Expression Of Specific Proteins Research Articles

Protein profiles reveal MSH6/MSH2 as a potential biomarker for hepatocellular carcinoma with microvascular invasion.

Microvascular invasion (MVI) is an independent risk factor for postoperative recurrence and metastasis in hepatocellular carcinoma (HCC). However, the specific protein expression profiles that differentiate HCC with MVI from those without MVI remain unclear. The profiles of proteins in early-stage HCC tissues and normal liver tissues were characterized by quantitative proteomics techniques. Immunohistochemical (IHC) staining was undertaken on tissue microarrays from 80 HCC patients to assess the expression of MSH2 and MSH6. Cell counting, colony formation, migration, and invasion assays were carried out in vitro. We identified 5164 proteins in both HCC tissues and adjacent normal liver tissues. Compared to HCC without MVI, 148 upregulated proteins and 97 downregulated proteins were found in HCC with MVI. Particularly noteworthy was the remarkable upregulation of MSH6/MSH2 among these dysregulated proteins in HCC with MVI. Further validation through bioinformatics prediction and IHC confirmed the elevated expression of MSH6/MSH2, which correlated with aggressive disease characteristics and poor prognosis. Receiver operating characteristic curve analyses revealed a substantial area under the curve of 0.761 (specificity 71.79%, sensitivity 73.17%) for the combined use of MSH6/MSH2. Knockdown of MSH6/MSH2 significantly inhibited HCC cell proliferation and invasion in vitro. Our study establishes MSH6 or MSH2 as an oncogene that is prominently overexpressed during HCC progression, which provides new targets for HCC with MVI.

Optimisation of a Method for the Differentiation of Human Umbilical Cord-derived Mesenchymal Stem Cells Toward Renal Epithelial-like Cells.

Human umbilical cord-derived mesenchymal stem cells (hucMSCs) can differentiate into multiple cell lineages, but few methods have been developed to generate kidney lineage cells. Due to their human origin, pluripotent nature and immunomodulatory properties, these stem cells are attractive candidates for clinical applications such as the repair or regeneration of damaged organs. This study evaluated the renal differentiation potential of hucMSCs, when exposed for 10 days to optimised concentrations of retinoic acid, activin-A and bone morphogenetic protein-7 (BMP-7) in various combinations, with and without the priming of the cells with a Wnt signalling pathway activator (CHIR99021). The hucMSCs were isolated and characterised according to surface marker expression (CD73, CD90, CD44, CD146 and CD8) and tri-lineage differentiation potential. The expression of key marker genes (OSR1, TBXT, HOXA13, SIX2, PAX2, KRT18 and ZO1) was examined by qRT-PCR. Specific marker protein expression (E-cadherin, cytokeratin-8 and cytokeratin-19) was analysed by immunocytochemistry. CHIR99021-primed cells treated with the retinoic acid, activin-A and BMP-7 cocktail showed epithelial cell-like differentiation - i.e. distinct phenotypic changes, as well as upregulated gene and protein expression, were observed that were consistent with an epithelial cell phenotype. Thus, our results showed that hucMSCs can efficiently differentiate into renal epithelial-like cells. This work may help in the development of focused therapeutic strategies, in which lineage-defined human stem cells can be used for renal regeneration.

Integrative Transcriptome and Proteome Analyses Elucidate the Mechanism of Lens-Induced Myopia in Mice.

The purpose of this study was to investigate the underlying molecular mechanism of lens-induced myopia (LIM) through transcriptome and proteome analyses with a modified mouse myopia model. Four-week-old C57BL/6J mice were treated with a homemade newly designed -25 diopter (D) lens mounting by a 3D printing pen before right eyes for 4 weeks. Refraction (RE) and axial dimensions were measured every 2 weeks. Retinas were analyzed by RNA-sequencing and data-independent acquisition liquid chromatography tandem mass spectrometry. Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) annotation, and STRING databases were used to identify significantly affected pathways in transcriptomic and proteomic data sets. Western blot was used to detect the expression of specific proteins. The modified model was accessible and efficient. Mice displayed a significant myopic shift (approximately 8 D) following 4 weeks' of lens treatment. Through transcriptomics and proteomics analysis, we elucidated 175 differently expressed genes (DEGs) and 646 differentially expressed proteins (DEPs) between binoculus. The transcriptomic and proteomic data showed a low correlation. Going over the mRNA protein matches, insulin like growth factor 2 mRNA binding protein 1 (Igf2bp1) was found to be a convincing biomarker of LIM, which was confirmed by Western blot. RNA-seq and proteome profiling confirmed that these two "omics" data sets complemented one another in KEGG pathways annovation. Among these, metabolic and human diseases pathways were considered to be correlated with the LIM forming process. The newly constructed LIM model provides a useful tool for future myopia research. Combining transcriptomic and proteomic analysis may potentially brighten the prospects of novel therapeutic targets for patients with myopia.

Nitric Oxide-Scavenging, Anti-Migration Effects, and Glycosylation Changes after Hemin Treatment of Human Triple-Negative Breast Cancer Cells: A Mechanistic Study.

The enhanced expression of nitric oxide (•NO) synthase predicts triple-negative breast cancer outcome and its resistance to different therapeutics. Our earlier work demonstrated the efficiency of hemin to scavenge the intra- and extracellular •NO, proposing its potency as a therapeutic agent for inhibiting cancer cell migration. In continuation, the present work evaluates the effects of •NO on the migration of MDA-MB-231 cells and how hemin modulates the accompanied cellular behavior, focusing on the corresponding expression of cellular glycoproteins, migration-associated markers, and mitochondrial functions. We demonstrated for the first time that while •NO induced cell migration, hemin contradicted that by •NO-scavenging. This was in combination with modulation of the •NO-enhanced glycosylation patterns of cellular proteins with inhibition of the expression of specific proteins involved in the epithelial-mesenchymal transition. These effects were in conjunction with changes in the mitochondrial functions related to both •NO, hemin, and its nitrosylated product. Together, these results suggest that hemin can be employed as a potential anti-migrating agent targeting •NO-scavenging and regulating the expression of migration-associated proteins.

α-Tocopherol reduces VLDL secretion through modulation of intracellular ER-to-Golgi transport of VLDL.

Avoiding hepatic steatosis is crucial for preventing liver dysfunction, and one mechanism by which this is accomplished is through synchronization of the rate of very low density lipoprotein (VLDL) synthesis with its secretion. Endoplasmic reticulum (ER)-to-Golgi transport of nascent VLDL is the rate-limiting step in its secretion and is mediated by the VLDL transport vesicle (VTV). Recent in vivo studies have indicated that α-tocopherol (α-T) supplementation can reverse steatosis in nonalcoholic fatty liver disease, but its effects on hepatic lipoprotein metabolism are poorly understood. Here, we investigated the impact of α-T on hepatic VLDL synthesis, secretion, and intracellular ER-to-Golgi VLDL trafficking using an in vitro model. Pulse-chase assays using [3H]-oleic acid and 100 µmol/L α-T demonstrated a disruption of early VLDL synthesis, resulting in enhanced apolipoprotein B-100 expression, decreased expression in markers for VTV budding, ER-to-Golgi VLDL transport, and reduced VLDL secretion. Additionally, an in vitro VTV budding assay indicated a significant decrease in VTV production and VTV-Golgi fusion. Confocal imaging of lipid droplet (LD) localization revealed a decrease in overall LD retention, diminished presence of ER-associated LDs, and an increase in Golgi-level LD retention. We conclude that α-T disrupts ER-to-Golgi VLDL transport by modulating the expression of specific proteins and thus reduces VLDL secretion.

The Role of Specificity Protein 1 (SP1) in Bladder Cancer Progression through PTEN-Mediated AKT/mTOR Pathway

Introduction: The aim of the study was to investigate the potential mechanism of specificity protein 1 (SP1) in bladder cancer progression through the PTEN-mediated AKT/mTOR pathway. Methods: Human bladder cancer cell lines (HT-1197, HT-1376, and T24) and normal ureteral epithelial cell line SV-HUC-1 were used. SP1 expression was detected via quantitative real-time PCR and Western blotting. Cell viability, migration, invasion, and apoptosis were assessed using CCK-8, transwell, and flow cytometry assays, respectively. The involvement of the PTEN-mediated AKT/mTOR pathway was evaluated by Western blot. A mouse xenograft model was built, and immunohistochemical staining was applied to visualize SP1 and Ki67 expression in tumor tissues. Results: SP1 was overexpressed in bladder cancer cells. SP1 knockdown inhibited viability, migration, and invasion and promoted apoptosis in bladder cancer cells. PTEN intervention increased cell viability, migration, and invasion and decreased apoptosis, which was reversed by SP1 knockdown. The activation of the AKT/mTOR pathway resulting from PTEN knockdown was attenuated by SP1 knockdown. In vivo results showed that SP1 knockdown suppressed tumor growth, increased PTEN expression, and decreased AKT/mTOR pathway-related protein levels. Conclusion: SP1 promotes bladder cancer progression by inhibiting the PTEN-mediated AKT/mTOR pathway. Targeting SP1 may be a potential therapeutic strategy for treating bladder cancer.

Regulation of Inflammation by IRAK-M Pathway Can Be Associated with nAchRalpha7 Activation and COVID-19.

In spite of the vaccine development and its importance, the SARS-CoV-2 pandemic is still impacting the world. It is known that the COVID-19 severity is related to the cytokine storm phenomenon, being inflammation a common disease feature. The nicotinic cholinergic system has been widely associated with COVID-19 since it plays a protective role in inflammation via nicotinic receptor alpha 7 (nAchRalpha7). In addition, SARS-CoV-2 spike protein (Spro) subunits can interact with nAchRalpha7. Moreover, Spro causes toll-like receptor (TLR) activation, leading to pro- and anti-inflammatory pathways. The increase and maturation of the IL-1 receptor-associated kinase (IRAK) family are mediated by activation of membrane receptors, such as TLRs. IRAK-M, a member of this family, is responsible for negatively regulating the activity of other active IRAKs. In addition, IRAK-M can regulate microglia phenotype by specific protein expression. Furthermore, there exists an antagonist influence of SARS-CoV-2 Spro and the cholinergic system action on the IRAK-M pathway and microglia phenotype. We discuss the overexpression and suppression of IRAK-M in inflammatory cell response to inflammation in SARS-CoV-2 infection when the cholinergic system is constantly activated via nAchRalpha7.

Wnt signaling pathway inhibitor promotes mesenchymal stem cells differentiation into cardiac progenitor cells in vitro and improves cardiomyopathy in vivo.

Cardiovascular diseases particularly myocardial infarction (MI) are the leading cause of mortality and morbidity around the globe. As cardiac tissue possesses very limited regeneration potential, therefore use of a potent small molecule, inhibitor Wnt production-4 (IWP-4) for stem cell differentiation into cardiomyocytes could be a promising approach for cardiac regeneration. Wnt pathway inhibitors may help stem cells in their fate determination towards cardiomyogenic lineage and provide better homing and survival of cells in vivo. Mesenchymal stem cells (MSCs) derived from the human umbilical cord have the potential to regenerate cardiac tissue, as they are easy to isolate and possess multilineage differentiation capability. IWP-4 may promote the differentiation of MSCs into the cardiac lineage. To evaluate the cardiac differentiation ability of IWP-4 and its subsequent in vivo effects. Umbilical cord tissue of human origin was utilized to isolate the MSCs which were characterized by their morphology, immunophenotyping of surface markers specific to MSCs, as well as by tri-lineage differentiation capability. Cytotoxicity analysis was performed to identify the optimal concentration of IWP-4. MSCs were treated with 5 μM IWP-4 at two different time intervals. Differentiation of MSCs into cardiomyocytes was evaluated at DNA and protein levels. The MI rat model was developed. IWP-4 treated as well as untreated MSCs were implanted in the MI model, then the cardiac function was analyzed via echocardiography. MSCs were labeled with 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) dye for tracking, while the regeneration of infarcted myocardium was examined by histology and immunohistochemistry. MSCs were isolated and characterized. Cytotoxicity analysis showed that IWP-4 was non-cytotoxic at 5 μM concentration. Cardiac specific gene and protein expression analyses exhibited more remarkable results in fourteen days treated group that was eventually selected for in vivo transplantation. Cardiac function was restored in the IWP-4 treated group in comparison to the MI group. Immunohistochemical analysis confirmed the homing of pre-differentiated MSCs that were labeled with DiI cell labeling dye. Histological analysis confirmed the significant reduction in fibrotic area, and improved left ventricular wall thickness in IWP-4 treated MSC group. Treatment of MSCs with IWP-4 inhibits Wnt pathway and promotes cardiac differentiation. These pre-conditioned MSCs transplanted in vivo improved cardiac function by cell homing, survival, and differentiation at the infarcted region, increased left ventricular wall thickness, and reduced infarct size.

Dragon blood resin ameliorates steroid-induced osteonecrosis of femoral head through osteoclastic pathways.

Objective: Dragon's Blood resin (DBR) is a traditional medicinal substance renowned for its diverse pharmacological effects, which consists of potent anti-inflammatory, antioxidant and angiogenic properties. This study aimed to elucidate its therapeutic mechanism in alleviating steroid-induced osteonecrosis of the femoral head (SIONFH). Methods: Techniques such as SPR and LC-MS were employed to identify and analyze the target proteins of DBR in bone marrow macrophages (BMMs). In vitro, BMMs were treated with RANKL and DBR, and TRAcP staining and actin belt staining were utilized to assess osteoclast activity. The inhibitory effects and underlying mechanisms of DBR on osteoclastogenesis and reactive oxygen species (ROS) generation were determined using real-time PCR, western blotting and immunofluorescence staining. An in vivo SIONFH rat model was set up to assess the curative impacts of DBR using micro-CT scanning and pathological staining. Results: Bioinformatic tools revealed a pivotal role of osteoclast differentiation in SIONFH. Proteomic analysis identified 164 proteins binding in BMMs. In vitro assessments demonstrated that DBR hindered osteoclastogenesis by modulating the expression of specific genes and proteins, along with antioxidant proteins including TRX1 and Glutathione Reductase. Notably, the resin effectively inhibited the expression of crucial proteins, such as the phosphorylation of JNK and the nuclear localization of p65 within the TRAF6/JNK and NFκB signaling pathways. In vivo experiments further confirmed that DBR mitigated the onset of SIONFH in rats by curbing osteoclast and ROS activities. Conclusion: These findings underscore the potential of Dragon's Blood as an effective administration for early-stage SIONFH, shedding light on its therapeutic influence on ROS-mediated osteoclastic signaling pathways.

Klebsiella pneumoniae manipulates human macrophages to acquire iron.

Klebsiella pneumoniae (KP) is a major cause of hospital-acquired infections, such as pneumonia. Moreover, it is classified as a pathogen of concern due to sprawling anti-microbial resistance. During infection, the gram-negative pathogen is capable of establishing an intracellular niche in macrophages by altering cellular metabolism. One factor critically affecting the host-pathogen interaction is the availability of essential nutrients, like iron, which is required for KP to proliferate but which also modulates anti-microbial immune effector pathways. We hypothesized, that KP manipulates macrophage iron homeostasis to acquire this crucial nutrient for sustained proliferation. We applied an in-vitro infection model, in which human macrophage-like PMA-differentiated THP1 cells were infected with KP (strain ATCC 43816). During a 24-h course of infection, we quantified the number of intracellular bacteria via serial plating of cell lysates and evaluated the effects of different stimuli on intracellular bacterial numbers and iron acquisition. Furthermore, we analyzed host and pathogen specific gene and protein expression of key iron metabolism molecules. Viable bacteria are recovered from macrophage cell lysates during the course of infection, indicative of persistence of bacteria within host cells and inefficient pathogen clearing by macrophages. Strikingly, following KP infection macrophages strongly induce the expression of the main cellular iron importer transferrin-receptor-1 (TFR1). Accordingly, intracellular KP proliferation is further augmented by the addition of iron loaded transferrin. The induction of TFR1 is mediated via the STAT-6-IL-10 axis, and pharmacological inhibition of this pathway reduces macrophage iron uptake, elicits bacterial iron starvation, and decreases bacterial survival. Our results suggest, that KP manipulates macrophage iron metabolism to acquire iron once confined inside the host cell and enforces intracellular bacterial persistence. This is facilitated by microbial mediated induction of TFR1 via the STAT-6-IL-10 axis. Mechanistic insights into immune metabolism will provide opportunities for the development of novel antimicrobial therapies.

Trade-offs between the instantaneous growth rate and long-term fitness: Consequences for microbial physiology and predictive computational models.

Microbial systems biology has made enormous advances in relating microbial physiology to the underlying biochemistry and molecular biology. By meticulously studying model microorganisms, in particular Escherichia coli and Saccharomyces cerevisiae, increasingly comprehensive computational models predict metabolic fluxes, protein expression, and growth. The modeling rationale is that cells are constrained by a limited pool of resources that they allocate optimally to maximize fitness. As a consequence, the expression of particular proteins is at the expense of others, causing trade-offs between cellular objectives such as instantaneous growth, stress tolerance, and capacity to adapt to new environments. While current computational models are remarkably predictive for E. coli and S. cerevisiae when grown in laboratory environments, this may not hold for other growth conditions and other microorganisms. In this contribution, we therefore discuss the relationship between the instantaneous growth rate, limited resources, and long-term fitness. We discuss uses and limitations of current computational models, in particular for rapidly changing and adverse environments, and propose to classify microbial growth strategies based on Grimes's CSRframework.

Abstract P3164: Podocalyxin, A Renal Podocyte Specific Protein Expression In Urine Exosome Acts As A Marker For Podocyte Injury During Sars-cov-2 Infection

COVID-19 attributed to SARS-CoV2 infection has been associated with cardiovascular disease and diabetes. COVID-19 has been associated with endothelial cell dysfunction (ECD), which is manifested by entities such as microvascular thrombosis. ECD is quite common in type 2 diabetes mellitus (T2DM) where the renal podocyte dysfunction is often an early manifestation of microvascular complication. We explored whether presence of hyperglycemia predisposes to increased SARS-CoV2 infection and whether SARS-CoV2 infection puts an individual at a higher risk of developing cardio-metabolic complications such as kidney disease (DKD), with podocyte damage. We evaluated albuminuria and podocyte protein in urine derived exosomes in urine exosomes from SARS-CoV2 patients at 10 days, 6 months and 12 months post SARS-CoV2 infection. Methods: Blood and Urine samples from SARS-CoV2 patients post-acute phase of infection were procured. Peripheral blood mononuclear cells (PBMNCs) and urine exosomes were isolated and podocyte protein marker Podocalyxin (PODXL) was identified by western blot analysis. Results: Podocyte specific proteins were examined in urine exosomal fraction such as Podocalyxin and Nephrin (normalized to CD9 exosomal protein band intensities) levels increased significantly compared to urine samples from type-2 diabetes subjects with documented evidence of nephropathy. Podocalyxin levels were significantly high at 12month time point (p=0.001; n=9) and Nephrin levels were noted to be high at 10week (p=0.01; n=8), 6months (p=0.04; n=14 ) and 12Months (p=0.001; n=6) time points. Podocalyxin and Nephrin obtained from urine exosome samples collected from non diabetic subjects who never had COVID-19, showed little or barely detectable corresponding western blot bands. Interestingly, there was no significant differences noted on urine albumin: creatinine (UAR) ratios between the two groups, indicating that urine exosomal protein estimation may be an ideal biomarker to monitor podocyte damage in early phases of CKD. Conclusion: A persistent high levels of podocyte specific proteins was noted in urinary exosomes even at 12 months post acute Covid which may be secondary to long-standing podocyte inflammation leading to long term renal damage.

Abstract P3126: Atrial Fibrillation Is Associated With Decreased Claudin-5 In Cardiomyocyte

Background: Although it is critically important to understand the underlying molecular and electrophysiological changes that predispose to the induction and maintenance of atrial fibrillation (Af), the underlying mechanism of Af is still poorly defined. Af is characterized as the electrophysiological and membrane integrity abnormality of the atrial cells, and claudin-5, a tight junction protein, may be involved in the pathophysiology of Af, however, the role of claudin-5 in Af is unknown. Methods and Results: Cardiac tissues from the left atrial appendage were obtained from AF patients with rheumatic valvular heart disease undergoing modified radiofrequency ablation maze procedure with approval from local human research ethic review and normal left atrial appendages were obtained from non-AF donors. Western blot, immunofluorescence and transmission electron microscope showed the expression of claudin-5 significantly decreased in cardiomyocytes from left atrium of AF patients compared to non-AF donors. Then proteomics analysis was performed to screen the specific protein expression and signal pathway changed in AF heart tissue vs. non-AF heart tissue, and showed that 83 proteins were downregulated and 102 proteins were upregulated in the left atrial appendage of AF patients. Among them, DAG, CACNA2D2, MYL2 were downregulated and MCU was upregulated. KEGG pathway analysis showed these changes would lead to hypertrophic and dilated cardiomyopathy. Finally, claudin-5 shRNA AAV was injected in mouse left ventricle to knockdown claudin-5 in cardiomyocytes to observe whether change of claudin-5 affects the protein expression in KEGG pathway analysis. We found claudin-5 deficiency caused severe ventricular dilation, ST elevation and arrythmia, and the protein level of DAG, CACNA2D2 and MYL2 were downregulated and MCU was upregulated. Co-immunoprecipitation also confirmed the specific protein-protein interaction between these targeted proteins and claudin-5. Conclusion: We demonstrated that the decrease of claudin-5 in the left atrium of AF patients with rheumatic valvular heart disease. The mechanism of AF might be associated with claudin-5 downregulation-induced decrease of DAG, CACNA2D2, MYL2 and increase of MCU in cardiomyocytes.

Abstract P3050: Cpne5 Is Necessary For Normal Murine Sinoatrial Node Function In Vivo

Cpne5 ( Copine 5 ) is a Ca 2+ -dependent phospholipid binding protein implicated in membrane trafficking but has no known role within the heart to date. In single-cell RNA-sequencing of the developing mouse heart, we previously found Cpne5 gene expression to be highly enriched in the cardiac conduction system (CCS). Further, variants in CPNE5 were identified in 6 independent GWAS related to heart rate variability in humans. We therefore hypothesized that Cpne5 is necessary for normal CCS function through Ca 2+ -dependent membrane trafficking of ion channels. Whole mount immunostaining with 3D volumetric imaging (iDISCO+) of intact WT hearts demonstrated robust and specific protein expression of Cpne5 throughout all CCS components, including the sinoatrial node (SAN) and SAN-transitional cells. Next, to assess the necessity of Cpne5 within the CCS, systemic knockout mice (Cpne5KO) were generated using CRISPR/Cas9. Successful deletion of Cpne5 in CRISPR-generated KO mice was confirmed by PCR and, at the protein level, immunofluorescence. Homozygous Cpne5KO mice were viable, showed normal growth and allele frequencies segregated in a normal Mendelian fashion. No gross anatomic defects of the CCS were observed in Cpne5KO mice and transthoracic echocardiogram studies showed a slight reduction in left ventricular fractional shortening but otherwise a structurally normal heart. Notably, while surface electrocardiogram (ECG) demonstrated normal intervals (PR, QRS, QTc), Cpne5KO mice exhibited pronounced sinus arrhythmia, with notable sinus pauses, sinus bradycardia, and increased heart rate variability as compared to WT littermate controls on long term telemetry. Systemic loss-of-function of Cpne5 in mice resulted in marked sinus node dysfunction, consistent with human GWAS data, implicating a novel role for Cpne5 in CCS function and/or development. Additional phenotyping and molecular studies, including CPNE5 deletion in human induced-pluripotent cell-derived SAN-like cells, are ongoing to further elucidate its underlying mechanism.

Comparative Functional Genomics Studies for Understanding the Hypothetical Proteins in Mycobacterium Tuberculosis Variant Microti 12

Comparative Functional Genomics Studies for Understanding the Hypothetical Proteins in Mycobacterium Tuberculosis Variant Microti 12

Proteomic Analysis of Dysfunctional Liver Sinusoidal Endothelial Cells Reveals Substantial Differences in Most Common Experimental Models of Chronic Liver Diseases.

Molecular markers of dedifferentiation of dysfunctional liver sinusoidal endothelial cells (LSEC) have not been fully elucidated. We aimed at deciphering the molecular profile of dysfunctional LSEC in different pathological scenarios. Flow cytometry was used to sort CD11b-/CD32b+ and CD11b-/CD32b- LSEC from three rat models of liver disease (bile duct ligation-BDL; inhaled carbon tetrachloride-CCl4; and high fat glucose/fructose diet-HFGFD). A full proteomic profile was performed applying nano-scale liquid chromatography tandem mass spectrometry (nLC-MS) and analyzed with PEAKS software. The percentage of CD32b- LSEC varied across groups, suggesting different capillarization processes. Both CD32+ and CD32b- LSEC from models are different from control LSEC, but differently expressed proteins in CD32b- LSEC are significantly higher. Heatmaps evidenced specific protein expression patterns for each model. Analysis of biological significance comparing dysfunctional CD32b- LSEC with specialized CD32b+ LSEC from controls showed central similarities represented by 45 common down-regulated proteins involved in the suppression of the endocytic machinery and 63 common up-regulated proteins associated with the actin-dependent cytoskeleton reorganization. In summary; substantial differences but also similarities in dysfunctional LSEC from the three most common models of liver disease were found, supporting the idea that LSEC may harbor different protein expression profiles according to the etiology or disease stage.

Phytochemicals Modify the Action of Cancer Cells Mitochondrial Drug-Resistance Mechanism

The genesis and evolution of cancer are known to be significantly influenced by mitochondria, and recent studies have indicated that mitochondrial modifications may potentially contribute to the emergence of treatment resistance. Additionally, drug-resistant cancer cells may also display modifications in mitochondrial metabolism, such as changes in the generation of reactive oxygen species (ROS), which are biological byproducts of mitochondrial respiration. These changes can alter the cell's sensitivity to chemotherapy drugs and contribute to drug resistance. The expression of specific genes or proteins that are crucial in the control of cell growth and survival may be altered by mitochondrial mutations, which may also contribute to medication resistance. Phytochemicals are naturally occurring, biologically active substances found in plants that have been demonstrated to offer a variety of health advantages, including anti-cancer effects. It has been demonstrated that phytochemicals target these altered mitochondrial pathways in cancer cells, increasing the potency of chemotherapy medications and overcoming drug resistance. For instance, it has been demonstrated that some phytochemicals, including curcumin, resveratrol, and quercetin, can block the function of mitochondrial membrane proteins that lead to drug resistance in cancer cells. Other phytochemicals, including berberine and epigallocatechin gallate (EGCG), have been demonstrated to directly interfere with mitochondrial activity, inducing apoptosis (programmed cell death) in cancer cells. Overall, the capacity of phytochemicals to modify the functioning of cancer cell mitochondrial drug-resistance systems is a viable strategy for the creation of novel anti-cancer treatments.

Selective Proteolysis of Activated Transcriptional Factor by NIR-Responsive Palindromic DNA Thalidomide Conjugate Inhibits the Canonical Smad Pathway.

Dysfunctional transcription factors that activate abnormal expressions of specific proteins are often associated with the progression of various diseases. Despite being attractive drug targets, the lack of druggable sites has dramatically hindered their drug development. The emergence of proteolysis targeting chimeras (PROTACs) has revitalized the drug development of many conventional hard-to-drug protein targets. Here, the use of a palindromic double-strand DNA thalidomide conjugate (PASTE) to selectively bind and induce proteolysis of targeted activated transcription factor (PROTAF) is reported. The selective proteolysis of the dimerized phosphorylated receptor-regulated Smad2/3 and inhibition of the canonical Smad pathway validates PASTE-mediated PROTAF. Further aptamer-guided active delivery of PASTE and near-infrared light-triggered PROTAF are demonstrated. Great potential in using PASTE for the selective degradation of the activated transcription factor is seen, providing a powerful tool for studying signaling pathways and developing precision medicines.

Analysis of Fatty Acid Metabolism in Fetal and Failing Hearts by Single-Cell RNA Sequencing Revealed SLC27A6 as a Critical Gene in Heart Maturation.

Heart failure is associated with shifts in substrate preferences and energy insufficiency. Although cardiac metabolism has been explored at the organ level, the metabolic changes at the individual cell level remain unclear. This study employed single-cell ribonucleic acid (RNA) sequencing to investigate the cell-type-specific characteristics of gene expression related to fatty acid metabolism. Single-cell RNA sequencing data from fetal hearts were processed to analyze gene expression patterns related to fatty acid metabolism. Immunofluorescence staining and Western blotting techniques were employed to validate the expression of specific proteins. Additionally, calcium recording and contractility measurements were performed to assess the functional implications of fatty acid metabolism in cardiomyocytes. Based on single-cell RNA sequencing data analysis, we found that a decrease in overall energy requirements underlies the downregulation of fatty acid oxidation-related genes in the later period of heart maturation and the compensatory increase of fatty acid metabolism in individual cardiomyocytes during heart failure. Furthermore, we found that solute carrier family 27 member 6 (SLC27A6), a fatty acid transport protein, is involved in cardiac maturation. SLC27A6 knockdown in human induced pluripotent stem cell-derived cardiomyocytes resulted in an immature cardiomyocyte transcriptional profile, abnormal morphology, impaired Ca2+ handling activity, and contractility. Overall, our study offers a novel perspective for exploring cardiac fatty acid metabolism in fetal and failing hearts along with new insights into the cellular mechanism underlying fatty acid metabolic alterations in individual cardiac cells. It thus facilitates further exploration of cardiac physiology and pathology.

P-540 Improper placentation due to skewed Oct4-Cdx2 expression may be a leading factor in idiopathic recurrent pregnancy loss (RPL) cases from Assam, India.

Abstract Study question Does the differential expression of key embryonic stem cell (ESC) markers due to upstream signaling cascade(s) deregulation influence RPL pathogenesis in the studied population? Summary answer Imbalanced Oct4-Cdx2 expression linked differential ESCs polarization due to β-catenin signaling deregulation during the peri-implantation period impacts RPL pathogenesis in the studied population. What is known already RPL affects approximately 2-5% of women globally and around 7% Indian women in reproductive age group; with 50% of case etiology being unclear. Proper placentation is indispensable for establishment, continuation, and success of a pregnancy; and it depends largely on a healthy balance between apoptosis and stem cell differentiation that aids proper trophoblast invasion. These differentiation events are tightly controlled by the interplay of oxygen tension, hormones, growth factors, other signaling molecules and most importantly by transcription factors including those deciding the stem cell lineage like Oct4, NANOG, and Cdx2, wherein, β-catenin is known to enhance Oct-4 activity in ESCs. Study design, size, duration The sample size was predicted using Raosoft software utilizing the prevalence rate of RPL. Overall 87 women with 2 or more pregnancy losses before completion of 20 weeks of gestation were enrolled as RPL cases with informed consent, whereas, for the control group, 107 women volunteers undergoing medical termination of pregnancy (MTP) were enrolled as gestation-week-matched comparative study group for experimental data analysis. Participants/materials, setting, methods Products of conception (POC) was collected from the MTP and RPL cases. POCs were collected in 2vials for every subject: (i) 4% buffered formalin for fixation and preparation of microscopic slides for immunofluorescence (IF) based protein expression of Oct4 and Cdx2; (ii) RNA later solution for site specific transcriptional analysis of Oct4, NANOG, Cdx2, and β-catenin utilizing Realtime PCR. The difference in expression was analyzed using SPSS statistical software. Main results and the role of chance IF based site specific protein expression data confirmed higher expression of OCT4 in RPL cases compared to MTP controls, whereas CDX2 expression was found to be low in RPL compared to MTP subjects. The Realtime PCR amplification of cDNA using β-actin as internal normalization control for Oct4 (25.76 ± 10.17 folds) and NANOG (18.32 ± 6.67folds) showed increased expression whereas the Cdx2 expression was downregulated (0.487 ± 0.186 folds) in RPL cases compared to MTP controls, indicating a shift towards pluripotency which is a non-favorable environment for the ongoing pregnancy since cell polarization for initiation of differentiation to different cell lineages is required at that time of gestation. The cdx2 and oct4 expression correlated inversely and significantly {Pearson’s correlation= −0.838, p = 0.009; Spearman’s rho= −0.786; p = 0.016}. The realtime PCR amplification of β-catenin, the key molecule involved in direct enhancing of Oct4 expression during gestation was found to be increased in RPL cases compared to MTP controls. Statistically, the β-catenin-Oct4 expression correlated positively significantly. Limitations, reasons for caution Being a patient based study, the present study is limited to being an associative study only; and therefore secondary validation of the data for causal association using cell lines or suitable model system futuristically may add valuable scientific inputs and aid clinical intervention modalities for controlling RPL. Wider implications of the findings As the majority of RPL pathogenesis etiology is undefined limiting the possibilities of clinical interventions therefore we presume the present data can layout possible therapeutic interventions by regulating key regulators influencing skewed ESC profile aiding proper placentation leading to successful pregnancy in a subpopulation of cases with RPL history. Trial registration number Not applicable