siRNA Knockdown Research Articles

WD-repeat containing protein-61 regulates endometrial epithelial cell adhesion indicating an important role in receptivity.

Endometrial receptivity is crucial for successful embryo implantation during early pregnancy. The human endometrium undergoes remodeling within each menstrual cycle to prepare or become receptive to an implanting blastocyst in the mid-secretory phase. However, the mechanisms behind these changes are not fully understood. Recently, using hormone treated endometrial organoids to model receptivity, we identified that the transcriptional regulator WD-repeat-containing protein-61 (WDR61) was reduced in organoids derived from infertile women. In this study, we aimed to determine the role of WDR61 in endometrial receptivity. Here we demonstrated that WDR61 immunolocalizes in the nuclei and cytosol of endometrial glandular epithelium, luminal epithelium and stroma. The staining intensity of WDR61 was significantly higher during the receptive mid-secretory phase compared to the non-receptive proliferative phase in fertile women. In a functional experiment to model blastocyst adhesion to the endometrial epithelium, we found that adhesion of cytotrophoblast progenitor spheroids was blocked when siRNA was used to knockdown WDR61 in primary endometrial epithelial cells. Similarly, in Ishikawa cells (a receptive human endometrial epithelial cell line), siRNA knockdown of WDR61 significantly reduced the cell adhesive and proliferative capacities. qPCR revealed that WDR61 knockdown reduced expression of key genes involved in receptivity including HOXD10, MMP2 and CD44. Chromatin immunoprecipitation sequencing demonstrated that WDR61 directly targeted 2,022 genes in Ishikawa cells, with functions including focal adhesion, intracellular signaling and epithelial-mesenchymal transition. Overall, these findings suggest that WDR61 promotes endometrial receptivity by modulating epithelial cell focal adhesions, proliferation and epithelial-mesenchymal transition.

IMMU-55. TUMOR-WIDE RNA SPLICING ABERRATIONS GENERATE IMMUNOGENIC PUBLIC NEOANTIGENS IN GLIOBLASTOMAS AND OTHER CANCER TYPES

Abstract BACKGROUND Immunotherapy in gliomas is limited by tumor heterogeneity and low mutational burden. Aberrant RNA-splicing (neojunctions) offers a new source for targets, and our neoantigen discovery platform (SNIPP) characterizes a novel class of clonally-expressed splicing-derived neoantigens that elicit a CD8+ T-cell-mediated tumor killing response. METHODS SNIPP identified public neojunctions expressed in TCGA RNA-seq (positive sample rate (PSR) > 10%) and not in GTEx normal tissue RNA-seq data (PSR < 1%) across 12 cancer types. To characterize intratumorally-conserved neojunctions, we performed maximally-distanced multi-site biopsies (n=535) within glioma patients (n=56) and obtained RNA-seq data from each intratumoral site. Two independent algorithms then predicted peptide processing likelihood and HLA-binding affinity of ASN candidates. Neoantigen-specific TCR sequences characterized from subsequent PBMC in vitro sensitization and 10x V(D)J scRNA-seq were transduced into CD8+ T-cells for immunogenicity and cytotoxicity assays against glioma cell lines. RESULTS Our pipeline identified 789 public neojunctions, with 32 neojunctions concurrently identified in transcriptomic and proteomic glioma data and predicted to be presented by HLA-A*02:01 with high confidence. IVS and subsequent 10x VDJ scRNA-seq identified TCR clonotypes reactive against neojunctions in RPL22 (n=7) and GNAS (n=1), the latter being highly intratumorally-conserved (detected in > 90% of spatially-mapped biopsies across 17/56 patients (26.78%)). TCR-transduced T-cells demonstrated recognition and immunogenic activation against endogenously processed and presented neoantigens in multiple GBM PDX cell lines, and co-culturing these T-cells against both glioblastoma and melanoma cell lines demonstrated tumor-specific cytotoxicity. Furthermore, IDH1-mutant oligodendroglioma samples demonstrated significantly elevated expression of neojunctions over IDH1-mutant astrocytoma and IDH1wt subtypes. Differential gene expression (DESeq2) identified decreased expression of splicing factors due to oligodendroglioma-specific co-deletion of Chromosomes 1p/19q. siRNA knockdown of these splicing factors (e.g. SF3A3, SNRPD2) in IDH1wt glioma cells resulted in significantly increased expression of corresponding neojunctions. CONCLUSION Our unique SNIPP neoantigen discovery platform identified novel public tumor-wide splice-derived neoantigens and reactive TCRs, and its pan-cancer application characterized candidates that are targetable across multiple diseases.

Single-cell sequencing combined with transcriptomics and invivo and invitro analysis reveals the landscape offerroptosis in myocardial ischemia-reperfusion injury.

Myocardial ischemia-reperfusion injury (MIRI) is a significant risk factor for acute myocardial infarction and is closely associated with ferroptosis. This study aimed to identify key ferroptosis-related genes as potential diagnostic markers for MIRI and to explore their roles in immune infiltration and therapeutic targeting in myocardial tissue. We obtained single-cell RNA sequencing (scRNA-seq) and RNA-seq data on MIRI from the GEO database, applied Seurat and UMAP for data processing and clustering, and analyzed ligand-receptor interactions using CellPhoneDB. By scoring ferroptosis in cardiomyocytes, we identified differentially expressed genes and conducted GO and KEGG pathway analyses. A protein interaction network was then constructed using the STRING database, and seven key genes (Atp5h, Vdac2, Pkm, Cycs, Hspa8, Tpi1, Ldha) were identified through Lasso regression modeling, showing significant associations with immune responses. Invivo experiments in a mouse ischemia-reperfusion model confirmed the roles of these seven genes in MIRI via RT-qPCR. To further investigate the role of Hspa8 in ferroptosis and MIRI, siRNA knockdown experiments were performed in H9C2 rat cardiomyocytes, and its involvement in ferroptosis was validated by JC-1 staining and PCR analysis. This study reveals the importance of ferroptosis-related genes in MIRI through integrated bioinformatics and experimental approaches, offering new insights into diagnostic markers and immune-related therapeutic targets for MIRI.

Biomimetic porphyrin-lipid nanoparticles - novel nanoscale theranostics for multimodal imaging and therapy in atherosclerotic cardiovascular disease

Abstract Background High-density lipoprotein (HDL) mimetic nanoagents have unrealized potential for atherosclerosis theranostics. Porphyrin-lipid HDL mimetic nanoparticles (Por-HDL-NPs) incorporate a porphyrin-lipid conjugate which permits near infrared fluorescence imaging and positron emission tomography (PET) through chelation of Copper-64 (64Cu). The outer shell contains apolipoprotein A-I mimetic peptide R4F designed to interact with scavenger receptor class B type I (SR-BI), enabling macrophage-targeting and therapeutic effects. Purpose To investigate the multimodal imaging, macrophage-targeting and therapeutic properties of Por-HDL-NPs and applications in atherosclerotic cardiovascular disease. Methods In vitro, the cholesterol efflux capacity of PBS (control), reconstituted HDL (rHDL) and Por-HDL-NPs was determined in immortalized bone marrow-derived macrophages (iBMDMs) loaded with [³H]-cholesterol. Real-time quantitative PCR (RT-qPCR) assessed mRNA levels of inflammatory mediators. ELISAs and Western blotting measured changes in protein levels. Apolipoprotein (Apo)e-/- mice were fed high-cholesterol diet (HCD) for 6 weeks, for early-stage plaque, and 13 weeks for the unstable plaque model in which two partial stenoses were made in the right carotid artery. PET and fluorescence (IVIS, flow cytometry, microscopy) imaging identified Por-HDL-NPs in plaques, circulating and aortic cells. Histological techniques assessed plaque area and composition in aortic sinus and carotid artery sections. Results Por-HDL-NPs were internalized by iBMDMs, visualized via fluorescence microscopy and flow cytometry. Por-HDL-NPs increased cholesterol efflux (49%, P<0.05), compared to rHDL. Por-HDL-NPs reduced macrophage mRNA levels of Il-1b (88%), Il-18 (54%) and Ccl5 (75%), and protein secretion of IL-1β (69%) and CCL5 (82%), P<0.05. Por-HDL-NPs suppressed inflammasome components Nlrp3 (69%) and Asc (36%), and activation of inflammatory transcription factor p65-NF-κB (53%), P<0.05. SR-BI siRNA knockdown and methyl-β-cyclodextrin, revealed that the anti-inflammatory properties of Por-HDL-NPs were independent of SR-BI and cholesterol efflux. In Apoe-/- mice, PET imaging showed 64Cu-Por-HDL-NPs localized in hearts and could detect increases in plaque size over time with HCD feeding. Por-HDL-NP fluorescence was detected within aortic sinus and unstable carotid plaques, co-localized with CD68+ macrophages. Por-HDL-NP-treated mice had smaller early-stage (22%) and unstable plaques (52%) than control PBS-treated mice, P<0.05. Por-HDL-NP mice had fewer circulating (32%) and aortic monocytes (81%), and a reduction in aortic arch Rela (26%), P<0.05. Conclusions Por-HDL-NPs present as potential nanoscale theranostics for atherosclerotic cardiovascular disease. Por-HDL-NPs can detect plaques using multiple imaging modalities and exhibit atheroprotective effects.Atherosclerosis imaging with Por-HDL-NPs

Modulating OCA2 Expression as a Promising Approach to Enhance Skin Brightness and Reduce Dark Spots.

The oculocutaneous albinism II (OCA2) gene encodes a melanosomal transmembrane protein involved in melanogenesis. Recent genome-wide association studies have identified several single nucleotide polymorphisms within OCA2 genes that are involved in skin pigmentation. Nevertheless, there have been no attempts to modulate this gene to improve skin discoloration. Accordingly, our aim was to identify compounds that can reduce OCA2 expression and to develop a formula that can improve skin brightness and reduce hyperpigmented spots. In this study, we investigated the effects of OCA2 expression reduction on melanin levels, melanosome pH, and autophagy induction through siRNA knockdown. Additionally, we identified several bioactives that effectively reduce OCA2 expression. Ultimately, in a clinical trial, we demonstrated that topical application of those compounds significantly improved skin tone and dark spots compared to vitamin C, a typical brightening agent. These findings demonstrate that OCA2 is a promising target for the development of efficacious cosmetics and therapeutics designed to treat hyperpigmentation.

CGAS deficient mice display premature aging associated with de-repression of LINE1 elements and inflammation.

Aging-associated inflammation, or 'inflammaging" is a driver of multiple age-associated diseases. Cyclic GMP-AMP Synthase (cGAS) is a cytosolic DNA sensor that functions to activate interferon response upon detecting viral DNA in the cytoplasm. cGAS contributes to inflammaging by responding to endogenous signals such as damaged DNA or LINE1 (L1) cDNA which forms in aged cells. While cGAS knockout mice are viable their aging has not been examined. Unexpectedly, we found that cGAS knockout mice exhibit accelerated aging phenotype associated with induction of inflammation. Transcription of L1 elements was increased in both cGAS knockout mice and in cGAS siRNA knockdown cells associated with high levels of cytoplasmic L1 DNA and expression of ORF1 protein. Cells from cGAS knockout mice showed increased chromatin accessibility and decreased DNA methylation on L1 transposons. Stimulated emission depletion microscopy (STED) showed that cGAS forms nuclear condensates that co-localize with H3K9me3 heterochromatin marks, and H3K9me3 pattern is disrupted in cGAS knockout cells. Taken together these results suggest a previously undescribed role for cGAS in maintaining heterochromatin on transposable elements. We propose that loss of cGAS leads to loss of chromatin organization, de-repression of transposable elements and induction of inflammation resulting in accelerated aging.

NWD1 influences the extension of neuronal axons by regulating microtubule stability

Proteins belonging to the STAND (signal transduction ATPases with numerous domains) family have been implicated in crucial functions across various signal transduction pathways, encompassing both apoptosis and innate immune responses. In this study, we have identified NWD1, a member of the STAND superfamily, as a gene that regulates neurite outgrowth. This was confirmed by siRNA knockdown assay in E18 neurons. A zebrafish model was utilized to create NWD1 knockdown using the NgAgo-gDNA system, revealing the significant role of NWD1 in neurogenesis. We further revealed that NWD1 siRNA reduced the acetylated tubulin protein, and changed the ratio of soluble and polymerized tubulin. Moreover, we investigated the mechanism underlying the regulation of NWD1-mediated microtubule dynamics, and MAP1B may be a target gene. This research unveiled, for the first time, the potential role of NWD1 in regulating axon outgrowth through modulating the ratio of acetylated tubulin.

Isoliquiritigenin mitigates intervertebral disc degeneration induced by oxidative stress and mitochondrial impairment through a PPARγ-dependent pathway

ObjectivesOxidative stress, mitochondrial dysfunction, and apoptosis play significant roles in the degradation of extracellular matrix (ECM) in nucleus pulposus cells (NPCs), ultimately contributing to intervertebral disc degeneration (IVDD). This study investigates the potential of isoliquiritigenin (ISL), a natural extract known for its antioxidant, anti-inflammatory, and anti-atherosclerotic properties, to alleviate IVDD. MethodsThe viability of NPCs treated with ISL and tert-butyl hydroperoxide (TBHP) was assessed using the CCK-8 assay. Various techniques, including Western blot, qRT-PCR, immunofluorescence (IF), and immunohistochemistry, were employed to measure the expression of ECM components, oxidative stress markers, and apoptosis-related proteins. Mitochondrial function was evaluated through Western blot and IF analyses. Network pharmacology predicted ISL targets, and the expression levels of PPARγ were assessed using the aforementioned methods. The role of PPARγ in the therapeutic effects of ISL on IVDD was examined through siRNA knockdown. The therapeutic impact of ISL on puncture-induced IVDD in rats was evaluated using X-ray, MRI, and histological staining techniques. ResultsIn vitro, ISL reduced oxidative stress in NPCs, restored mitochondrial function, inhibited apoptosis, and improved the ECM phenotype. In vivo, ISL slowed the progression of IVDD in a rat model. Further analysis revealed that ISL enhances PPARγ activity and promotes its expression by direct binding, contributing to the delay of IVDD progression. ConclusionThis study demonstrates that ISL effectively treats puncture-induced IVDD in rats by inhibiting oxidative stress, restoring mitochondrial function, and reducing NPC apoptosis through a PPARγ-dependent mechanism. By balancing ECM synthesis and degradation, ISL presents a novel therapeutic approach for IVDD and identifies a promising target for treatment.

Regulation of Enterocyte Brush Border Membrane Primary Na-Absorptive Transporters in Human Intestinal Organoid-Derived Monolayers.

In the small intestine, sodium (Na) absorption occurs primarily via two apical transporters, Na-hydrogen exchanger 3 (NHE3) and Na-glucose cotransporter 1 (SGLT1). The two primary Na-absorptive pathways were previously shown to compensatorily regulate each other in rabbit and rat intestinal epithelial cells. However, whether NHE3 and SGLT1 regulate one another in normal human enterocytes is unknown, mainly due to a lack of appropriate experimental models. To investigate this, we generated 2D enterocyte monolayers from human jejunal 3D organoids and used small interfering RNAs (siRNAs) to knock down NHE3 or SGLT1. Molecular and uptake studies were performed to determine the effects on NHE3 and SGLT1 expression and activity. Knockdown of NHE3 by siRNA in enterocyte monolayers was verified by qPCR and Western blot analysis and resulted in reduced NHE3 activity. However, in NHE3 siRNA-transfected cells, SGLT1 activity was significantly increased. siRNA knockdown of SGLT1 was confirmed by qPCR and Western blot analysis and resulted in reduced SGLT1 activity. However, in SGLT1 siRNA-transfected cells, NHE3 activity was significantly increased. These results demonstrate for the first time the functionality of siRNA in patient-derived organoid monolayers. Furthermore, they show that the two primary Na absorptive pathways in human enterocytes reciprocally regulate one another.

RTP4 Enhances Corneal HSV-1 Infection in Mice With Type 2 Diabetes Mellitus.

The purpose of this study was to investigate whether corneal lesions in mice with type 2 diabetes mellitus (T2D) infected with herpes simplex virus (HSV)-1 are more severe, and to elucidate the specific underlying mechanism. The corneas of control mice and T2D mice induced by a high-fat diet combined with streptozotocin were infected with the HSV-1 Mckrae strain to assess corneal infection, opacity, and HSV-1 replication. RNA sequencing of the corneal epithelium from wild-type and db/db mice (a genetic T2D mouse model) was conducted to identify the key gene affecting T2D infection. Immunofluorescence staining was performed on corneal sections from T2D mice and patients with T2D. The effect of small interfering RNA (siRNA) knockdown on corneal HSV-1 infection was evaluated in both in vitro and in vivo models. T2D mice exhibited a more severe infection phenotype following HSV-1 infection, characterized by augmented corneal opacity scores, elevated viral titers, and transcripts compared to control mice. Transcriptome analysis of corneal epithelium revealed a hyperactive viral response in T2D mice, highlighting the differentially expressed gene Rtp4 (encoding receptor transporter protein 4). Receptor transporter protein 4 (RTP4) expression was enhanced in the corneal epithelium of T2D mice and patients with T2D. Virus binding assays demonstrated that RTP4 facilitated HSV-1 binding to human corneal epithelial cells. Silencing RTP4 alleviated HSV-1 infection in both in vitro and in vivo T2D models. The findings indicate that elevated RTP4 exacerbates HSV-1 infection by enhancing its binding to corneal epithelial cells, whereas Rtp4 knockdown mitigated corneal lesions in T2D mice. This implies RTP4 as a potential target for intervention in diabetic HSV-1 infection.

NONRATT000538.2 promotes vascular smooth muscle cell phenotypic switch and in-stent restenosis

Vascular smooth muscle cell (VSMC) excessive proliferation and migration are considered the main pathological process in in-stent restenosis (ISR) following vascular intervention. Certain long noncoding RNAs play vital roles in this process. Therefore, this study aimed to explore novel regulators for ISR and further uncover the mechanism. Using a rat abdominal aorta stent implantation model, we observed that NONRATT000538.2 (NR538.2) served as a positive regulator for VSMC proliferation and migration. By manipulating NR538.2 expression via adenoviral overexpression or siRNA knockdown, we noted that NR538.2 promoted VSMC phenotypic switching, thereby inducing proliferation and migration. Significantly, the local delivery of siRNA of NR538.2 via adeno-associated virus vector suppressed balloon injury-induced neointima formation. Our study demonstrated for the first time that NR538.2 positively influenced VSMC proliferation during ISR.

Characterization of the angiomodulatory effects of Interleukin 11 cis- and trans-signaling in the retina

BackgroundThe IL-6 cytokine family, with its crucial and pleiotropic intracellular signaling pathway STAT3, is a promising target for treating vasoproliferative retinal diseases. Previous research has shown that IL-6 cis-signaling (via membrane-bound receptors) and trans-signaling (via soluble receptors) can have distinct effects on target cells, leading to their application in various disease treatments. While IL-6 has been extensively studied, less is known about the angiogenic effects of IL-11, another member of the IL-6 family, in the retina. Therefore, the aim of this study was to characterize the effects of IL-11 on retinal angiogenesis.Main textIn vitreous samples from proliferative diabetic retinopathy (PDR) patients, elevated levels of IL-11Rα, but not IL-11, were detected. In vitro studies using vascular endothelial cells revealed distinct effects of cis- and trans-signaling: cis-signaling (IL-11 alone) had antiangiogenic effects, while trans-signaling (IL-11 + sIL-11Rα) had proangiogenic and pro-migratory effects. These differences can be attributed to their individual signaling responses and associated transcriptomic changes. Notably, no differences in cis- and trans-signaling were detected in primary mouse Müller cell cultures. STAT3 and STAT1 siRNA knockdown experiments revealed opposing effects on IL-11 signaling, with STAT3 functioning as an antiproliferative and proapoptotic player while STAT1 acts in opposition to STAT3. In vivo, both IL-11 and IL-11 + sIL-11Rα led to a reduction in retinal neovascularization. Immunohistochemical staining revealed Müller cell activation in response to treatment, suggesting that IL-11 affects multiple retinal cell types in vivo beyond vascular endothelial cells.ConclusionsCis- and trans-signaling by IL-11 have contrasting angiomodulatory effects on endothelial cells in vitro. In vivo, cis- and trans-signaling also influence Müller cells, ultimately determining the overall angiomodulatory impact on the retina, highlighting the intricate interplay between vascular and glial cells in the retina.

Revealing the mechanisms of RAC3 in tumor aggressiveness, the immunotherapy response, and drug resistance in bladder cancer.

Bladder cancer (BLCA) is a prevalent urinary tract malignancy with a high propensity for recurrence and chemoresistance. The molecular mechanisms underlying its progression and response to therapy have not been fully elucidated. We conducted a multifaceted analysis, integrating immunohistochemical (IHC) staining, bioinformatics evaluation using TCGA and CCLE databases, and in vitro assays using the BLCA cell lines 5637 and T24. RAC3 expression was assessed relative to clinical and pathological features. Functional enrichment analyses and gene set enrichment analysis (GSEA) were performed to identify associated biological processes and pathways. The impacts of RAC3 on cell proliferation, migration, invasion, and the immune microenvironment were evaluated using siRNA knockdown, CCK-8, Transwell, wound healing and colony formation assays. Elevated RAC3 expression was significantly correlated with an advanced tumor stage, lymph node metastasis, and poor prognosis for BLCA patients. The functional enrichment analysis implicated RAC3 in immune cell infiltration and immune checkpoint mechanisms. Notably, RAC3 knockdown significantly reduced the proliferative, migratory, and invasive capabilities of BLCA cells. These effects were reversed by the overexpression of RAC3. Additionally, RAC3 expression was linked to chemoresistance, with high RAC3 expression predicting resistance to certain therapeutic agents. The TIDE algorithm indicated that RAC3 expression could be a predictive biomarker for the immunotherapy response. RAC3 was identified as a potential therapeutic target and biomarker of BLCA, as its expression significantly influenced tumor progression, the immune response, and chemosensitivity. Targeting RAC3 may provide a novel strategy for the management of BLCA, particularly for patients resistant to conventional therapies. Further research is essential to elucidate the detailed mechanisms of RAC3 in BLCA and explore its clinical application in precision medicine.

Re-Evaluating PIN1 as a Therapeutic Target in Oncology Using Neutral Inhibitors and PROTACs.

Peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (PIN1) has emerged as a promising therapeutic target for cancer treatment. However, the current PIN1 inhibitors have shown limited efficacy in animal models, leaving the question of whether PIN1 is a proper oncologic target still unanswered. By screening a 1 trillion DNA-encoded library (DEL), we identified novel nonacidic compounds. Among resynthesized DEL compounds, DEL1067-56-469 (A0) is the most potent one (KD = 430 nM, IC50 = 420 nM). Further optimization of A0 resulted in compound C10 with much improved potency (KD = 25 nM, IC50 = 150 nM). As an alternative approach, C10 was then converted into proteolysis targeting chimeras (PROTACs) in order to achieve deeper downregulation of the PIN1 protein in cancer cell lines. Unfortunately, neither PIN1 inhibitors nor PIN1 PROTACs demonstrated meaningful antiproliferation activity. In addition, siRNA knock-down experiments provided unfavorable evidence of PIN1 as an oncologic target. Our findings highlight the complexity of targeting PIN1 for cancer therapy.

Paramyxovirus matrix proteins modulate host cell translation via exon-junction complex interactions in the cytoplasm.

Viruses have evolved myriad strategies to exploit the translation machinery of host cells to potentiate their replication. However, how paramyxovirus (PMVs) modulate cellular translation for their own benefit has not been systematically examined. Utilizing puromycylation labeling, overexpression of individual viral genes, and infection with wild-type virus versus its gene-deleted counterpart, we found that PMVs significantly inhibit host cells' nascent peptide synthesis during infection, with the viral matrix being the primary contributor to this effect. Using the rNiV-NPL replicon system, we discovered that the viral matrix enhances viral protein translation without affecting viral mRNA transcription and suppresses host protein expression at the translational level. Polysome profile analysis revealed that the HPIV3 matrix promotes the association of viral mRNAs with ribosomes, thereby enhancing their translation efficiency during infection. Intriguingly, our NiV-Matrix interactome identified the core exon-junction complex (cEJC), critical for mRNA biogenesis, as a significant component that interacts with the paramyxoviral matrix predominantly in the cytoplasm. siRNA knockdown of eIF4AIII simulated the restriction of cellular functions by the viral matrix, leading to enhanced viral gene translation and a reduction in host protein synthesis. Moreover, siRNA depletion of cEJC resulted in a 2-3 log enhancement in infectious virus titer for various PMVs but not SARS-CoV-2, enterovirus D68, or influenza virus. Our findings characterize a host translational interference mechanism mediated by viral matrix and host cEJC interactions. We propose that the PMV matrix redirects ribosomes to translate viral mRNAs at the expense of host cell transcripts, enhancing viral replication, and thereby enhancing viral replication. These insights provide a deeper understanding of the molecular interactions between paramyxoviruses and host cells, highlighting potential targets for antiviral strategies.

The Role of αvβ3 Integrin in Lamina Cribrosa Cell Mechanotransduction in Glaucoma.

Purpose: Glaucoma, one of the leading causes of irreversible blindness, is a common progressive optic neuropathy characterised by visual field defects and structural changes to the optic nerve head (ONH). There is extracellular matrix (ECM) accumulation and fibrosis of the lamina cribrosa (LC) in the ONH, and consequently increased tissue stiffness of the LC connective tissue. Integrins are cell surface proteins that provide the key molecular link connecting cells to the ECM and serve as bidirectional sensors transmitting signals between cells and their environment to promote cell adhesion, proliferation, and remodelling of the ECM. Here, we investigated the expression of αVβ3 integrin in glaucoma LC cell, and its effect on stiffness-induced ECM gene transcription and cellular proliferation rate in normal (NLC) and glaucoma (GLC) LC cells, by down-regulating αVβ3 integrin expression using cilengitide (a known potent αVβ3 and αVβ5 inhibitor) and β3 integrin siRNA knockdown. Methods: GLC cells were compared to age-matched controls NLC to determine differential expression levels of αVβ3 integrin, ECM genes (Col1A1, α-SMA, fibronectin, vitronectin), and proliferation rates. The effects of αVβ3 integrin blockade (with cilengitide) and silencing (with a pool of four predesigned αVβ3 integrin siRNAs) on ECM gene expression and proliferation rates were evaluated using both reverse transcription quantitative polymerase chain reaction (RT-qPCR) and Western blotting in the human NLC cells cultured on soft (4 kPa) and stiff (100 kPa) substrate and in GLC cells grown on standard plastic plates. Results: αVβ3 integrin gene and protein expression were enhanced (p < 0.05) in GLC cells as compared to NLC. Both cilengitide and siRNA significantly reduced αVβ3 expression in GLC. When NLC were grown in the stiff substrate, cilengitide and siRNA also significantly reduced the increased expression in αVβ3, ECM components, and proliferation rate. Conclusions: Here, we provide evidence of cilengitide- and siRNA-mediated silencing of αVβ3 integrin expression, and inhibition of ECM synthesis in LC cells. Therefore, αVβ3 integrin may be a promising target for the development of novel anti-fibrotic therapies for treating the LC cupping of the ONH in glaucoma.

Cystathionine γ lyase Attenuates Vascular Smooth Muscle Cell Senescence via Foxm1-Gas1 Pathway to Mediate Arterial Stiffness.

Aims Arterial stiffness, a hallmark of vascular aging, significantly contributes to hypertension and impaired organ perfusion. Vascular smooth muscle cell (VSMC) dysfunction, particularly VSMC senescence and its interaction with stiffness, is crucial in the pathogenesis of arterial stiffness. Although hydrogen sulfide (H2S) and its key enzyme cystathionine γ-lyase (CSE) are known to play roles in cardiovascular diseases, their effects on arterial stiffness are not well understood. Methods & Results First, we observed a downregulation of CSE/H2S in the aortic media during biological aging and Angiotensin II (AngII)-induced aging. The VSMC-specific CSE knockout mice were created by loxp-cre (Tagln-cre) system, and which exacerbated AngII-induced aortic aging and stiffness in vivo and VSMC senescence and stiffness in vitro. Conversely, the CSE agonist norswertianolin mitigated these effects. Next, we identified growth arrest-specific 1 (Gas1) as a crucial target of CSE/H2S and found it to be a downstream target gene of forkhead box protein M1 (Foxm1). siRNA knockdown Foxm1 increased Gas1 transcription and reduced the protective effects of H2S on VSMC senescence and stiffness. Finally, we demonstrated that CSE/H2S sulfhydrates Foxm1 at the C210 site, regulating its nuclear translocation and activity, thus reducing VSMC senescence and stiffness. Innovation Our findings highlight the protective role of CSE/H2S in arterial stiffness, emphasizing the novel contributions of CSE, Gas1, and Foxm1 to VSMC senescence and stiffness. Conclusion Endogenous CSE/H2S in VSMCs reduces VSMC senescence and stiffness, thereby attenuating arterial stiffness and aging, partly through sulfhydration-mediated activation of Foxm1 and subsequent inhibition of Gas1 signaling pathways.

4-Methyl-2,4-bis(4-hydroxyphenyl)pent-1-ene (MBP) exposure induces hepatotoxicity and nephrotoxicity - role of oxidative stress, mitochondrial dysfunction and pathways of cytotoxicity.

Bisphenol A (BPA) is a ubiquitous pollutant worldwide and 4-Methyl-2,4-bis(4-hydroxyphenyl)pent-1-ene (MBP) is considered a major active metabolite of BPA with a wide range of potent toxicological properties. However, its adverse outcome pathway (AOP) on the hepatic and renal system has not yet been explored. Hence, the current study evaluated its effect on cell survival, oxidative stress, and apoptosis. In addition, the influence of signalling pathways on cytotoxicity and ROS generating enzymes (NOX2 and XO) on oxidative stress was explored by siRNA knockdown experiments. Further, its molecular interaction with SOD, CAT, and HSA (molecular docking and dynamics) was evaluated and validated with spectroscopy (fluorescence and FTIR) based methods. The outcome indicates that MBP exposure dose dependently increased the cytotoxic response, oxidative stress, and apoptosis in both hepatocytes and kidney cells. Further, MAPK signalling pathways and oxidative stress influenced the overall cytotoxic response in both cells. In addition, the stimulatory (NOX2 and XO) and inhibitory (SOD and CAT) effects of MBP were observed, along with a robust interaction with HSA. The overall observation illustrates that MBP exposure adversely impacts hepatic and renal cells through oxidative stress and relevant molecular pathways which may connect the missing links during risk assessment of BPA.

Effect of Pterostilbene on Regulating LncRNA-Linc00511 Targeting MiR-184 to Promote the Proliferation and Invasion of Non-Small Cell Lung Cancer Under Hypoxic Environment

Non-small cell lung cancer (NSCLC) is one of the most common lung cancers, accounting for more than 85% of lung cancer incidence rates and seriously endangering human health. Increasing evidence shows that some long non-coding RNAs (lncRNAs) act as tumor suppressors and some promote cancer. Pterostilbene-regulated lncRNA-linc00511 has been confirmed to be an oncogenic gene in a variety of tumors. This study aimed to determine the biological function of pterostilbene-regulated lncRNA-linc00511 (LINC00511) in non-small cell lung cancer and provide new diagnostic and therapeutic targets for it. Lung cancer A549 cells were randomly divided into control group and hypoxic group. siRNA knockdown and LINC00511 overexpression plasmid were constructed under hypoxic conditions. Pterostilbene was used to intervene with lncRNA-linc00511. Real time PCR was used to detect the expression changes of LINC00511 and MiR-184. Analyze and detect the effect on the proliferation and invasion of non-small cell lung cancer cells under hypoxic conditions. Real time PCR analysis was used to detect the expression changes of EMT molecules E-cadherin and Vimentin. Western blot detected changes in HIF-1α expression. The expression of LINC00511 increased and the expression of MiR-184 decreased in lung cancer A549 cells, and hypoxic environment led to more significant changes in both. After siRNA knocked down the expression of LINC00511 under hypoxic conditions, pterostilbene was used to intervene with lncRNA-linc00511. The results showed that it promoted the expression of MiR-184, inhibited the proliferation and invasion of lung cancer cells, upregulated the expression of EMT molecules E-cadherin, and increased the expression of Vimentin. The expression is reduced and the expression of HIF-1α is downregulated. Overexpression of LINC00511 can reverse the above changes. Under hypoxic conditions, pterostilbene was used to interfere with lncRNA-linc00511 to promote cell proliferation in non-small cell lung cancer cells. Pterostilbene’s intervention with lncRNA-linc00511 could target the expression of MiR-184 to promote the proliferation and invasion of non-small cell lung cancer. Knocking down the expression of LINC00511 can target down-regulate the expression of MiR-184, change the occurrence of EMT, and alleviate the occurrence and progression of non-small cell lung cancer.

A new method for network bioinformatics identifies novel drug targets for mucinous ovarian carcinoma.

Mucinous ovarian carcinoma (MOC) is a subtype of ovarian cancer that is distinct from all other ovarian cancer subtypes and currently has no targeted therapies. To identify novel therapeutic targets, we developed and applied a new method of differential network analysis comparing MOC to benign mucinous tumours (in the absence of a known normal tissue of origin). This method mapped the protein-protein network in MOC and then utilised structural bioinformatics to prioritise the proteins identified as upregulated in the MOC network for their likelihood of being successfully drugged. Using this protein-protein interaction modelling, we identified the strongest 5 candidates, CDK1, CDC20, PRC1, CCNA2 and TRIP13, as structurally tractable to therapeutic targeting by small molecules. siRNA knockdown of these candidates performed in MOC and control normal fibroblast cell lines identified CDK1, CCNA2, PRC1 and CDC20, as potential drug targets in MOC. Three targets (TRIP13, CDC20, CDK1) were validated using known small molecule inhibitors. Our findings demonstrate the utility of our pipeline for identifying new targets and highlight potential new therapeutic options for MOC patients.