International Journal of Molecular Sciences

Immunostaining has emerged as one of the most common and valuable techniques that allow the localization of proteins at a quantitative level within cells and tissues using antibodies coupled to enzymes, fluorochromes, or colloidal nanogold particles. The application of fluorochromes during immunolabeling is referred to as immunofluorescence, a method coupled to widefield or confocal microscopy and extensively applied in basic research and clinical diagnosis. Notwithstanding, there are still disadvantages associated with the application of this technique due to technical challenges in the process, such as sample fixation, permeabilization, antibody incubation times, and fluid exchange, etc. These disadvantages call for continuous updates and improvements to the protocols extensively described in the literature. This review contributes to protocol optimization, outlining 10 current methods for improving sample processing in different stages of immunofluorescence, including a section with further recommendations. Additionally, we have extended our own antibody signal enhancer method, which was reported to significantly increase antibody signals and is useful for cervical cancer detection, to improve the signals of fluorochrome-conjugated staining reagents in fibrous tissues. In summary, this review is a valuable tool for experienced researchers and beginners when planning or troubleshooting the immunofluorescence assay. Full article

The clinical breakthrough of bone tissue engineering (BTE) depends on the ability to provide patients routinely with BTE products of consistent pharmacological quality. The bottleneck of this approach is the availability of stem cells. To avoid this, we suggest immobilization of random-donor-derived heterologous osteoinductive MSCs onto osteoconductive matrices. Such BTE products could then be frozen and, after thawing, could be released as ready-to-use products for permanent implantation during surgery. For this purpose, we developed a simple protocol for cryopreservation of BTE constructs and evaluated the effects of this procedure on human MSC (hMSCs) metabolic and osteogenic activity in vitro. Our findings show that hMSCs can be freeze-thawed on a β-TCP scaffold through a technically simple procedure. Treated cells sustained their metabolic activity and showed favorable osteogenic potential. Mechanistically, HIF1α and YBX1 genes were activated after freeze-thawing, and supposed to be linked to enhanced osteogenesis. However, the detailed mechanisms as to how the cryopreservation procedure beneficially affects the osteogenic potential of hMSCs remains to be evaluated. Additionally, we demonstrated that our BTE products could be stored for 3 days on dry ice; this could facilitate the supply chain management of cryopreserved BTE constructs from the site of manufacture to the operating room. Full article

The purpose of this study was to establish the clinical breakpoint (CBP) of apramycin (APR) against Salmonella in swine and evaluate its effect on intestinal microbiota. The CBP was established based on three cutoff values of wild-type cutoff value (CO_WT), pharmacokinetic-pharmadynamic (PK/PD) cutoff value (CO_PD) and clinical cutoff value (CO_CL). The effect of the optimized dose regimen based on ex vivo PK/PD study. The evolution of the ileum flora was determined by the 16rRNA gene sequencing and bioinformatics. This study firstly established the CO_WT, CO_PD in ileum, and CO_CL of APR against swine Salmonella, the value of these cutoffs were 32 µg/mL, 32 µg/mL and 8 µg/mL, respectively. According to the guiding principle of the Clinical Laboratory Standards Institute (CLSI), the final CBP in ileum was 32 µg/mL. Our results revealed the main evolution route in the composition of ileum microbiota of diarrheic piglets treated by APR. The change of the abundances of Bacteroidetes and Euryarchaeota was the most obvious during the evolution process. Methanobrevibacter, Prevotella, S24-7 and Ruminococcaceae were obtained as the highest abundance genus. The abundance of Methanobrevibacter increased significantly when APR treatment carried and decreased in cure and withdrawal period groups. The abundance of Prevotella in the tested groups was significantly lower than that in the healthy group. A decreased of abundance in S24-7 was observed after Salmonella infection and increased slightly after cure. Ruminococcaceae increased significantly after Salmonella infection and decreased significantly after APR treatment. In addition, the genera of Methanobrevibacter and Prevotella were defined as the key node. Valine, leucine and isoleucine biosynthesis, D-Glutamine and D-glutamate metabolism, D-Alanine metabolism, Peptidoglycan and amino acids biosynthesis were the top five Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways in the ileum microbiota of piglets during the Salmonella infection and APR treatment process. Our study extended the understanding of dynamic shift of gut microbes during diarrheic piglets treated by APR. Full article

Anthocyanins, as the most important chromogenic substances in flavonoids, are responsible for the red, purple, and blue coloration of flowers. Anthocyanins are synthesized in the cytoplasmic surface of the endoplasmic reticulum (ER) but accumulate predominantly in the vacuole, while glutathione S-transferases (GSTs) are considered to be mainly responsible for the transport process. Our previous studies showed that the expression of PsGSTF3 was positively correlated with anthocyanin content in tree peony tissues, which is a key candidate gene for anthocyanin accumulation. Here, we successfully cloned and characterized full-length PsGSTF3 containing three exons and two introns. Subcellular localization showed that PsGSTF3 was localized in the nucleus and ER membrane. Functional complementation of the Arabidopsis transparent testa19 (tt19) mutant indicated that PsGSTF3 was responsible for the transport of anthocyanins but not of proanthocyanidins (PAs). Virus-induced gene silencing (VIGS) of PsGSTF3 not only led to a decrease in anthocyanin accumulation but also caused a reduction of structural genes in the anthocyanin biosynthesis pathway (ABP) to varying degrees. Heterologous overexpression of PsGSTF3 was found to increase the anthocyanin accumulation in tobacco petals. Furthermore, the yeast two-hybrid (Y2H) assay showed that PsGSTF3 interacted with PsDFR, which together contributed to the coloration of petals. In conclusion, these results demonstrate that PsGSTF3 encodes an important GST transporter of anthocyanin in tree peony petals and provides a new perspective for the associated transport and regulatory mechanisms. Full article

The complexity of microglia phenotypes and their related functions compels the continuous study of microglia in diseases animal models. We demonstrated that oxygen-glucose deprivation (OGD) induced rapid, time- and space-dependent phenotypic microglia modifications in CA1 stratum pyramidalis (SP) and stratum radiatum (SR) of rat organotypic hippocampal slices as well as the degeneration of pyramidal neurons, especially in the outer layer of SP. Twentyfour h following OGD, many rod microglia formed trains of elongated cells spanning from the SR throughout the CA1, reaching the SP outer layer where they acquired a round-shaped amoeboid phagocytic head and phagocytosed most of the pyknotic, damaged neurons. NIR-laser treatment, known to preserve neuronal viability after OGD, prevented rod microglia formation. In CA3 SP, pyramidal neurons were less damaged, no rod microglia were found. Thirty six h after OGD, neuronal damage was more pronounced in SP outer and inner layers of CA1, rod microglia cells were no longer detectable, and most microglia were amoeboid/phagocytic. Damaged neurons, more numerous 36 h after OGD, were phagocytosed by amoeboid microglia in both inner and outer layers of CA1. In response to OGD, microglia can acquire different morphofunctional phenotypes which depend on the time after the insult and on the subregion where microglia are located. Full article

In recent years, some exogenous bioactive peptides have been shown to have promising anti-aging effects. These exogenous peptides may have a mechanism similar to endogenous peptides, and some can even regulate the release of endogenous active peptides and play a synergistic role with endogenous active peptides. Most aging studies use rodents that are easy to maintain in the laboratory and have relatively homogenous genotypes. Moreover, many of the anti-aging studies using bioactive peptides in rodent models only focus on the activity of single endogenous or exogenous active peptides, while the regulatory effects of exogenous active peptides on endogenous active peptides remain largely under-investigated. Furthermore, the anti-aging activity studies only focus on the effects of these bioactive peptides in individual organs or systems. However, the pathological changes of one organ can usually lead to multi-organ complications. Some anti-aging bioactive peptides could be used for rescuing the multi-organ damage associated with aging. In this paper, we review recent reports on the anti-aging effects of bioactive peptides in rodents and summarize the mechanism of action for these peptides, as well as discuss the regulation of exogenous active peptides on endogenous active peptides. Full article

The histamine H₁ receptor (H₁R) is a G protein-coupled receptor (GPCR) and represents a main target in the treatment of allergic reactions as well as inflammatory reactions and depressions. Although the overall effect of antagonists on H₁ function has been extensively investigated, rather little is known about the potential modulatory effect of ions or sequence variants on antagonist binding. We investigated the dynamics of a phosphate ion present in the crystal structure and of a sodium ion, for which we determined the position in the allosteric pocket by metadynamics simulations. Both types of ions exhibit significant dynamics within their binding site; however, some key contacts remain stable over the simulation time, which might be exploited to develop more potent drugs targeting these sites. The dynamics of the ions is almost unaffected by the presence or absence of doxepin, as also reflected in their small effect (less than 1 kcal·mol⁻¹) on doxepin binding affinity. We also examined the effect of four H₁R sequence variants observed in the human population on doxepin binding. These variants cause a reduction in doxepin affinity of up to 2.5 kcal·mol⁻¹, indicating that personalized medical treatments that take into account individual mutation patterns could increase precision in the dosage of GPCR-targeting drugs. Full article

Local extracellular acidification occurs at sites of inflammation. Proton-sensing ovarian cancer G-protein-coupled receptor 1 (OGR1, also known as GPR68) responds to decreases in extracellular pH. Our previous studies show a role for OGR1 in the pathogenesis of mucosal inflammation, suggesting a link between tissue pH and immune responses. Additionally, pH-dependent signalling is associated with the progression of intestinal fibrosis. In this study, we aimed to investigate OGR1 expression and OGR1-mediated signalling in patients with inflammatory bowel disease (IBD). Our results show that OGR1 expression significantly increased in patients with IBD compared to non-IBD patients, as demonstrated by qPCR and immunohistochemistry (IHC). Paired samples from non-inflamed and inflamed intestinal areas of IBD patients showed stronger OGR1 IHC staining in inflamed mucosal segments compared to non-inflamed mucosa. IHC of human surgical samples revealed OGR1 expression in macrophages, granulocytes, endothelial cells, and fibroblasts. OGR1-dependent inositol phosphate (IP) production was significantly increased in CD14+ monocytes from IBD patients compared to healthy subjects. Primary human and murine fibroblasts exhibited OGR1-dependent IP formation, RhoA activation, F-actin, and stress fibre formation upon an acidic pH shift. OGR1 expression and signalling increases with IBD disease activity, suggesting an active role of OGR1 in the pathogenesis of IBD. Full article

Carotenoid cleavage dioxygenases (CCDs) catalyzes the cleavage of various carotenoids into smaller apocarotenoids which are essential for plant growth and development and response to abiotic stresses. CCD family is divided into two subfamilies: 9-cis epoxycarotenoid dioxygenases (NCED) family and CCD family. A better knowledge of carotenoid biosynthesis and degradation could be useful for regulating carotenoid contents. Here, 23 CCD genes were identified from the Populus trichocarpa genome, and their characterizations and expression profiling were validated. The PtCCD members were divided into PtCCD and PtNCED subfamilies. The PtCCD family contained the PtCCD1, 4, 7, and 8 classes. The PtCCDs clustered in the same clade shared similar intron/exon structures and motif compositions and distributions. In addition, the tandem and segmental duplications resulted in the PtCCD gene expansion based on the collinearity analysis. An additional integrated collinearity analysis among poplar, Arabidopsis, rice, and willow revealed the gene pairs between poplar and willow more than that between poplar and rice. Identifying tissue-special expression patterns indicated that PtCCD genes display different expression patterns in leaves, stems, and roots. Abscisic acid (ABA) treatment and abiotic stress suggested that many PtCCD genes are responsive to osmotic stress regarding the comprehensive regulation networks. The genome-wide identification of PtCCD genes may provide the foundation for further exploring the putative regulation mechanism on osmotic stress and benefit poplar molecular breeding. Full article

The ribosome CAR interaction surface behaves as an extension of the decoding center A site and has H-bond interactions with the +1 codon, which is next in line to enter the A site. Through molecular dynamic simulations, we investigated the codon sequence specificity of this CAR–mRNA interaction and discovered a strong preference for GCN codons, suggesting that there may be a sequence-dependent layer of translational regulation dependent on the CAR interaction surface. Dissection of the CAR–mRNA interaction through nucleotide substitution experiments showed that the first nucleotide of the +1 codon dominates over the second nucleotide position, consistent with an energetically favorable zipper-like activity that emanates from the A site through the CAR–mRNA interface. Moreover, the CAR/+1 codon interaction is affected by the identity of nucleotide 3 of +1 GCN codons, which influences the stacking of G and C. Clustering analysis suggests that the A-site decoding center adopts different neighborhood substates that depend on the identity of the +1 codon. Full article

The prevention of cardiac life-threatening ventricular fibrillation and stroke-provoking atrial fibrillation remains a serious global clinical issue, with ongoing need for novel approaches. Numerous experimental and clinical studies suggest that oxidative stress and inflammation are deleterious to cardiovascular health, and can increase heart susceptibility to arrhythmias. It is quite interesting, however, that various cardio-protective compounds with antiarrhythmic properties are potent anti-oxidative and anti-inflammatory agents. These most likely target the pro-arrhythmia primary mechanisms. This review and literature-based analysis presents a realistic view of antiarrhythmic efficacy and the molecular mechanisms of current pharmaceuticals in clinical use. These include the sodium‑glucose cotransporter-2 inhibitors used in diabetes treatment, statins in dyslipidemia and naturally protective omega-3 fatty acids. This approach supports the hypothesis that prevention or attenuation of oxidative and inflammatory stress can abolish pro-arrhythmic factors and the development of an arrhythmia substrate. This could prove a powerful tool of reducing cardiac arrhythmia burden. Full article

Hydrogels from biopolymers are readily synthesized, can possess various characteristics for different applications, and have been widely used in biomedicine to help with patient treatments and outcomes. Polysaccharides, polypeptides, and nucleic acids can be produced into hydrogels, each for unique purposes depending on their qualities. Examples of polypeptide hydrogels include collagen, gelatin, and elastin, and polysaccharide hydrogels include alginate, cellulose, and glycosaminoglycan. Many different theories have been formulated to research hydrogels, which include Flory-Rehner theory, Rubber Elasticity Theory, and the calculation of porosity and pore size. All these theories take into consideration enthalpy, entropy, and other thermodynamic variables so that the structure and pore sizes of hydrogels can be formulated. Hydrogels can be fabricated in a straightforward process using a homogeneous mixture of different chemicals, depending on the intended purpose of the gel. Different types of hydrogels exist which include pH-sensitive gels, thermogels, electro-sensitive gels, and light-sensitive gels and each has its unique biomedical applications including structural capabilities, regenerative repair, or drug delivery. Major biopolymer-based hydrogels used for cell delivery include encapsulated skeletal muscle cells, osteochondral muscle cells, and stem cells being delivered to desired locations for tissue regeneration. Some examples of hydrogels used for drug and biomolecule delivery include insulin encapsulated hydrogels and hydrogels that encompass cancer drugs for desired controlled release. This review summarizes these newly developed biopolymer-based hydrogel materials that have been mainly made since 2015 and have shown to work and present more avenues for advanced medical applications. Full article

Doxorubicin (DOX) is the most widely used anthracycline anticancer agent; however, its cardiotoxicity limits its clinical efficacy. Numerous studies have elucidated the mechanisms underlying DOX-induced cardiotoxicity, wherein apoptosis has been reported as the most common final step leading to cardiomyocyte death. However, in the past two years, the involvement of ferroptosis, a novel programmed cell death, has been proposed. The purpose of this review is to summarize the historical background that led to each form of cell death, focusing on DOX-induced cardiotoxicity and the molecular mechanisms that trigger each form of cell death. Furthermore, based on this understanding, possible therapeutic strategies to prevent DOX cardiotoxicity are outlined. DNA damage, oxidative stress, intracellular signaling, transcription factors, epigenetic regulators, autophagy, and metabolic inflammation are important factors in the molecular mechanisms of DOX-induced cardiomyocyte apoptosis. Conversely, the accumulation of lipid peroxides, iron ion accumulation, and decreased expression of glutathione and glutathione peroxidase 4 are important in ferroptosis. In both cascades, the mitochondria are an important site of DOX cardiotoxicity. The last part of this review focuses on the significance of the disruption of mitochondrial homeostasis in DOX cardiotoxicity. Full article

(1) Background: As a novel type of non-coding RNA with a stable closed-loop structure, circular RNA (circRNA) can interact with microRNA (miRNA) and influence the expression of miRNA target genes. However, circRNA involved in pituitary growth hormone (GH) regulation is poorly understood. Our previous study revealed protein kinase C alpha (PRKCA) as the target gene of miR-709. Currently, the expression and function of rno_circRNA_0001004 in the rat pituitary gland is not clarified; (2) Methods: In this study, both bioinformatics analysis and dual-luciferase report assays showed a target relationship between rno_circRNA_0001004 and miR-709. Furthermore, the rno_circRNA_0001004 overexpression vector and si-circ_0001004 were constructed and transfected into GH₃ cells; (3) Results: We found that rno_circRNA_0001004 expression was positively correlated with the PRKCA gene and GH expression levels, while it was negatively correlated with miR-709. In addition, overexpression of rno-circ_0001004 also promoted proliferation and relieved the inhibition of miR-709 in GH₃ cells; (4) Conclusions: Our findings show that rno_circ_0001004 acts as a novel sponge for miR-709 to regulate GH synthesis and cell proliferation, and are the first case of discovery of the regulatory role of circRNA_0001004 in pituitary GH. Full article

The SARS-CoV-2 virus is currently the most serious challenge to global public health. Its emergence has severely disrupted the functioning of health services and the economic and social situation worldwide. Therefore, new diagnostic and therapeutic tools are urgently needed to allow for the early detection of the SARS-CoV-2 virus and appropriate treatment, which is crucial for the effective control of the COVID-19 disease. The ideal solution seems to be the use of aptamers—short fragments of nucleic acids, DNA or RNA—that can bind selected proteins with high specificity and affinity. They can be used in methods that base the reading of the test result on fluorescence phenomena, chemiluminescence, and electrochemical changes. Exploiting the properties of aptamers will enable the introduction of rapid, sensitive, specific, and low-cost tests for the routine diagnosis of SARS-CoV-2. Aptamers are excellent candidates for the development of point-of-care diagnostic devices and are potential therapeutic tools for the treatment of COVID-19. They can effectively block coronavirus activity in multiple fields by binding viral proteins and acting as carriers of therapeutic substances. In this review, we present recent developments in the design of various types of aptasensors to detect and treat the SARS-CoV-2 infection. Full article

Thrombosis is one of the major causes of mortality worldwide. Notably, it is not only implicated in cardiovascular diseases, such as myocardial infarction (MI), stroke, and pulmonary embolism (PE), but also in cancers. Understanding the cellular and molecular mechanisms involved in platelet thrombus formation is a major challenge for scientists today. For this purpose, new imaging technologies (such as confocal intravital microscopy, electron microscopy, holotomography, etc.) coupled with animal models of thrombosis (mouse, rat, rabbit, etc.) allow a better overview of this complex physiopathological process. Each of the cellular components is known to participate, including the subendothelial matrix, the endothelium, platelets, circulating cells, and, notably, neutrophils. Initially known as immune cells, neutrophils have been considered to be part of the landscape of thrombosis for more than a decade. They participate in this biological process through their expression of tissue factor (TF) and protein disulfide isomerase (PDI). Moreover, highly activated neutrophils are described as being able to release their DNA and thus form chromatin networks known as “neutrophil extracellular traps” (NETs). Initially, described as “dead sacrifices for a good cause” that prevent the dissemination of bacteria in the body, NETs have also been studied in several human pathologies, such as cardiovascular and respiratory diseases. Many articles suggest that they are involved in platelet thrombus formation and the activation of the coagulation cascade. This review presents the models of thrombosis in which neutrophils and NETs are involved and describes their mechanisms of action. We have even highlighted the medical diagnostic advances related to this research. Full article

Vitamin D3, known to regulate bone homeostasis, has recently been shown to have many pleiotropic effects in different tissues and organs due to the presence of its receptor in a wide range of cells. Our previous study demonstrated that vitamin D3 was able to increase the wound healing respect to the control sample, 24 h after cutting, without however leading to a complete repair. The aim of the study was to combine vitamin D3 with silver nanoparticles to possibly enable a faster reparative effect. The results showed that this association was capable of inducing a complete wound healing only after 18 h. Moreover, a treatment of vitamin D3 + silver nanoparticles yielded a small percentage of keratinocytes vimentin-positive, suggesting the possibility that the treatment was responsible for epithelial to mesenchymal transition of the cells, facilitating wound healing repair. Since vitamin D3 acts via sphingolipid metabolism, we studied the expression of gene encoding for the metabolic enzymes and protein level. We found an increase in neutral sphingomyelinase without involvement of neutral ceramidase or sphingosine kinase2. In support, an increase in ceramide level was identified by Ultrafast Liquid Chromatography–Tandem Mass Spectrometry, suggesting a possible involvement of ceramides in wound healing process. Full article

The formation and isomerization of disulfide bonds mediated by protein disulfide isomerase (PDI) in the endoplasmic reticulum (ER) is of fundamental importance in eukaryotes. Canonical PDI structure comprises four domains with the order of a-b-b’-a’. In Arabidopsis thaliana, the PDI-S subgroup contains only one member, AtPDI11, with an a-a’-D organization, which has no orthologs in mammals or yeast. However, the expression pattern of AtPDI11 and the functioning mechanism of AtPDI11 D domain are currently unclear. In this work, we found that PDI-S is evolutionarily conserved between land plants and algal organisms. AtPDI11 is expressed in various tissues and its induction by ER stress is disrupted in bzip28/60 and ire1a/b mutants that are null mutants of key components in the unfolded protein response (UPR) signal transduction pathway, suggesting that the induction of AtPDI11 by ER stress is mediated by the UPR signaling pathway. Furthermore, enzymatic activity assays and genetic evidence showed that the D domain is crucially important for the activities of AtPDI11. Overall, this work will help to further understand the working mechanism of AtPDI11 in catalyzing disulfide formation in plants. Full article

One important role of epigenetic regulation is controlling gene expression in development and homeostasis. However, little is known about epigenetics’ role in regulating opsin expression. Cell cultures (HEK 293, Y79, and WERI) producing different levels of opsins were treated with 5-aza-2’-deoxycytidine (5-Aza-dc) and/or sodium butyrate (SB) or suberoylanilide hydroxamic acid (SAHA) for 72 h. Global DNA methylation, site-specific methylation, and expressions of opsins were measured by LUMA assay, bisulfite pyrosequencing, and qPCR, respectively. Mouse retinal explants from wild-type P0/P1 pups were ex vivo cultured with/without 5-Aza-dc or SAHA for 6 days. The morphology of explants, DNA methylation, and expressions of opsins was examined. The drugs induced global DNA hypomethylation or increased histone acetylation in cells, including DNA hypomethylation of rhodopsin (RHO) and L-opsin (OPN1LW) and a concomitant increase in their expression. Further upregulation of RHO and/or OPN1LW in HEK 293 or WERI cells was observed with 5-Aza-dc and either SB or SAHA combination treatment. Mouse retinal explants developed normally but had drug-dependent differential DNA methylation and expression patterns of opsins. DNA methylation and histone acetylation directly regulate opsin expression both in vitro and ex vivo. The ability to manipulate opsin expression using epigenetic modifiers enables further study into the role of epigenetics in eye development and disease. Full article