International Journal of Molecular Sciences

PolyPurine Reverse Hoogsteen Hairpins (PPRHs) are gene-silencing DNA-oligonucleotides developed in our laboratory that are formed by two antiparallel polypurine mirror repeat domains bound intramolecularly by Hoogsteen bonds. The aim of this work was to explore the feasibility of using viral vectors to deliver PPRHs as a gene therapy tool. After treatment with synthetic RNA, plasmid transfection, or viral infection targeting the survivin gene, viability was determined by the MTT assay, mRNA was determined by RT-qPCR, and protein levels were determined by Western blot. We showed that the RNA-PPRH induced a decrease in cell viability in a dose-dependent manner and an increase in apoptosis in PC-3 and HeLa cells. Both synthetic RNA-PPRH and RNA-PPRH intracellularly generated upon the transfection of a plasmid vector were able to reduce survivin mRNA and protein levels in PC-3 cells. An adenovirus type-5 vector encoding the PPRH against survivin was also able to decrease survivin mRNA and protein levels, leading to a reduction in HeLa cell viability. In this work, we demonstrated that PPRHs can also work as RNA species, either chemically synthesized, transcribed from a plasmid construct, or transcribed from viral vectors. Therefore, all these results are the proof of principle that viral vectors could be considered as a delivery system for PPRHs. Full article

TGF-β family signaling pathways, including TGF-β and BMP pathways, are widely involved in the regulation of health and diseases through downstream SMADs, which are also regulated by multiple validated mechanisms, such as genetic regulation, epigenetic regulation, and feedback regulation. However, it is still unclear whether R-SMADs or Co-SMAD can feedback regulate the TGF-β family signaling pathways in granulosa cells (GCs). In this study, we report a novel mechanism underlying the feedback regulation of TGF-β family signaling pathways, i.e., SMAD4, the only Co-SMAD, positive feedback activates the TGF-β family signaling pathways in GCs with a basal level of TGF-β ligands by interacting with the core promoters of its upstream receptors. Mechanistically, SMAD4 acts as a transcription factor, and feedback activates the transcription of its upstream receptors, including ACVR1B, BMPR2, and TGFBR2, of the canonical TGF-β signaling pathways by interacting with three coactivators (c-JUN, CREB1, and SP1), respectively. Notably, three different interaction modes between SMAD4 and coactivators were identified in SMAD4-mediated feedback regulation of upstream receptors through reciprocal ChIP assays. Our findings in the present study indicate for the first time that SMAD4 feedback activates the canonical TGF-β family signaling pathways in GCs, which improves and expands the regulatory mechanism, especially the feedback regulation modes of TGF-β family signaling pathways in ovarian GCs. Full article

Sarcopenia is a loss of muscle mass and function in elderly people and can lead to physical frailty and fall-related injuries. Sarcopenia is an inevitable event of the aging process that substantially impacts a person’s quality of life. Recent studies to improve muscle function through the intake of various functional food materials are attracting attention. However, it is not yet known whether probiotics can improve muscle mass and muscle strength and affect physical performance. Lactobacillus plantarum HY7715 (HY7715) is a lactic acid bacteria isolated from kimchi. The present research shows that L. plantarum HY7715 increases physical performance and skeletal muscle mass in 80-week-old aged Balb/c male mice. HY7715 not only induces myoblast differentiation and mitochondrial biogenesis but also inhibits the sarcopenic process in skeletal muscle. In addition, HY7715 recovers the microbiome composition and beta-diversity shift. Therefore, HY7715 has promise as a functional probiotic supplement to improve the degeneration of muscle function that is associated with aging. Full article

CXC Chemokine signaling plays an important role in wound healing. The four-eyed sleeper (Bostrychus sinensis) is a commercially important marine fish, which is prone to suffer skin ulceration at high temperature seasons, leading to mass mortality of fish in aquaculture farms. The genetic background related to skin ulceration and wound healing has remained unknown in this fish. Herein, we identified 10 differentially expressed Bostrychus sinensis CXC chemokine receptors (BsCXCRs) in skin ulcerated fish by de novo transcriptome sequencing. The transcripts of these BsCXCRs were classified in seven types, including BsCXCR1a/1b, BsCXCR2, BsCXCR3a1/3a2, BsCXCR4a/4b, and BsCXCR5-7, and BsCXCR6 was the first CXCR6 homologue experimentally identified in teleost fish. These BsCXCRs were further characterized in gene and protein structures, as well as phylogenetics, and the results revealed that BsCXCRs have expanded to divergent homologues. Our results showed that, in healthy fish, the BsCXCR transcripts was mainly distributed in the muscle and immune related organs, and that BsCXCR1a/1b proteins located in the cytomembrane, BsCXCR4a/4b/5/6 in the cytomembrane and perinuclear region, and BsCXCR3a1/3a2/7 in the cytomembrane, perinuclear region, and nuclear membrane, respectively. In skin injured fish, the transcripts of all BsCXCRs were transiently increased within one hour after injury, suggesting the involvement of BsCXCRs into the early inflammatory response to skin injury in the four-eyed sleeper. These results are valuable for understanding the evolutionary events of fish CXCR genes and provide insights into the roles of CXCR family in fish skin injury. Full article

Although it has been over 20 years since Neural Cell Adhesion Molecule 2 (NCAM2) was identified as the second member of the NCAM family with a high expression in the nervous system, the knowledge of NCAM2 is still eclipsed by NCAM1. The first studies with NCAM2 focused on the olfactory bulb, where this protein has a key role in axonal projection and axonal/dendritic compartmentalization. In contrast to NCAM1, NCAM2’s functions and partners in the brain during development and adulthood have remained largely unknown until not long ago. Recent studies have revealed the importance of NCAM2 in nervous system development. NCAM2 governs neuronal morphogenesis and axodendritic architecture, and controls important neuron-specific processes such as neuronal differentiation, synaptogenesis and memory formation. In the adult brain, NCAM2 is highly expressed in dendritic spines, and it regulates synaptic plasticity and learning processes. NCAM2’s functions are related to its ability to adapt to the external inputs of the cell and to modify the cytoskeleton accordingly. Different studies show that NCAM2 interacts with proteins involved in cytoskeleton stability and proteins that regulate calcium influx, which could also modify the cytoskeleton. In this review, we examine the evidence that points to NCAM2 as a crucial cytoskeleton regulation protein during brain development and adulthood. This key function of NCAM2 may offer promising new therapeutic approaches for the treatment of neurodevelopmental diseases and neurodegenerative disorders. Full article

Antibody-cytokine fusion proteins (immunocytokines) are gaining importance for cancer therapy, but those products are often limited by systemic toxicity related to the activity of the cytokine payload in circulation and in secondary lymphoid organs. Tumor necrosis factor (TNF) is used as a pro-inflammatory payload to trigger haemorrhagic necrosis and boost anti-cancer immunity at the tumor site. Here we describe a depotentiated version of TNF (carrying the single point mutation I97A), which displayed reduced binding affinity to its cognate receptor tumor necrosis factor receptor 1 (TNFR-1) and lower biocidal activity. The fusion of the TNF(I97A) mutant to the L19 antibody promoted restoration of anti-tumor activity upon accumulation on the cognate antigen, the alternatively spliced EDB domain of fibronectin. In vivo administration of high doses (375 μg/Kg) of the fusion protein showed a potent anti-tumor effect without apparent toxicity compared with the wild type protein. L19-TNF^I97A holds promise for the targeted delivery of TNF activity to neoplastic lesions, helping spare normal tissues. Full article

Functional annotation of unknown function genes reveals unidentified functions that can enhance our understanding of complex genome communications. A common approach for inferring gene function involves the ortholog-based method. However, genetic data alone are often not enough to provide information for function annotation. Thus, integrating other sources of data can potentially increase the possibility of retrieving annotations. Network-based methods are efficient techniques for exploring interactions among genes and can be used for functional inference. In this study, we present an analysis framework for inferring the functions of Plasmodium falciparum genes based on connection profiles in a heterogeneous network between human and Plasmodium falciparum proteins. These profiles were fed into a hybrid deep learning algorithm to predict the orthologs of unknown function genes. The results show high performance of the model’s predictions, with an AUC of 0.89. One hundred and twenty-one predicted pairs with high prediction scores were selected for inferring the functions using statistical enrichment analysis. Using this method, PF3D7_1248700 and PF3D7_0401800 were found to be involved with muscle contraction and striated muscle tissue development, while PF3D7_1303800 and PF3D7_1201000 were found to be related to protein dephosphorylation. In conclusion, combining a heterogeneous network and a hybrid deep learning technique can allow us to identify unknown gene functions of malaria parasites. This approach is generalized and can be applied to other diseases that enhance the field of biomedical science. Full article

C3G (RAPGEF1) is a guanine nucleotide exchange factor (GEF) for GTPases from the Ras superfamily, mainly Rap1, although it also acts through GEF-independent mechanisms. C3G regulates several cellular functions. It is expressed at relatively high levels in specific brain areas, playing important roles during embryonic development. Recent studies have uncovered different roles for C3G in cancer that are likely to depend on cell context, tumour type, and stage. However, its role in brain tumours remained unknown until very recently. We found that C3G expression is downregulated in GBM, which promotes the acquisition of a more mesenchymal phenotype, enhancing migration and invasion, but not proliferation. ERKs hyperactivation, likely induced by FGFR1, is responsible for this pro-invasive effect detected in C3G silenced cells. Other RTKs (Receptor Tyrosine Kinases) are also dysregulated and could also contribute to C3G effects. However, it remains undetermined whether Rap1 is a mediator of C3G actions in GBM. Various Rap1 isoforms can promote proliferation and invasion in GBM cells, while C3G inhibits migration/invasion. Therefore, other RapGEFs could play a major role regulating Rap1 activity in these tumours. Based on the information available, C3G could represent a new biomarker for GBM diagnosis, prognosis, and personalised treatment of patients in combination with other GBM molecular markers. The quantification of C3G levels in circulating tumour cells (CTCs) in the cerebrospinal liquid and/or circulating fluids might be a useful tool to improve GBM patient treatment and survival. Full article

Metabolic syndrome results from multiple risk factors that arise from insulin resistance induced by abnormal fat deposition. Chronic inflammation owing to obesity primarily results from the recruitment of pro-inflammatory M1 macrophages into the adipose tissue stroma, as the adipocytes within become hypertrophied. During obesity-induced inflammation in adipose tissue, pro-inflammatory cytokines are produced by macrophages and recruit further pro-inflammatory immune cells into the adipose tissue to boost the immune response. Here, we provide an overview of the biology of macrophages in adipose tissue and the relationship between other immune cells, such as CD4+ T cells, natural killer cells, and innate lymphoid cells, and obesity and type 2 diabetes. Finally, we discuss the link between the human pathology and immune response and metabolism and further highlight potential therapeutic targets for the treatment of metabolic disorders. Full article

Schizophrenia is a neuropsychiatric disorder characterized by various symptoms including autonomic imbalance. These disturbances involve almost all autonomic functions and might contribute to poor medication compliance, worsened quality of life and increased mortality. Therefore, it has a great importance to find a potential therapeutic solution to improve the autonomic disturbances. The altered level of kynurenines (e.g., kynurenic acid), as tryptophan metabolites, is almost the most consistently found biochemical abnormality in schizophrenia. Kynurenic acid influences different types of receptors, most of them involved in the pathophysiology of schizophrenia. Only few data suggest that kynurenines might have effects on multiple autonomic functions. Publications so far have discussed the implication of kynurenines and the alteration of the autonomic nervous system in schizophrenia independently from each other. Thus, the coupling between them has not yet been addressed in schizophrenia, although their direct common points, potential interfaces indicate the consideration of their interaction. The present review gathers autonomic disturbances, the impaired kynurenine pathway in schizophrenia, and the effects of kynurenine pathway on autonomic functions. In the last part of the review, the potential interaction between the two systems in schizophrenia, and the possible therapeutic options are discussed. Full article

The transcription factor GATA2 regulates gene expression in several cells and tissues, including hematopoietic tissues and the central nervous system. Recent studies revealed that loss-of-function mutations in GATA2 are associated with hematological disorders. Our earlier in vitro studies showed that GATA2 plays an essential role in the hypothalamus–pituitary–thyroid axis (HPT axis) by regulating the genes encoding prepro-thyrotropin-releasing hormone (preproTRH) and thyroid-stimulating hormone β (TSHβ). However, the effect of GATA2 mutants on the transcriptional activity of their promoters remains unelucidated. In this study, we created five human GATA2 mutations (R308P, T354M, R396Q, R398W, and S447R) that were reported to be associated with hematological disorders and analyzed their functional properties, including transactivation potential and DNA-binding capacity toward the preproTRH and the TSHβ promoters. Three mutations (T354M, R396Q, and R398W) within the C-terminal zinc-finger domain reduced the basal GATA2 transcriptional activity on both the preproTRH and the TSHβ promoters with a significant loss of DNA binding affinity. Interestingly, only the R398W mutation reduced the GATA2 protein expression. Subsequent analysis demonstrated that the R398W mutation possibly facilitated the GATA2 degradation process. R308P and S447R mutants exhibited decreased transcriptional activity under protein kinase C compared to the wild-type protein. In conclusion, we demonstrated that naturally occurring GATA2 mutations impair the HPT axis through differential functional mechanisms in vitro. Full article

Eye-drop recombinant human nerve growth factor (ed-rhNGF) has proved to recover the retina and optic nerve damage in animal models, including the unilateral optic nerve crush (ONC), and to improve visual acuity in humans. These data, associated with evidence that ed-rhNGF stimulates the brain derived neurotrophic factor (BDNF) in retina and cortex, suggests that NGF might exert retino-fugal effects by affecting BDNF and its receptor TrkB. To address these questions, their expression and relationship with the GABAergic and glutamatergic transmission markers, GAD65 and GAD67, vesicular inhibitory amino acid transporter (VGAT), and vesicular glutamate transporters 1 and 2 (VGLUT-1 and VGLUT-2) were investigated in adult ONC rats contralateral and ipsilateral visual cortex (VCx). Ed-rhNGF recovers the ONC-induced alteration of GABAergic and glutamatergic markers in contralateral VCx, induces an upregulation of TrkB, which is positively correlated with BDNF precursor (proBDNF) decrease in both VCx sides, and strongly enhances TrkB+ cell soma and neuronal endings surrounded by GAD65 immuno-reactive afferents. These findings contribute to enlarging the knowledge on the mechanism of actions and cellular targets of exogenously administrated NGF, and suggest that ed-rhNGF might act by potentiating the activity-dependent TrkB expression in GAD+ cells in VCx following retina damage and/or ONC. Full article

It has been established that enhancement of serotonergic transmission contributes to improvement of major depression; however, several post-mortem studies and experimental depression rodent models suggest that functional abnormalities of astrocytes play important roles in the pathomechanisms/pathophysiology of mood disorders. Direct effects of serotonin (5-HT) transporter inhibiting antidepressants on astroglial transmission systems has never been assessed in this context. Therefore, to explore the effects of antidepressants on transmission associated with astrocytes, the present study determined the effects of the selective 5-HT transporter inhibitor, escitalopram, and the 5-HT partial agonist reuptake inhibitor, vortioxetine, on astroglial L-glutamate release through activated hemichannels, and the expression of connexin43 (Cx43), type 1A (5-HT1AR) and type 7 (5-HT7R) 5-HT receptor subtypes, and extracellular signal-regulated kinase (ERK) in astrocytes using primary cultured rat cortical astrocytes in a 5-HT-free environment. Both escitalopram and 5-HT1AR antagonist (WAY100635) did not affect basal astroglial L-glutamate release or L-glutamate release through activated hemichannels. Subchronic (for seven days) administrations of vortioxetine and the 5-HT7R inverse agonist (SB269970) suppressed both basal L-glutamate release and L-glutamate release through activated hemichannels, whereas 5-HT1AR agonist (BP554) inhibited L-glutamate release through activated hemichannels, but did not affect basal L-glutamate release. In particular, WAY100635 did not affect the inhibitory effects of vortioxetine on L-glutamate release. Subchronic administration of vortioxetine, BP554 and SB269970 downregulated 5-HT1AR, 5-HT7R and phosphorylated ERK in the plasma membrane fraction, but escitalopram and WAY100635 did not affect them. Subchronic administration of SB269970 decreased Cx43 expression in the plasma membrane but did not affect the cytosol; however, subchronic administration of BP554 increased Cx43 expression in the cytosol but did not affect the plasma membrane. Subchronic vortioxetine administration increased Cx43 expression in the cytosol and decreased it in the plasma membrane. WAY100635 prevented an increased Cx43 expression in the cytosol induced by vortioxetine without affecting the reduced Cx43 expression in the plasma membrane. These results suggest that 5-HT1AR downregulation probably increases Cx43 synthesis, but 5-HT7R downregulation suppresses Cx43 trafficking to the plasma membrane. These results also suggest that the subchronic administration of therapeutic-relevant concentrations of vortioxetine inhibits both astroglial L-glutamate and Cx43 expression in the plasma membrane via 5-HT7R downregulation but enhances Cx43 synthesis in the cytosol via 5-HT1AR downregulation. This combination of the downregulation of 5-HT1AR, 5-HT7R and Cx43 in the astroglial plasma membrane induced by subchronic vortioxetine administration suggest that astrocytes is possibly involved in the pathophysiology of depression. Full article

Background: The STEAP1 is a cell-surface antigen over-expressed in prostate cancer, which contributes to tumor progression and aggressiveness. However, the molecular mechanisms underlying STEAP1 and its structural determinants remain elusive. Methods: The fraction capacity of Butyl- and Octyl-Sepharose matrices on LNCaP lysates was evaluated by manipulating the ionic strength of binding and elution phases, followed by a Co-Immunoprecipitation (Co-IP) polishing. Several potential stabilizing additives were assessed, and the melting temperature (Tm) values ranked the best/worst compounds. The secondary structure of STEAP1 was identified by circular dichroism. Results: The STEAP1 was not fully captured with 1.375 M (Butyl), in contrast with interfering heterologous proteins, which were strongly retained and mostly eluted with water. This single step demonstrated higher selectivity of Butyl-Sepharose for host impurities removal from injected crude samples. Co-IP allowed recovering a purified fraction of STEAP1 and contributed to unveil potential physiologically interacting counterparts with the target. A Tm of ~55 °C was determined, confirming STEAP1 stability in the purification buffer. A predominant α-helical structure was identified, ensuring the protein’s structural stability. Conclusions: A method for successfully isolating human STEAP1 from LNCaP cells was provided, avoiding the use of detergents to achieve stability, even outside a membrane-mimicking environment. Full article

Antimicrobial peptides (AMPs) represent a skilled class of new antibiotics, due to their broad range of activity, rapid killing, and low bacterial resistance. Many efforts have been made to discover AMPs with improved performances, i.e., high antimicrobial activity, low cytotoxicity against human cells, stability against proteolytic degradation, and low costs of production. In the design of new AMPs, several physicochemical features, such as hydrophobicity, net positive charge, propensity to assume amphipathic conformation, and self-assembling properties, must be considered. Starting from the sequence of the dodecapeptide 1018-K6, we designed a new 10-aminoacid peptide, namely RiLK1, which is highly effective against both fungi and Gram-positive and -negative bacteria at low micromolar concentrations without causing human cell cytotoxicity. In order to find the structural reasons explaining the improved performance of RiLK1 versus 1018-K6, a comparative analysis of the two peptides was carried out with a combination of CD, NMR, and fluorescence spectroscopies, while their self-assembling properties were analyzed by optical and atomic force microscopies. Interestingly, the different spectroscopic and microscopic profiles exhibited by the two peptides, including the propensity of RiLK1 to adopt helix arrangements in contrast to 1018-K6, could explain the improved bactericidal, antifungal, and anti-biofilm activities shown by the new peptide against a panel of food pathogens. Full article

Global data correlate severe vitamin D deficiency with COVID-19-associated coagulopathy, further suggesting the presence of a hypercoagulable state in severe COVID-19 patients, which could promote thrombosis in the lungs and in other organs. The feedback loop between COVID-19-associated coagulopathy and vitamin D also involves platelets (PLTs), since vitamin D deficiency stimulates PLT activation and aggregation and increases fibrinolysis and thrombosis. Vitamin D and PLTs share and play specific roles not only in coagulation and thrombosis but also during inflammation, endothelial dysfunction, and immune response. Additionally, another ‘fil rouge’ between vitamin D and PLTs is represented by their role in mineral metabolism and bone health, since vitamin D deficiency, low PLT count, and altered PLT-related parameters are linked to abnormal bone remodeling in certain pathological conditions, such as osteoporosis (OP). Hence, it is possible to speculate that severe COVID-19 patients are characterized by the presence of several predisposing factors to bone fragility and OP that may be monitored to avoid potential complications. Here, we hypothesize different pervasive actions of vitamin D and PLT association in COVID-19, also allowing for potential preliminary information on bone health status during COVID-19 infection. Full article

A variety of innate immune cells such as macrophages, dendritic cells, myeloid-derived suppressor cells, natural killer cells, and neutrophils in the tumor microenvironments, contribute to tumor progression. However, while several recent reports have studied the use of immune checkpoint-based cancer immunotherapy, little work has focused on modulating the innate immune cells. This review focuses on the recent studies and challenges of using nanoparticles to target innate immune cells. In particular, we also examine the immunosuppressive properties of certain innate immune cells that limit clinical benefits. Understanding the cross-talk between tumors and innate immune cells could contribute to the development of strategies for manipulating the nanoparticles targeting tumor microenvironments. Full article

Hydrogen sulfide (H₂S) plays several physiological roles in plants. Despite the evidence, the role of H₂S on cell wall disassembly and its implications on fleshy fruit firmness remains unknown. In this work, the effect of H₂S treatment on the shelf-life, cell wall polymers and cell wall modifying-related gene expression of Chilean strawberry (Fragaria chiloensis) fruit was tested during postharvest storage. The treatment with H₂S prolonged the shelf-life of fruit by an effect of optimal dose. Fruit treated with 0.2 mM H₂S maintained significantly higher fruit firmness than non-treated fruit, reducing its decay and tripling its shelf-life. Additionally, H₂S treatment delays pectin degradation throughout the storage period and significantly downregulated the expression of genes encoding for pectinases, such as polygalacturonase, pectate lyase, and expansin. This evidence suggests that H₂S as a gasotransmitter prolongs the post-harvest shelf-life of the fruit and prevents its fast softening rate by a downregulation of the expression of key pectinase genes, which leads to a decreased pectin degradation. Full article

D-psicose 3-epimerase (DPEase) catalyzes the isomerization of D-fructose to D-psicose (aka D-allulose, a low-calorie sweetener), but its industrial application has been restricted by the poor thermostability of the naturally available enzymes. Computational rational design of disulfide bridges was used to select potential sites in the protein structure of DPEase from Clostridium bolteae to engineer new disulfide bridges. Three mutants were engineered successfully with new disulfide bridges in different locations, increasing their optimum catalytic temperature from 55 to 65 °C, greatly improving their thermal stability and extending their half-lives (t_1/2) at 55 °C from 0.37 h to 4–4.5 h, thereby greatly enhancing their potential for industrial application. Molecular dynamics simulation and spatial configuration analysis revealed that introduction of a disulfide bridge modified the protein hydrogen–bond network, rigidified both the local and overall structures of the mutants and decreased the entropy of unfolded protein, thereby enhancing the thermostability of DPEase. Full article