Application of Proteomics Technology Based on LC-MS Combined with Western Blotting and Co-IP in Antiviral Innate Immunity.

As an interferon-stimulating factor protein, STING plays a role in the response and downstream liaison in antiviral natural immunity. Upon viral invasion, the immediate response of STING protein leads to a series of changes in downstream proteins, which ultimately leads to an antiviral immune response in the form of proinflammatory cytokines and type I interferons, thus triggering an innate immune response, an adaptive immune response in vivo, and long-term protection of the host. In the field of antiviral natural immunity, it is particularly important to rigorously and sequentially probe the dynamic changes in the antiviral natural immunity connector protein STING caused by the entire anti-inflammatory and anti-pathway mechanism and the differences in upstream and downstream proteins. Traditionally, proteomics technology has been validated by detecting proteins in a 2D platform, for which it is difficult to sensitively identify changes in the nature and abundance of target proteins. With the development of mass spectrometry (MS) technology, MS-based proteomics has made important contributions to characterizing the dynamic changes in the natural immune proteome induced by viral infections. MS analytical techniques have several advantages, such as high throughput, rapidity, sensitivity, accuracy, and automation. The most common techniques for detecting complex proteomes are liquid chromatography (LC) and mass spectrometry (MS). LC-MS (Liquid Chromatography-Mass Spectrometry), which combines the physical separation capability of LC and the mass analysis capability of MS, is a powerful technique mainly used for analyzing the proteome of cells, tissues, and body fluids. To explore the combination of traditional proteomics techniques such as Western blotting, Co-IP (co-Immunoprecipitation), and the latest LC-MS methods to probe the anti-inflammatory pathway and the differential changes in upstream and downstream proteins induced by the antiviral natural immune junction protein STING.

A guide to selecting high-performing antibodies for Protein-glutamine gamma-glutamyltransferase 2 (TGM2) for use in western blot, immunoprecipitation and immunofluorescence.

Protein-glutamine gamma-glutamyltransferase 2 (TGM2) is a Ca 2+ dependent enzyme that catalyzes transglutaminase cross-linking modifications. TGM2 is involved in various diseases, either in a protective or contributory manner, making it a crucial protein to study and determine its therapeutic potential. Identifying high-performing TGM2 antibodies would facilitate these investigations. Here we have characterized seventeen TGM2 commercial antibodies for western blot and sixteen for immunoprecipitation, and immunofluorescence. The implemented standardized experimental protocol is based on comparing read-outs in knockout cell lines against their isogenic parental controls. This study is part of a larger, collaborative initiative seeking to address antibody reproducibility issues by characterizing commercially available antibodies for human proteins and publishing the results openly as a resource for the scientific community. While the use of antibodies and protocols vary between laboratories, we encourage readers to use this report as a guide to select the most appropriate antibodies for their specific needs.

Knockdown of HSPA13 Inhibits TGFß1-Induced Epithelial-Mesenchymal Transition of RPE by Suppressing the PI3K/Akt Signaling Pathway.

Purpose: This study aimed to explore the impact of HSPA13 on epithelial-mesenchymal transition (EMT) in retinal pigment epithelial (RPE) cells and proliferative vitreoretinopathy (PVR) development, along with its associated molecular mechanisms. Methods: HSPA13 expression was evaluated in epiretinal membranes (ERMs) from patients with PVR using immunohistochemistry. The effects of HSPA13 knockdown on TGFß1-induced EMT in hESC-RPE cells were studied through quantitative PCR (qPCR), Western blot, and wound healing assays. Intracellular Ca2+ levels were measured using Fluo-8/AM incubation. A rat PVR model was induced by the intravitreal injection of RPE cells combined with platelet-rich plasma (PRP). RNA-seq was applied to study the molecular mechanism of HSPA13 knockdown-mediated EMT inhibition. Results: HSPA13 was found in human ERMs and its expression increased with TGFß1 treatment in hESC-RPE cells. Knockdown of HSPA13 inhibited TGFß1-induced EMT and migration. In the PVR rat model, HSPA13 was expressed in the ERMs and its knockdown in RPE cells reduced the development of PVR. Consistent with these observations, RNA-seq showed a global suppression of TGFß1-induced EMT and migration by shHSPA13 in RPE cells. Mechanistically, TGFß1 treatment increased intracellular Ca2+ levels, leading to an upregulation of HSPA13 expression. Downregulation of HSPA13 hindered the phosphorylation of PI3K/Akt in TGFß1-induced RPE cells. Conclusions: Our study revealed the involvement of HSPA13 in PVR development, as well as in TGFß1-induced EMT of RPE through the PI3K/Akt signaling pathway. Targeting HSPA13-related pathways involved in regulating EMT in RPE cells could serve as a novel therapeutic approach for patients with PVR.

Silencing the long noncoding RNA MALAT1 inhibits vitreous-induced epithelial-mesenchymal transition in RPE cells by regulating the PDGFRs/AKT axis.

PURPOSE: Epithelial-mesenchymal transition (EMT) is a crucial pathological process that contributes to proliferative vitreoretinopathy (PVR), and research indicates that factors present in the vitreous that target cells play pivotal roles in regulating EMT. Experimental studies have confirmed that rabbit vitreous (RV) promotes EMT in human retinal pigment epithelial (RPE) cells. The long noncoding RNA (lncRNA) MALAT1 has been implicated in EMT in various diseases. Thus, this study aimed to investigate the involvement of lncRNA MALAT1 in vitreous-induced EMT in RPE cells. METHODS: MALAT1 was knocked down in ARPE-19 cells by short hairpin RNA (shRNA) transfection. Reverse transcription PCR (RT‒PCR) was used to evaluate MALAT1 expression, and Western blotting analysis was used to measure the expression of EMT-related proteins. Wound-healing, Transwell, and cell contraction assays were conducted to assess cell migration, invasion, and contraction, respectively. Additionally, cell proliferation was assessed using the CCK-8 assay, and cytoskeletal changes were examined by immunofluorescence. RESULTS: MALAT1 expression was significantly increased in ARPE-19 cells cultured with RV. Silencing MALAT1 effectively suppressed EMT and downregulated the associated factors snail1 and E-cadherin. Furthermore, silencing MALAT1 inhibited the RV-induced migration, invasion, proliferation, and contraction of ARPE-19 cells. Silencing MALAT1 also decreased RV-induced AKT and P53 phosphorylation. CONCLUSIONS: In conclusion, lncRNA MALAT1 participates in regulating vitreous-induced EMT in human RPE cells; these results provide new insight into the pathogenesis of PVR and offer a potential direction for the development of antiproliferative drugs.

Changes in Intrinsically Photosensitive Retinal Ganglion Cells, Dopaminergic Amacrine Cells, and Their Connectivity in the Retinas of Lid Suture Myopia.

Purpose: This study investigates alterations in intrinsically photosensitive retinal ganglion cells (ipRGCs) and dopaminergic amacrine cells (DACs) in lid suture myopia (LSM) rats. Methods: LSM was induced in rats by suturing the right eyes for 4 weeks. Double immunofluorescence staining of ipRGCs and DACs in whole-mount retinas was performed to analyze changes in the density and morphology of control, LSM, and fellow eyes. Real-time quantitative PCR and Western blotting were used to detect related genes and protein expression levels. Results: Significant myopia was induced in the lid-sutured eye, but the fellow eye was not different to control. Decreased ipRGC density with paradoxically increased overall melanopsin expression and enlarged dendritic beads was observed in both the LSM and fellow eyes of the LSM rat retinas. In contrast, DAC changes occurred only in the LSM eyes, with reduced DAC density and tyrosine hydroxylase (TH) expression, sparser dendritic processes, and fewer varicosities. Interestingly, contacts between ipRGCs and DACs in the inner plexiform layer (IPL) and the expression of pituitary adenylate cyclase-activating polypeptide (PACAP) and vesicular monoamine transporter protein 2 (VMAT2) mRNA were decreased in the LSM eyes. Conclusions: The ipRGCs and DACs in LSM rat retinas undergo multiple alterations in density, morphology, and related molecule expressions. However, the ipRGC changes alone appear not to be required for the development of myopia, given that myopia is only induced in the lid-sutured eye, and they are unlikely alone to drive the DAC changes. Reduced contacts between ipRGCs and DACs in the LSM eyes may be the structural foundation for the impaired signaling between them. PACAP and VMAT2, strongly associated with ipRGCs and DACs, may play important roles in LSM through complex mechanisms.

GSK3ß Inhibitors Inhibit TGFß Signaling in the Human Trabecular Meshwork.

Purpose: Primary open-angle glaucoma (POAG) is a leading cause of blindness, and its primary risk factor is elevated intraocular pressure (IOP) due to pathologic changes in the trabecular meshwork (TM). We previously showed that there is a cross-inhibition between TGFß and Wnt signaling pathways in the TM. In this study, we determined if activation of the Wnt signaling pathway using small-molecule Wnt activators can inhibit TGFß2-induced TM changes and ocular hypertension (OHT). Methods: Primary human TM (pHTM) cells and transduced SBE-GTM3 cells were treated with or without Wnt and/or TGFß signaling activators and used for luciferase assays; for the extraction of whole-cell lysate, conditioned medium, cytosolic proteins, and nuclear proteins for Western immunoblotting (WB); or for immunofluorescent staining. Human donor eyes were perfusion cultured to study the effect of Wnt activators on IOP. Results: We found that the small-molecule Wnt activators (GSK3ß inhibitors) (BIO, SB216763, and CHIR99021) activated canonical Wnt signaling in pHTM cells without toxicity at tested concentrations. This activation inhibited TGFß signaling as well as TGFß2-induced extracellular matrix deposition and formation of cross-linked actin networks in pHTM cells or SBE-GTM3 cells. We also observed nuclear translocation of both Smad4 and ß-catenin in pHTM cells, which suggested that the cross-inhibition between the TGFß and Wnt signaling pathways may occur in the nucleus. Using our ex vivo model, we found that CHIR99021 inhibited TGFß2-induced OHT in perfusion-cultured human eyes. Conclusions: Our results showed that small-molecule Wnt activators have the potential for treating TGFß signaling-induced OHT in patients with POAG.

Iron Chelator Deferiprone Restores Iron Homeostasis and Inhibits Retinal Neovascularization in Experimental Neovascular Age-Related Macular Degeneration.

Purpose: Retinal neovascularization is a significant feature of advanced age-related macular degeneration (AMD) and a major cause of blindness in patients with AMD. However, the underlying mechanism of this pathological neovascularization remains unknown. Iron metabolism has been implicated in various biological processes. This study was conducted to investigate the effects of iron metabolism on retinal neovascularization in neovascular AMD (nAMD). Methods: C57BL/6J and very low-density lipoprotein receptor (VLDLR) knockout (Vldlr-/-) mice, a murine model of nAMD, were used in this study. Bulk-RNA sequencing was used to identify differentially expressed genes. Western blot analysis was performed to test the expression of proteins. Iron chelator deferiprone (DFP) was administrated to the mice by oral gavage. Fundus fluorescein angiography was used to evaluate retinal vascular leakage. Immunofluorescence staining was used to detect macrophages and iron-related proteins. Results: RNA sequencing (RNA-seq) results showed altered transferrin expression in the retina and RPE of Vldlr-/- mice. Disrupted iron homeostasis was observed in the retina and RPE of Vldlr-/- mice. DFP mitigated iron overload and significantly reduced retinal neovascularization and vascular leakage. In addition, DFP suppressed the inflammation in Vldlr-/- retinas. The reduced signals of macrophages were observed at sites of neovascularization in the retina and RPE of Vldlr-/- mice after DFP treatment. Further, the IL-6/JAK2/STAT3 signaling pathway was activated in the retina and RPE of Vldlr-/- mice and reversed by DFP treatment. Conclusions: Disrupted iron metabolism may contribute to retinal neovascularization in nAMD. Restoring iron homeostasis by DFP could be a potential therapeutic approach for nAMD.

Hyaluronic acid-mediated epithelial-mesenchymal transition of human lens epithelial cells via CD44 and TGF-ß2.

PURPOSE: The epithelial-mesenchymal transition of human lens epithelial cells plays a role in posterior capsule opacification, a fibrotic process that leads to a common type of cataract. Hyaluronic acid has been implicated in this fibrosis. Studies have investigated the role of transforming growth factor (TGF)-ß2 in epithelial-mesenchymal transition. However, the role of TGF-ß2 in hyaluronic acid-mediated fibrosis of lens epithelial cell remains unknown. We here examined the role of TGF-ß2 in the hyaluronic acid-mediated epithelial-mesenchymal transition of lens epithelial cells. METHODS: Cultured human lens epithelial cells (HLEB3) were infected with CD44-siRNA by using the Lipofectamine 3000 transfection reagent. The CCK-8 kit was used to measure cell viability, and the scratch assay was used to determine cell migration. Cell oxidative stress was analyzed in a dichloro-dihydro-fluorescein diacetate assay and by using a flow cytometer. The TGF-ß2 level in HLEB3 cells was examined through immunohistochemical staining. The TGF-ß2 protein level was determined through western blotting. mRNA expression levels were determined through quantitative real-time polymerase chain reaction. RESULTS: Treatment with hyaluronic acid (1.0 µM, 24 h) increased the epithelial-mesenchymal transition of HLEB3 cells. The increase in TGF-ß2 levels corresponded to an increase in CD44 levels in the culture medium. However, blocking the CD44 function significantly reduced the TGF-ß2-mediated epithelial-mesenchymal transition response of HLEB3 cells. CONCLUSIONS: Our study showed that both CD44 and TGF-ß2 are critical contributors to the hyaluronic acid-mediated epithelial-mesenchymal transition of lens epithelial cells, and that TGF-ß2 in epithelial-mesenchymal transition is regulated by CD44. These results suggest that CD44 could be used as a target for preventing hyaluronic acid-induced posterior capsule opacification. Our findings suggest that CD44/TGF-ß2 is crucial for the hyaluronic acid-induced epithelial-mesenchymal transition of lens epithelial cells.

Methods for Monitoring ER-Phagy.

The endoplasmic reticulum (ER) serves as a central hub for protein synthesis, folding, and lipid biosynthesis in eukaryotic cells. Maintaining ER homeostasis is essential for optimal cellular function, and one mechanism that has garnered attention is endoplasmic reticulum-specific autophagy, or ER-phagy. ER-phagy selectively removes specific ER portions, playing a pivotal role in cellular health and adaptation to environmental stressors. ER-phagy can be induced by diverse cellular conditions such as amino acid starvation, disruption of ER quality control mechanisms, and accumulation of misfolded ER protein, highlighting cellular adaptability and the significance of ER-phagy in stress responses. Clinically relevant mutations in ER-phagy receptors are implicated in various diseases, underlining the fundamental importance of ER-phagy in ER homeostasis. Here, we provide comprehensive protocols and general considerations while investigating ER-phagy using three fundamental techniques-Western blotting, immunofluorescence, and flow cytometry-commonly used in ER-phagy detection and quantitation.

The role of the SIRT1/NF-κB/NLRP3 pathway in the pyroptosis of lens epithelial cells under shortwave blue light radiation.

Cataracts are the world's number one blinding eye disease. Cataracts can only be effectively treated surgically, although there is a chance of surgical complications. One of the pathogenic processes of cataracts is oxidative stress, which closely correlated with pyroptosis. SIRT1 is essential for the regulation of pyroptosis. Nevertheless, the role of SIRT1 in formation of cataracts is unclear. In this work, we developed an in vitro model of shortwave blue light (SWBL)-induced scotomization in human lens epithelial cells (HLECs) and an in vivo model of SWBL-induced cataracts in rats. The study aimed to understand how the SIRT1/NF-κB/NLRP3 pathway functions. Additionally, the evaluation included cell death and the release of lactate dehydrogenase (LDH), a cytotoxicity marker, from injured cells. First, we discovered that SWBL exposure resulted in lens clouding in Sprague- Dawley (SD) rats and that the degree of clouding was positively linked to the duration of irradiation. Second, we discovered that SIRT1 exhibited antioxidant properties and was connected to the NF-κB/NLRP3 pathway. SWBL irradiation inhibited SIRT1 expression, exacerbated oxidative stress, and promoted nuclear translocation of NF-κB and the activation of the NLRP3 inflammasome, which caused LEC pyroptosis and ultimately led to cataract formation. Transient transfection to increase the expression of SIRT1 decreased the protein expression levels of NF-κB, NLRP3, caspase-1, and GSDMD, inhibited HLEC pyroptosis, and reduced the release of LDH, providing a potential method for cataract prevention and treatment.

Expression of sirtuins 1 in placenta, umbilical cord, and maternal serum of patients diagnosed with placenta accreta spectrum.

OBJECTIVE: Placenta accreta spectrum (PAS) is defined as the attachment of the placenta to the uterine wall in varying degrees. However, the studies have explored that the underlying molecular mechanisms of the PAS are very limited. Sirtuins 1 (SIRT1) is associated with placental development by controlling trophoblast cell invasion and remodeling of spiral arteries. We aimed to determine the expression level of SIRT1 in placentas, and maternal and umbilical cord serum of patients with PAS. METHODS: In total, 30 individuals in control, 20 patients in the placenta previa group, and 30 patients in the PAS group were included in this study. The expression levels of SIRT1 in the placentas were determined by Western blot and immunohistochemistry. Serum levels of SIRT1 in maternal and umbilical cord blood were determined by ELISA. RESULTS: SIRT1 was significantly lower in placentas of the PAS. However, maternal and umbilical cord serum samples were not significantly different between groups. CONCLUSION: SIRT1 may play an important role in the pathogenesis of the PAS.

pJoseph2: a family of plasmids as positive controls for bacterial protein expression, transfections, and western blots.

Epitope tagging represents a powerful strategy for expedited identification, isolation, and characterization of proteins in molecular biological studies, including protein-protein interactions. We aimed to improve the reproducibility of epitope-tagged protein expression and detection by developing a range of plasmids as positive controls. The pJoseph2 family of expression plasmids functions in diverse cellular environments and cell types to enable the evaluation of transfection efficiency and antibody staining for epitope detection. The expressed green fluorescent proteins harbor five unique epitope tags, and their efficient expression in Escherichia coli, Drosophila Schneider's line 2 cells, and human SKOV3 and HEK293T cells was demonstrated by fluorescence microscopy and western blotting. The pJoseph2 plasmids provide versatile and valuable positive controls for numerous experimental applications.

Epitope tagging, a fundamental technique in molecular biology, involves attaching short amino acid sequences (epitope tags) to target proteins for their efficient identification and study. This technique has evolved since its inception, enabling diverse applications in protein research. Notably, CRISPR/Cas9 gene editing has enhanced epitope tagging by enabling the tagging of endogenous genes, expanding its versatility. However, reproducibility challenges exist, demanding positive controls for troubleshooting. The pJoseph2 family of plasmids was developed to address this need, providing robust positive controls for various epitope-based experiments, from bacterial expression to Drosophila and mammalian cell studies. This resource enhances the reliability and accuracy of epitope tagging, benefiting researchers across disciplines.

Pigment Epithelium-Derived Factor Binding to VEGFR-1 (Flt-1) Increases the Survival of Retinal Neurons.

Purpose: The purpose of this study was to examine possible involvement of vascular endothelial growth factor (VEGF) receptor (VEGFR)-1/Flt-1 in pigment epithelium-derived factor (PEDF)-promoted survival of retinal neurons. Methods: Survival of growth factor-deprived retinal ganglion cells (RGCs) and R28 cells and activation of ERK-1/-2 MAP kinases were assessed in the presence of PEDF, placental growth factor (PlGF), and VEGF using cell cultures, viability assays and quantitation of ERK-1/-2 phosphorylation. VEGFR-1/Flt-1 expression was determined using quantitative PCR (qPCR) and Western blotting. VEGFR-1/Flt-1 was knocked down in R28 cells by small interfering RNA (siRNA). Binding of a PEDF-IgG Fc fusion protein (PEDF-Fc) to retinal neurons, immobilized VEGFR-1/Flt-1 and VEGFR-1/Flt-1-derived peptides was studied using binding assays and peptide scanning. Results: PEDF in combination with PlGF stimulated increased cell survival and ERK-1/-2 MAP kinase activation compared to effects of either factor alone. VEGFR-1/Flt-1 expression in RGCs and R28 cells was significantly upregulated by hypoxia, VEGF, and PEDF. VEGFR-1/Flt-1 ligands (VEGF and PlGF) or soluble VEGFR-1 (sflt-1) competed with PEDF-Fc for binding to R28 cells. Depleting R28 cells of VEGFR-1/Flt-1 resulted in reduced PEDF-Fc binding when comparing VEGFR-1/Flt-1 siRNA- and control siRNA-treated cells. PEDF-Fc interacted with immobilized sflt-1, which was specifically blocked by VEGF and PlGF. PEDF-Fc binding sites were mapped to VEGFR-1/Flt-1 extracellular domains D3 and D4. Peptides corresponding to D3 and D4 specifically inhibited PEDF-Fc binding to R28 cells. These peptides and sflt-1 significantly inhibited PEDF-promoted survival of R28 cells. Conclusions: These results suggest that PEDF can target VEGFR-1/Flt-1 and this interaction plays a significant role in PEDF-mediated neuroprotection in the retina.

Inhibition of Heme Oxygenase 1 Suppresses Growth, Migration, and Invasion, and Regulates Tumor-Infiltrating CD8+ T Cells and in Uveal Melanoma.

Purpose: Metastatic uveal melanoma (UM) treatment is difficult, and effective treatments are urgently needed. We aimed to explore the role of heme oxygenase 1 (HO-1) in UM and provide new therapeutic strategies for UM. Methods: Bioinformatics was used to analyze the relationship between HMOX1 and immunity in UM and other tumors. Cell Counting Kit-8, Western blot, immunofluorescence staining, wound healing, and Transwell assays were used. A subcutaneous transplanted UM tumor model was used in mice to verify the therapeutic effect. Results: In UM, the expression level of HMOX1 was strongly correlated with the immune score and the infiltration level of various immune cells. ZnPP can inhibit the growth of UM cells, promote cell apoptosis, and block the cell cycle at G0/G1 phase in vitro. HO-1 knockout can effectively inhibit the proliferation of UM cells. ZnPP effectively inhibited the growth of UM and promoted the infiltration of CD8+ T cells in a subcutaneous tumor transplantation model. Conclusions: These results indicate that targeting HO-1 in UM has the potential for independent targeted immunotherapy or adjuvant immunotherapy.

Identification and validation of autophagy-related genes influenced by paris polyphylla in tongue cancer using network pharmacology.

BACKGROUND: Tongue squamous cell carcinoma (TSCC) represents the most prevalent form of head and neck squamous cell carcinomas, comprising approximately one-third of all oral cancers. Paris polyphylla(PP) exhibit promising anti-tumor properties, yet their underlying mechanisms remain elusive. This study offers novel insights into the molecular mechanisms underlying TSCC treatment with PP and establishes a theoretical basis for their clinical application. METHODS: Employing transcriptomics and network pharmacology methodologies, we identified autophagy-related key genes associated with the effects of PP. These genes were subjected to KEGG and GO enrichment analyses to determine their related functions. In vitro, CAL-27 cells were treated with 10, 30, and 60 µg/ml of PP for 24 h to assess tumor cell proliferation, apoptosis, and autophagy-related markers. KEY FINDINGS: Molecular docking of MAPK3 and PSEN1 with PP revealed stable hydrogen bond interactions, indicating the therapeutic potential of these saponins in TSCC through the autophagy pathway. In vitro experiments demonstrated significant inhibition of proliferative activity in tongue squamous carcinoma CAL-27 cells and promotion of tumor cell apoptosis by PP. Western blot analysis confirmed alterations in the expression of autophagy markers P62, LC3B, and Beclin1 following treatment, suggesting activation of the autophagy pathway. CONCLUSIONS: Our results suggest that PP inhibits tumor cells through the autophagy pathway, in which MAPK3 and PSEN1 play a role as potential functional molecules.

Detection of Antibodies against Feline Morbillivirus by Recombinant Matrix Enzyme-Linked Immunosorbent Assay.

Feline morbillivirus (FeMV) has been associated with feline health, although its exact role in pathogenesis is still debated. In this study, an indirect enzyme-linked immunosorbent assay (i-ELISA) targeting a recombinant matrix protein of FeMV (rFeMV-M) was developed and assessed in comparison to a Western blotting (WB) assay. The i-ELISA was evaluated using blood samples from 136 cats that were additionally tested with real-time reverse-transcription PCR (RT-qPCR). The i-ELISA exhibited a sensitivity of 90.1%, specificity of 75.6%, positive predictive value of 88.2%, and negative predictive value of 79.1%. The agreement between i-ELISA and WB analyses was substantial (a κ coefficient of 0.664 with a 95% confidence interval of 0.529 to 0.799). Within the study group, 68.4% (93/136) of the cats were serologically positive in the i-ELISA and 66.9% (91/136) in the WB assay, with 11.8% (11/93) of false positivity with the i-ELISA. However, only 8.1% (11/136) of the cats tested positive for FeMV using RT-qPCR (p < 0.001). The developed i-ELISA proved effective in identifying FeMV-infected cats and indicated the prevalence of FeMV exposure. Combining FeMV antibody detection through i-ELISA with FeMV RT-qPCR could offer a comprehensive method to determine and monitor FeMV infection status. Nevertheless, this assay still requires refinement due to a significant number of false positive results, which can lead to the misdiagnosis of cats without antibodies as having antibodies. This study also provided the first evidence of seroprevalence against FeMV among cat populations in Thailand, contributing valuable insights into the geographic distribution and prevalence of this virus.

Proteomic Analysis and Immunoprofiling of Persian Horned Viper Venom, Pseudocerastes Persicus, from Central Part of Iran.

Numerous species of venomous snakes of medical importance exist in Iran. Pseudocerastes persicus (P. persicus), one of the medically important snakes, also called the Persian horned viper, has a geographical spread that extends to the east, southwest, and central areas of Iran and is endemic across the wider region. As a result, this species is responsible for many snakebite occurrences. Venom from P. persicus found in the central province of Semnan contains phospholipase A2 and L-amino acid oxidase activities, and high toxic potency. The venom was fractionated by reverse-phase high-performance liquid chromatography (HPLC) and analyzed by Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), Western blotting and two-dimensional electrophoresis. Using liquid chromatography with tandem mass spectrometry (LC-MS/MS), a range of components were identified, consistent with the biochemical and toxicological properties of the venom. Proteins identified from 2D electrophoresis and shotgun methods included metallo- and serine proteases, phospholipases, oxidases, and Kunitz trypsin inhibitors, along with many other components at lower qualitative abundance. This study provides a more detailed understanding of the protein profile of Iranian P. persicus venom, which can be effective in the production of an effective antidote against it. The analysis of the resulting data shows that there is a wide range of proteins in the venom of the Persian horned viper. This information can provide a better understanding of how venom is neutralized by polyclonal antivenom. Considering the wide presence of this snake and its related species in Iran and surrounding countries, knowing the venom protein profile of this family can be of great support to antivenom producers such as Razi Vaccine & Serum Research Institute in the preparation of regional antivenoms.

PRELP inhibits the progression of oral squamous cell carcinoma via inactivation of the NF-κB pathway.

OBJECTIVES: The aim of this study was to investigate the role and molecular mechanism of proline/arginine-rich end leucine-rich repeat protein (PRELP), a secreted protein in extracellular matrix, in oral squamous cell carcinoma (OSCC) progression. DESIGN: PRELP expression in OSCC was analyzed in the Gene Set Enrichment (GSE) 138206, GSE37991, and GSE23558 datasets as well as cell lines. Also, PRELP expression and its relationship with prognosis and immune infiltration in head and neck squamous cell carcinoma (HNSCC) were confirmed by bioinformatics analysis. The proliferation, apoptosis, invasion, epithelial-to-mesenchymal transition (EMT) and NF-κB activation were detected after alteration of PRELP expression in OSCC cells using CCK-8, EdU, flow cytometry, Transwell, real-time PCR, immunofluorescence and Western blot. Additionally, an NF-κB inhibitor PDTC was used to confirm the regulation mechanism of PRELP. RESULTS: The expression of PRELP in OSCC tissues, cells and in HNSCC samples was low. HNSCC patients with higher PRELP expression was associated with longer overall survival. A positive correlation between PRELP expression and immune cell infiltration was found in HNSCC. Upregulation of PRELP inhibited, whereas PRELP silencing promoted, the proliferation, invasion and EMT of OSCC cells. Also, overexpression of PRELP promoted cell apoptosis. Mechanistically, PRELP suppressed p65 phosphorylation and nuclear translocation. And PDTC treatment partially reversed the influences of PRELP knockdown on the malignant behaviors in OSCC cells. CONCLUSION: PRELP suppressed OSCC progression via inactivation of the NF-κB pathway. Targeting PRELP may be a potential approach for OSCC treatment.

A guide to selecting high-performing antibodies for Synaptotagmin-1 (Uniprot ID P21579) for use in western blot, immunoprecipitation, immunofluorescence and flow cytometry.

Synaptotagmin-1 is a synaptic vesicle transmembrane protein that senses calcium influx via its tandem C2-domains, triggering synchronous neurotransmitter release. Disruption to SYT1 is associated with neurodevelopmental disorders, highlighting the importance of identifying high-quality research reagents to enhance understanding of Synaptotagmin-1 in health and disease. Here we have characterized thirteen Synaptotagmin-1 commercial antibodies for western blot, immunoprecipitation, immunofluorescence and flow cytometry using a standardized experimental protocol based on comparing read-outs in knockout cell lines and isogenic parental controls. These studies are part of a larger, collaborative initiative seeking to address antibody reproducibility issues by characterizing commercially available antibodies for human proteins and publishing the results openly as a resource for the scientific community. While use of antibodies and protocols vary between laboratories, we encourage readers to use this report as a guide to select the most appropriate antibodies for their specific needs.

Protein profile of purified plasma- and aqueous humor-derived extracellular vesicles from patients with ocular toxoplasmosis.

PURPOSE: To characterize the extracellular vesicle protein cargo in the aqueous humor and plasma of patients with ocular toxoplasmosis. METHODS: Aqueous humor and plasma were collected from six patients with active ocular toxoplasmosis and six patients with cataract. Extracellular vesicles were isolated, and western blotting and mass spectrometry were performed for protein analysis. RESULTS: All plasma samples from patients with ocular toxoplasmosis and cataract were positive for the tetraspanins CD63 and TSG101. However, the aqueous humor from patients with ocular toxoplasmosis was positive only for CD63. Sixty-seven new unreported proteins were identified in the aqueous humor and plasma of patients with the ocular toxoplasmosis and cataract. Of the 67 proteins, 10 and 7 were found only in the cataract and ocular toxoplasmosis groups, respectively. In general, these proteins were involved in immune system activation and retina homeostasis and were related to infections and retina-associated diseases. CONCLUSION: The distinct protein signatures between ocular toxoplasmosis and cataract may be helpful in the differential diagnosis of ocular toxoplasmosis. However, more studies are needed to better understand the role of these proteins in the pathogenesis of ocular toxoplasmosis.