclear mediators. Some estrogen effects can be mediated by sphingolipids. Estrogen activates sphingosine kinase 1 (SphK1) and amplifies the intracellular concentration of sphingosine-1-phosphate (S1P) in breast cancer cells during stimulation of proliferation and survival. Specifically, Estrogen activates S1P receptors (S1PR) and induces growth factor receptor transactivation. SphK, S1P, and S1PR expression are causally associated with endocrine resistance and progression to advanced tumor stages in ER-positive breast cancers in vivo. Recently, the network of SphK/S1PR was shown to promote the development of ER-negative cancers and breast cancer stem cells, as well as stimulating angiogenesis. Novel findings confirm and broaden our knowledge about the cross-talk between sphingolipids and estrogen network in normal and malignant cells. Current S1PRs therapeutic inhibition was indicated as a promising chemotherapy approach in non-responsive and advanced malignancies. Considering that sphingolipid signaling has a prominent role in terminally differentiated cells, the impact should be considered when designing specific SphK/S1PR inhibitors. This study analyzes the dynamic of the transformation of sphingolipid axis during a transition from normal to pathological condition on the level of the whole organism. The sphingolipid-based mediation and facilitation of global effects of estrogen were critically accented as a bridging mechanism that should be explored in cancer prevention. Full article

N-glycosylation profoundly affects the biological stability and function of therapeutic proteins, which explains the recent interest in glycoengineering technologies as methods to develop biobetter therapeutics. In current manufacturing processes, N-glycosylation is host-specific and remains difficult to control in a production environment that changes with scale and production batches leading to glycosylation heterogeneity and inconsistency. On the other hand, in vitro chemoenzymatic glycan remodeling has been successful in producing homogeneous pre-defined protein glycoforms, but needs to be combined with a cost-effective and scalable production method. An efficient chemoenzymatic glycan remodeling technology using a plant expression system that combines in vivo deglycosylation with an in vitro chemoenzymatic glycosylation is described. Using the monoclonal antibody rituximab as a model therapeutic protein, a uniform Gal2GlcNAc2Man3GlcNAc2 (A2G2) glycoform without α-1,6-fucose, plant-specific α-1,3-fucose or β-1,2-xylose residues was produced. When compared with the innovator product Rituxan^®, the plant-made remodeled afucosylated antibody showed similar binding affinity to the CD20 antigen but significantly enhanced cell cytotoxicity in vitro. Using a scalable plant expression system and reducing the in vitro deglycosylation burden creates the potential to eliminate glycan heterogeneity and provide affordable customization of therapeutics’ glycosylation for maximal and targeted biological activity. This feature can reduce cost and provide an affordable platform to manufacture biobetter antibodies. Full article

Persistent cervical infection with high-risk human papillomaviruses (hrHPVs) is a necessary, but not sufficient, condition for the development of cervical cancer. Therefore, there are other co-factors facilitating the hrHPV carcinogenic process, one of which is smoking. To assess the effect of smoking on high-risk (hr) HPV DNA positivity and on the expression of HPV E7 oncoprotein, as a surrogate of persistent hrHPV infection, we used data from women recruited for the PIPAVIR project, which examined the role of E7 protein detection in cervical cancer screening. Women were tested for hrHPV DNA, using Multiplex Genotyping (MPG), and E7 protein, using a novel sandwich ELISA method, and gave information on their smoking habits. Among 1473 women, hrHPV prevalence was 19.1%. The odds ratio (OR) for hrHPV positivity of smokers compared to non-smokers was 1.785 (95% confidence intervals (CI): 1.365–2.332, p < 0.001). The ORs for E7 positivity, concerning hrHPV positive women, ranged from 0.720 to 1.360 depending on the E7 detection assay used, but this was not statistically significant. Smoking increases the probability of hrHPV infection, and smoking intensity is positively associated to this increase. Smoking is not related to an increased probability of E7 protein positivity for hrHPV positive women. Full article

Amyotrophic lateral sclerosis (ALS) is a multifactorial disease with limited therapeutic options. Numerous intrinsic and extrinsic factors are involved in ALS motor neuron degeneration. One possible effector accelerating motor neuron death in ALS is damage to the blood-Central Nervous System barrier (B-CNS-B), mainly due to endothelial cell (EC) degeneration. Although mechanisms of EC damage in ALS are still unknown, vascular impairment may be initiated by various humoral inflammatory factors and other mediators. Systemic IL-6-mediated inflammation is a possible early extrinsic effector leading to the EC death causing central nervous system (CNS) barrier damage. In this review, we discuss the potential role of humoral factors in triggering EC alterations in ALS. A specific focus was on humoral IL-6 cytokine mediating EC inflammation via the trans-signaling pathway. Our preliminary in vitro studies demonstrated a proof of principle that short term exposure of human bone marrow endothelial cells to plasma from ALS patient leads to cell morphological changes, significantly upregulated IL-6R immunoexpression, and pro-inflammatory cell response. Our in-depth understanding of specific molecular mechanisms of this humoral cytokine in EC degeneration may facilitate an endothelial-IL-6-targeting therapy for restoring cell homeostasis and eventually reestablishing B-CNS-B integrity in ALS. Full article

Coronary artery disease (CAD) is one of the most common causes of death worldwide. In the last decade, significant advancements in CAD treatment have been made. The existing treatment is medical, surgical or a combination of both depending on the extent, severity and clinical presentation of CAD. The collaboration between different science disciplines such as biotechnology and tissue engineering has led to the development of novel therapeutic strategies such as stem cells, nanotechnology, robotic surgery and other advancements (3-D printing and drugs). These treatment modalities show promising effects in managing CAD and associated conditions. Research on stem cells focuses on studying the potential for cardiac regeneration, while nanotechnology research investigates nano-drug delivery and percutaneous coronary interventions including stent modifications and coatings. This article aims to provide an update on the literature (in vitro, translational, animal and clinical) related to these novel strategies and to elucidate the rationale behind their potential treatment of CAD. Through the extensive and continued efforts of researchers and clinicians worldwide, these novel strategies hold the promise to be effective alternatives to existing treatment modalities. Full article

Glucose oxidase (GOD, EC.1.1.3.4) specifically catalyzes the reaction of β-d-glucose to gluconic acid and hydrogen peroxide in the presence of oxygen, which has become widely used in the food industry, gluconic acid production and the feed industry. However, the poor thermostability of the current commercial GOD is a key limiting factor preventing its widespread application. In the present study, amino acids closely related to the thermostability of glucose oxidase from Penicillium notatum were predicted with a computer-aided molecular simulation analysis, and mutant libraries were established following a saturation mutagenesis strategy. Two mutants with significantly improved thermostabilities, S100A and D408W, were subsequently obtained. Their protein denaturing temperatures were enhanced by about 4.4 °C and 1.2 °C, respectively, compared with the wild-type enzyme. Treated at 55 °C for 3 h, the residual activities of the mutants were greater than 72%, while that of the wild-type enzyme was only 20%. The half-lives of S100A and D408W were 5.13- and 4.41-fold greater, respectively, than that of the wild-type enzyme at the same temperature. This work provides novel and efficient approaches for enhancing the thermostability of GOD by reducing the protein free unfolding energy or increasing the interaction of amino acids with the coenzyme. Full article

This study aims to evaluate the putative roles of a single acute dose of resveratrol (RVT) in preventing cerebral oxidative stress induced by bilateral common carotid artery occlusion, followed by reperfusion (BCCAO/R) and to investigate RVT’s ability to preserve the neuronal structural integrity. Frontal and temporal-occipital cortices were examined in two groups of adult Wistar rats, sham-operated and submitted to BCCAO/R. In both groups, 6 h before surgery, half the rats were gavage-fed with a single dose of RVT (40 mg/per rat in 300 µL of sunflower oil as the vehicle), while the second half received the vehicle alone. In the frontal cortex, RVT pre-treatment prevented the BCCAO/R-induced increase of lipoperoxides, augmented concentrations of palmitoylethanolamide and docosahexaenoic acid, increased relative levels of the cannabinoid receptors type 1 (CB1) and 2 (CB2), and peroxisome-proliferator-activated-receptor (PPAR)-α proteins. Increased expression of CB1/CB2 receptors mirrored that of synaptophysin and post-synaptic density-95 protein. No BCCAO/R-induced changes occurred in the temporal-occipital cortex. Collectively, our results demonstrate that, in the frontal cortex, RVT pre-treatment prevents the BCCAO/R-induced oxidative stress and modulates the endocannabinoid and PPAR-α systems. The increased expression of synaptic structural proteins further suggests the possible efficacy of RVT as a dietary supplement to preserve the nervous tissue metabolism and control the physiological response to the hypoperfusion/reperfusion challenge. Full article

Using femur explants from mice as an in vitro model, we investigated the effect of the physiological polymer, inorganic polyphosphate (polyP), on differentiation of the cells of the bone marrow in their natural microenvironment into the osteogenic and chondrogenic lineages. In the form of amorphous Ca-polyP nano/microparticles, polyP retains its function to act as both an intra- and extracellular metabolic fuel and a stimulus eliciting morphogenetic signals. The method for synthesis of the nano/microparticles with the polyanionic polyP also allowed the fabrication of hybrid particles with the bisphosphonate zoledronic acid, a drug used in therapy of bone metastases in cancer patients. The results revealed that the amorphous Ca-polyP particles promote the growth/viability of mesenchymal stem cells, as well as the osteogenic and chondrogenic differentiation of the bone marrow cells in rat femur explants, as revealed by an upregulation of the expression of the transcription factors SOX9 (differentiation towards osteoblasts) and RUNX2 (chondrocyte differentiation). In parallel to this bone anabolic effect, incubation of the femur explants with these particles significantly reduced the expression of the gene encoding the osteoclast bone-catabolic enzyme, cathepsin-K, while the expression of the tartrate-resistant acid phosphatase remained unaffected. The gene expression data were supported by the finding of an increased mineralization of the cells in the femur explants in response to the Ca-polyP particles. Finally, we show that the hybrid particles of polyP complexed with zoledronic acid exhibit both the cytotoxic effect of the bisphosphonate and the morphogenetic and mineralization inducing activity of polyP. Our results suggest that the Ca-polyP nano/microparticles are not only a promising scaffold material for repairing long bone osteo-articular damages but can also be applied, as a hybrid with zoledronic acid, as a drug delivery system for treatment of bone metastases. The polyP particles are highlighted as genuine, smart, bioinspired nano/micro biomaterials. Full article

Protic ionic liquids (PILs) have been established as effective solvents for the selective extraction and recovery of lignin from lignocellulosic biomass. In this study, we utilize extensive analytical techniques to characterize the PIL-extracted lignins to (1) expand on the physical/chemical structure, and to (2) develop a better understanding of the mechanism behind the lignin dissolution process. The PIL-lignins were characterized using elemental and FT-IR analyses, alongside molecular weight distribution and chemical modeling via MM2. For the more ionic pyrrolidinium acetate ([Pyrr][Ac]), there is an increase in the fragmentation of lignin, resulting in lignin with a smaller average molecular weight and a more uniform dispersity. This lends better understanding to previous findings indicating that higher ionicity in a PIL leads to increased lignin extraction. Full article

Most effective nematicides for the control of root-knot nematodes are banned, which demands a better understanding of the plant-nematode interaction. Understanding how gene expression in the nematode-feeding sites relates to morphological features may assist a better characterization of the interaction. However, nematode-induced galls resulting from cell-proliferation and hypertrophy hinders such observation, which would require tissue sectioning or clearing. We demonstrate that a method based on the green auto-fluorescence produced by glutaraldehyde and the tissue-clearing properties of benzyl-alcohol/benzyl-benzoate preserves the structure of the nematode-feeding sites and the plant-nematode interface with unprecedented resolution quality. This allowed us to obtain detailed measurements of the giant cells’ area in an Arabidopsis line overexpressing CHITINASE-LIKE-1 (CTL1) from optical sections by confocal microscopy, assigning a role for CTL1 and adding essential data to the scarce information of the role of gene repression in giant cells. Furthermore, subcellular structures and features of the nematodes body and tissues from thick organs formed after different biotic interactions, i.e., galls, syncytia, and nodules, were clearly distinguished without embedding or sectioning in different plant species (Arabidopsis, cucumber or Medicago). The combination of this method with molecular studies will be valuable for a better understanding of the plant-biotic interactions. Full article

Galectins are glycan-binding proteins that contain one or two carbohydrate domains and mediate multiple biological functions. By analyzing clinical tumor samples, the abnormal expression of galectins is known to be linked to the development, progression and metastasis of cancers. Galectins also have diverse functions on different immune cells that either promote inflammation or dampen T cell-mediated immune responses, depending on cognate receptors on target cells. Thus, tumor-derived galectins can have bifunctional effects on tumor and immune cells. This review focuses on the biological effects of galectin-1, galectin-3 and galectin-9 in various cancers and discusses anticancer therapies that target these molecules. Full article

Altered expression of secreted factors by tumor cells or cells of the tumor microenvironment is a key event in cancer development and progression. In the last decade, emerging evidences supported the autocrine and paracrine activity of the members of the Angiopoietin-like (ANGPTL) protein family in angiogenesis, inflammation and in the regulation of different steps of carcinogenesis and metastasis development. Thus, ANGPTL proteins become attractive either as prognostic or predictive biomarkers, or as novel target for cancer treatment. Here, we outline the current knowledge about the functions of the ANGPTL proteins in angiogenesis, cancer progression and metastasis. Moreover, we discuss the most recent evidences sustaining their role as prognostic or predictive biomarkers for cancer therapy. Although the role of ANGPTL proteins in cancer has not been fully elucidated, increasing evidence suggest their key effects in the proliferative and invasive properties of cancer cells. Moreover, given the common overexpression of ANGPTL proteins in several aggressive solid tumors, and their role in tumor cells and cells of the tumor microenvironment, the field of research about ANGPTL proteins network may highlight new potential targets for the development of future therapeutic strategies. Full article

Jatropha curcas L. seeds an oilseed plant with great potential for biodiesel production. However, low seed yield, which was limited by its lower female flowers, was a major drawback for its utilization. Our previous study found that the flower number and female-to-male ratio were increased by gibberellin treatment. Here, we compared the transcriptomic profiles of inflorescence meristem at different time points after gibberellic acid A3 (GA₃) treatment. The present study showed that 951 differentially expressed genes were obtained in response to gibberellin treatment, compared with control samples. The 6-h time point was an important phase in the response to exogenous gibberellin. Furthermore, the plant endogenous gibberellin, auxin, ethylene, abscisic acid, and brassinolide-signaling transduction pathways were repressed, whereas the genes associated with cytokinin and jasmonic acid signaling were upregulated for 24-h time point following GA₃ treatment. In addition, the floral meristem determinacy genes (JcLFY, JcSOC1) and floral organ identity genes (JcAP3, JcPI, JcSEP1-3) were significantly upregulated, but their negative regulator (JcSVP) was downregulated after GA₃ treatment. Moreover, the effects of phytohormone, which was induced by exogenous plant growth regulator, mainly acted on the female floral differentiation process. To the best of our knowledge, this data is the first comprehensive analysis of the underlying transcriptional response mechanism of floral differentiation following GA₃ treatment in J. curcas, which helps in engineering high-yielding varieties of Jatropha. Full article

This in vitro study evaluated the compositional, crystalline, and morphological effects promoted by Nd:YAG laser on root dentin, and verified the effects of laser and topical acidulated phosphate fluoride application (APF-gel) on dentin erosion. 180 bovine dentin slabs were randomized into 4 groups (n = 45): G1–untreated, G2–APF-gel (1.23% F⁻, 4 min), G3–Nd:YAG (1064 nm, 84.9 J/cm², 10 Hz), and G4–APF-gel application followed by Nd:YAG laser irradiation. The compositional, crystalline, and morphological effects promoted by treatments were investigated on five samples of each experimental group. The other samples were submitted to a 5-day, 10-day, or 15-day erosive and abrasive demineralization and remineralization cycling in order to create erosion lesions. The area and depth of lesions, as well as the optical attenuation coefficient, were assessed, and all data were statistically analysed (p < 0.05). Nd:YAG laser promoted the reduction of carbonate, the formation of tetracalcium phosphate, as well as the melting and recrystallization of the dentin surface. Laser significantly decreased the area and depth of erosion lesions and altered the optical attenuation coefficient when compared to untreated and APF-gel groups, but the association of APF-gel and laser did not promote an additional effect. Nd:YAG laser irradiation can be a promissory treatment to prevent dentin erosion and the abrasion process. Full article

Photodynamic inactivation (PDI) has been shown to be a potential treatment modality against Candida infection. However, limited light penetration might leave some cells alive and undergoing regrowth. In this study, we explored the possibility of combining PDI and antifungal agents to enhance the therapeutic efficacy of Candida albicans and drug-resistant clinical isolates. We found that planktonic cells that had survived toluidine blue O (TBO)-mediated PDI were significantly susceptible to fluconazole within the first 2 h post PDI. Following PDI, the killing efficacy of antifungal agents relates to the PDI dose in wild-type and drug-resistant clinical isolates. However, only a 3-log reduction was found in the biofilm cells, suggesting limited therapeutic efficacy under the combined treatment of PDI and azole antifungal drugs. Using confocal microscopic analysis, we showed that TBO-mediated PDI could partially remove the extracellular polymeric substance (EPS) of biofilm. Finally, we showed that a combination of PDI with caspofungin could result in the complete killing of biofilms compared to those treated with caspofungin or PDI alone. These results clearly indicate that the combination of PDI and antifungal agents could be a promising treatment against C. albicans infections. Full article

Articular cartilage is a tissue characterized by its poor intrinsic capacity for self-repair. This tissue is frequently altered upon trauma or in osteoarthritis (OA), a degenerative disease that is currently incurable. Similar musculoskeletal disorders also affect horses and OA incurs considerable economic loss for the equine sector. In the view to develop new therapies for humans and horses, significant progress in tissue engineering has led to the emergence of new generations of cartilage therapy. Matrix-associated autologous chondrocyte implantation is an advanced 3D cell-based therapy that holds promise for cartilage repair. This study aims to improve the autologous chondrocyte implantation technique by using equine mesenchymal stem cells (MSCs) from bone marrow differentiated into chondrocytes that can be implanted in the chondral lesion. The optimized protocol relies on culture under hypoxia within type I/III collagen sponges. Here, we explored three parameters that influence MSC differentiation: culture times, growth factors and RNA interference strategies. Our results suggest first that an increase in culture time from 14 to 28 or 42 days lead to a sharp increase in the expression of chondrocyte markers, notably type II collagen (especially the IIB isoform), along with a concomitant decrease in HtrA1 expression. Nevertheless, the expression of type I collagen also increased with longer culture times. Second, regarding the growth factor cocktail, TGF-β3 alone showed promising result but the previously tested association of BMP-2 and TGF-β1 better limits the expression of type I collagen. Third, RNA interference targeting Col1a2 as well as Col1a1 mRNA led to a more significant knockdown, compared with a conventional strategy targeting Col1a1 alone. This chondrogenic differentiation strategy showed a strong increase in the Col2a1:Col1a1 mRNA ratio in the chondrocytes derived from equine bone marrow MSCs, this ratio being considered as an index of the functionality of cartilage. These data provide evidence of a more stable chondrocyte phenotype when combining Col1a1 and Col1a2 siRNAs associated to a longer culture time in the presence of BMP-2 and TGF-β1, opening new opportunities for preclinical trials in the horse. In addition, because the horse is an excellent model for human articular cartilage disorders, the equine therapeutic approach developed here can also serve as a preclinical step for human medicine. Full article

As a substantial part of the brain tumor microenvironment (TME), glioma-associated microglia/macrophages (GAMs) have an emerging role in tumor progression and in controlling anti-tumor immune responses. We review challenges and improvements of cell models and highlight the contribution of this highly plastic cell population to an immunosuppressive TME, besides their well-known functional role regarding glioma cell invasion and angiogenesis. Finally, we summarize first therapeutic interventions to target GAMs and their effect on the immunobiology of gliomas, focusing on their interaction with T cells. Full article

Experimental and computational simulation methods are used to investigate the adsorption behavior of the surfactant nonylphenol ethoxylate (NPEO10), which contains 10 ethylene oxide groups, on the lignite surface. The adsorption of NPEO10 on lignite follow a Langmuir-type isotherm. The thermodynamic parameters of the adsorption process show that the whole process is spontaneous. X-ray photoelectron spectroscopic (XPS) analysis indicates that a significant fraction of the oxygen-containing functional groups on the lignitic surface were covered by NPEO10. Molecular dynamics (MD) simulations show that the NPEO10 molecules were found to adsorb at the water-coal interface. Moreover, polar interactions are the main effect in the adsorption process. The density distributions of coal, NPEO10, and water molecules along the Z axis show that the remaining hydrophobic portions of the surfactant extend into the solution, creating a more hydrophobic coal surface that repels water molecules. The negative interaction energy calculated from the density profiles of the head and tail groups along the three spatial directions between the surfactant and the lignitic surface suggest that the adsorption process is spontaneous. The self-diffusion coefficients show that the presence of NPEO10 causes higher water mobility by improving the hydrophobicity of lignite. Full article

Cohesin complex components exert fundamental roles in animal cells, both canonical in cell cycle and non-canonical in gene expression regulation. Germline mutations in genes coding for cohesins result in developmental disorders named cohesinopaties, of which Cornelia de Lange syndrome (CdLS) is the best-known entity. However, a basic description of mammalian expression pattern of cohesins in a physiologic condition is still needed. Hence, we report a detailed analysis of expression in murine and human tissues of cohesin genes defective in CdLS. Using both quantitative and qualitative methods in fetal and adult tissues, cohesin genes were found to be ubiquitously and differentially expressed in human tissues. In particular, abundant expression was observed in hematopoietic and central nervous system organs. Findings of the present study indicate tissues which should be particularly sensitive to mutations, germline and/or somatic, in cohesin genes. Hence, this expression analysis in physiological conditions may represent a first core reference for cohesinopathies. Full article

A distinct G-protein coupled receptor that senses changes in extracellular Zn²⁺, ZnR/GPR39, was found in cells from tissues in which Zn²⁺ plays a physiological role. Most prominently, ZnR/GPR39 activity was described in prostate cancer, skin keratinocytes, and colon epithelial cells, where zinc is essential for cell growth, wound closure, and barrier formation. ZnR/GPR39 activity was also described in neurons that are postsynaptic to vesicular Zn²⁺ release. Activation of ZnR/GPR39 triggers Gαq-dependent signaling and subsequent cellular pathways associated with cell growth and survival. Furthermore, ZnR/GPR39 was shown to regulate the activity of ion transport mechanisms that are essential for the physiological function of epithelial and neuronal cells. Thus, ZnR/GPR39 provides a unique target for therapeutically modifying the actions of zinc in a specific and selective manner. Full article

Natural killer T cells (NKT) are a subset of T lymphocytes bridging innate and adaptive immunity. These cells recognize self and microbial glycolipids bound to non-polymorphic and highly conserved CD1d molecules. Three NKT cell subsets, type I, II, and NKT-like expressing different antigen receptors (TCR) were described and TCR activation promotes intracellular events leading to specific functional activities. NKT can exhibit different functions depending on the secretion of soluble molecules and the interaction with other cell types. NKT cells act as regulatory cells in the defense against infections but, on the other hand, their effector functions can be involved in the pathogenesis of several inflammatory disorders due to their exposure to different microbial or self-antigens, respectively. A deep understanding of the biology and functions of type I, II, and NKT-like cells as well as their interplay with cell types acting in innate (neuthrophils, innate lymphoid cells, machrophages, and dendritic cells) and adaptive immunity (CD4⁺,CD8⁺, and double negative T cells) should be important to design potential immunotherapies for infectious and autoimmune diseases. Full article

Fiber quality and yield improvement are crucial for cotton domestication and breeding. With the transformation in spinning techniques and multiplicity needs, the development of cotton fiber quality and yield is of great importance. A genetic map of 5178 Single Nucleotide Polymorphism (SNP) markers were generated using 277 F_2:3 population, from an intra-specific cross between two upland cotton accessions, CCRI35 a high fiber quality as female and Nan Dan Ba Di Da Hua (NH), with good yield properties as male parent. The map spanned 4768.098 cM with an average distance of 0.92 cM. A total of 110 Quantitative Traits Loci (QTLs) were identified for 11 traits, but only 30 QTLs were consistent in at least two environments. The highest percentage of phenotypic variance explained by a single QTL was 15.45%. Two major cluster regions were found, cluster 1 (chromosome17-D03) and cluster 2 (chromosome26-D12). Five candidate genes were identified in the two QTL cluster regions. Based on GO functional annotation, all the genes were highly correlated with fiber development, with functions such as protein kinase and phosphorylation. The five genes were associated with various fiber traits as follows: Gh_D03G0889 linked to qFM-D03_cb, Gh_D12G0093, Gh_D12G0410, Gh_D12G0435 associated with qFS-D12_cb and Gh_D12G0969 linked to qFY-D12_cb. Further structural annotation and fine mapping is needed to determine the specific role played by the five identified genes in fiber quality and yield related pathway. Full article