Nectin-4-directed antibody-drug conjugates (ADCs): Spotlight on preclinical and clinical evidence.

Nectin-4 (Nectin cell adhesion molecule 4), a type I transmembrane cell adhesion protein, was demonstrated to be overexpressed in a variety of tumors, making it an attractive antigen for targeted therapies such as antibody-drug conjugates (ADCs). Of great note, the US Food and Drug Administration (FDA)-approval of the first Nectin-4-directed ADC, enfortumab vedotin (EV), in urothelial cancer (UC) not only introduced Nectin-4 as a clinically validated and reliable target antigen but also confirmed the evolving role of Nectin-4-directed ADCs as novel and promising cancer therapeutics. In addition to EV, there have been or are currently being seven and eleven Nectin-4-directed ADCs, respectively, in various stages of clinical trials and preclinical development, offering a promising future for the treatment of Nectin-4-positive cancer patients. This study reviewed clinical- and preclinical-stage Nectin-4-directed ADCs.

Pasteurized form of a potential probiotic lactobacillus brevis IBRC-M10790 exerts anti-inflammatory effects on inflammatory bowel disease in vitro.

BACKGROUND: Inflammatory bowel disease (IBD) is a chronic, relapsing inflammatory disorder of the gastrointestinal system. So far, no treatment has been identified that can completely cure IBD. Lactobacillus brevis is hypothesized to be beneficial in preventing inflammation. This study aimed to evaluate the potential probiotic effects of live and pasteurized L. brevis IBRC-M10790 on the in vitro cell co-culture model of IBD. METHODS: An in vitro intestinal model was established using a transwell co-culture system of Caco-2 intestinal epithelial cells and RAW264.7 macrophages. Inflammatory conditions were induced in RAW264.7 cells using lipopolysaccharide. The effects of live and pasteurized L. brevis IBRC-M10790 on inflammatory mediators and epithelial barrier markers were investigated. RESULTS: L. brevis IBRC-M10790 was able to significantly decrease the proinflammatory cytokines (IL-6, IL-1ß, and TNF-α) and increase the anti-inflammatory cytokine (IL-10) in the in vitro co-culture system. In addition, L. brevis increased adherens and tight junction (TJ) markers (ZO-1, E-cadherin, and Occludin) in Caco-2 intestinal epithelial cells. Based on the results, pasteurized L. brevis showed a higher protective effect than live L. brevis. CONCLUSIONS: Our findings suggest that live and pasteurized forms of L. brevis possess probiotic properties and can mitigate inflammatory conditions in IBD.

Long non-coding RNAs as pathophysiological regulators, therapeutic targets and novel extracellular vesicle biomarkers for the diagnosis of inflammatory bowel disease.

Inflammatory bowel disease (IBD) is a chronic relapsing disease of the gastrointestinal (GI) system that includes two groups, Crohn's disease (CD) and ulcerative colitis (UC). To cope with these two classes of IBD, the investigation of pathogenic mechanisms and the discovery of new diagnostic and therapeutic approaches are crucial. Long non-coding RNAs (lncRNAs) which are non-coding RNAs with a length of longer than 200 nucleotides have indicated significant association with the pathology of IBD and strong potential to be used as accurate biomarkers in diagnosing and predicting responses to the IBD treatment. In the current review, we aim to investigate the role of lncRNAs in the pathology and development of IBD. We first describe recent advances in research on dysregulated lncRNAs in the pathogenesis of IBD from the perspective of epithelial barrier function, intestinal immunity, mitochondrial function, and intestinal autophagy. Then, we highlight the possible translational role of lncRNAs as therapeutic targets, diagnostic biomarkers, and predictors of therapeutic response in colon tissues and plasma samples. Finally, we discuss the potential of extracellular vesicles and their lncRNA cargo in the pathophysiology, diagnosis, and treatment of IBD.

Long non-coding RNAs in biomarking COVID-19: a machine learning-based approach.

BACKGROUND: The coronavirus pandemic that started in 2019 has caused the highest mortality and morbidity rates worldwide. Data on the role of long non-coding RNAs (lncRNAs) in coronavirus disease 2019 (COVID-19) is scarce. We aimed to elucidate the relationship of three important lncRNAs in the inflammatory states, H19, taurine upregulated gene 1 (TUG1), and colorectal neoplasia differentially expressed (CRNDE) with key factors in inflammation and fibrosis induction including signal transducer and activator of transcription3 (STAT3), alpha smooth muscle actin (α-SMA), tumor necrosis factor-alpha (TNF-α), and interleukin-6 (IL-6) in COVID-19 patients with moderate to severe symptoms. METHODS: Peripheral blood mononuclear cells from 28 COVID-19 patients and 17 healthy controls were collected. The real-time quantitative polymerase chain reaction (RT-qPCR) was performed to evaluate the expression of RNAs and lncRNAs. Western blotting analysis was also performed to determine the expression levels of STAT3 and α-SMA proteins. Machine learning and receiver operating characteristic (ROC) curve analysis were carried out to evaluate the distinguishing ability of lncRNAs. RESULTS: The expression levels of H19, TUG1, and CRNDE were significantly overexpressed in COVID-19 patients compared to healthy controls. Moreover, STAT3 and α-SMA expression levels were remarkedly increased at both transcript and protein levels in patients with COVID-19 compared to healthy subjects and were correlated with Three lncRNAs. Likewise, IL-6 and TNF-α were considerably upregulated in COVID-19 patients. Machine learning and ROC curve analysis showed that CRNDE-H19 panel has the proper ability to distinguish COVID-19 patients from healthy individuals (area under the curve (AUC) = 0.86). CONCLUSION: The overexpression of three lncRNAs in COVID-19 patients observed in this study may align with significant manifestations of COVID-19. Furthermore, their co-expression with STAT3 and α-SMA, two critical factors implicated in inflammation and fibrosis induction, underscores their potential involvement in exacerbating cardiovascular, pulmonary and common symptoms and complications associated with COVID-19. The combination of CRNDE and H19 lncRNAs seems to be an impressive host-based biomarker panel for screening and diagnosis of COVID-19 patients from healthy controls. Research into lncRNAs can provide a robust platform to find new viral infection-related mediators and propose novel therapeutic strategies for viral infections and immune disorders.

Progress of antibody-drug conjugates (ADCs) targeting c-Met in cancer therapy; insights from clinical and preclinical studies.

The cell-surface receptor tyrosine kinase c-mesenchymal-epithelial transition factor (c-Met) is overexpressed in a wide range of solid tumors, making it an appropriate target antigen for the development of anticancer therapeutics. Various antitumor c-Met-targeting therapies (including monoclonal antibodies [mAbs] and tyrosine kinases) have been developed for the treatment of c-Met-overexpressing tumors, most of which have so far failed to enter the clinic because of their efficacy and complications. Antibody-drug conjugates (ADCs), a new emerging class of cancer therapeutic agents that harness the target specificity of mAbs to deliver highly potent small molecules to the tumor with the minimal damage to normal cells, could be an attractive therapeutic approach to circumvent these limitations in patients with c-Met-overexpressing tumors. Of great note, there are currently nine c-Met-targeting ADCs being examined in different phases of clinical studies as well as eight preclinical studies for treating various solid tumors. The purpose of this study is to present a broad overview of clinical- and preclinical-stage c-Met-targeting ADCs.

Predictive three-biomarker panel in peripheral blood mononuclear cells for detecting hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) ranks among the most prevalent cancers and accounts for a significant proportion of cancer-associated deaths worldwide. This disease, marked by multifaceted etiology, often poses diagnostic challenges. Finding a reliable and non-invasive diagnostic method seems to be necessary. In this study, we analyzed the gene expression profiles of 20 HCC patients, 12 individuals with chronic hepatitis, and 15 healthy controls. Enrichment analysis revealed that platelet aggregation, secretory granule lumen, and G-protein-coupled purinergic nucleotide receptor activity were common biological processes, cellular components, and molecular function in HCC and chronic hepatitis B (CHB) compared to healthy controls, respectively. Furthermore, pathway analysis demonstrated that "estrogen response" was involved in the pathogenesis of HCC and CHB conditions, while, "apoptosis" and "coagulation" pathways were specific for HCC. Employing computational feature selection and logistic regression classification, we identified candidate genes pivotal for diagnostic panel development and evaluated the performance of these panels. Subsequent machine learning evaluations assessed these panels' performance in an independent cohort. Remarkably, a 3-marker panel, comprising RANSE2, TNF-α, and MAP3K7, demonstrated the best performance in qRT-PCR-validated experimental data, achieving 98.4% accuracy and an area under the curve of 1. Our findings highlight this panel's promising potential as a non-invasive approach not only for detecting HCC but also for distinguishing HCC from CHB patients.

Evaluation of long non-coding RNAs EGOT, NRAV, NRIR and mRNAs ISG15 and IFITM3 expressions in COVID-19 patients.

Individuals with Coronavirus Disease 2019 (COVID-19) may show no symptoms to moderate or severe complications. This variation may be due to differences in the strength of the immune response, including a delayed interferon (IFN) response in asymptomatic patients and higher IFN levels in severe patients. Some long non-coding RNAs (lncRNAs), as regulators of the IFN pathway, may contribute to the emergence of different COVID-19 symptoms. This study aimed to comparatively investigate the relationship between lncRNAs (eosinophil granule ontogeny transcript (EGOT), negative regulator of antiviral response (NRAV), and negative regulator of interferon response (NRIR)), alongside interferon-stimulated genes (ISGs) like ISG-15 and interferon-induced transmembrane protein 3 (IFITM3) in COVID-19 patients with asymptomatic, moderate, and severe symptoms. Buffy coat samples were collected from 17 asymptomatic, 23 moderate, 22 severe patients, and 44 healthy controls. Quantitative real-time PCR was utilized to determine the expression levels. In a comparison between COVID-19 patients and healthy individuals, higher expression levels of EGOT and NRAV were observed in severe and moderate patients. NRIR expression was increased across all patient groups. Meanwhile, ISG15 expression decreased in all patient groups, and the moderate group showed a significant decrease in IFITM3 expression. Comparing COVID-19 patient groups, EGOT expression was significantly higher in moderate COVID-19 patients compared to asymptomatic patients. NRAV was higher in moderate and severe patients compared to asymptomatic. NRIR levels did not differ significantly between the COVID-19 patient groups. ISG15 was higher in moderate and severe patients compared to asymptomatic. IFITM3 expression was significantly higher in severe patients compared to the moderate group. In severe COVID-19 patients, EGOT expression was positively correlated with NRAV levels. EGOT and NRAV showed a significant positive correlation in asymptomatic patients, and both were positively correlated with IFITM3 expression. This study suggests that EGOT, NRAV, NRIR, ISG15, and IFITM3 may serve as diagnostic biomarkers for COVID-19. The lncRNA NRAV may be a good biomarker in a prognostic panel between asymptomatic and severe patients in combination with other high-sensitivity biomarkers. EGOT, NRAV, and ISG15 could also be considered as specific biomarkers in a prognostic panel comparing asymptomatic and moderate patients with other high-sensitivity biomarkers.

Prediction of anti-TNF therapy failure in ulcerative colitis patients by ensemble machine learning: A prospective study.

Nowadays, anti-TNF therapy remarkably improves the medical management of ulcerative colitis (UC), but approximately 40 % of patients do not respond to this treatment. In this study, we used 79 anti-TNF-naive patients with moderate-to-severe UC from four cohorts to discover alternative therapeutic targets and develop a personalized medicine approach that can diagnose UC non-responders (UCN) prior to receiving anti-TNF therapy. To this end, two microarray data series were integrated to create a discovery cohort with 35 UC samples. A comprehensive gene expression and functional analysis was performed and identified 313 significantly altered genes, among which IL6 and INHBA were highlighted as overexpressed genes in the baseline mucosal biopsies of UCN, whose cooperation may lead to a decrease in the Tregs population. Besides, screening the abundances of immune cell subpopulations showed neutrophils' accumulation increasing the inflammation. Furthermore, the correlation of KRAS signaling activation with unresponsiveness to anti-TNF mAb was observed using network analysis. Using 50x repeated 10-fold cross-validation LASSO feature selection and a stack ensemble machine learning algorithm, a five-mRNA prognostic panel including IL13RA2, HCAR3, CSF3, INHBA, and MMP1 was introduced that could predict the response of UC patients to anti-TNF antibodies with an average accuracy of 95.3 %. The predictive capacity of the introduced biomarker panel was also validated in two independent cohorts (44 UC patients). Moreover, we presented a distinct immune cell landscape and gene signature for UCN to anti-TNF drugs and further studies should be considered to make this predictive biomarker panel and therapeutic targets applicable in the clinical setting.

Plasma Extracellular Vesicle LncRNA H19 as a Potential Diagnostic Biomarker for Inflammatory Bowel Diseases.

BACKGROUND: Inflammatory bowel disease (IBD) is a complex gastrointestinal disease with 2 main subtypes of Crohn's disease (CD) and ulcerative colitis (UC), whose diagnosis mainly depends on the medical history, clinical symptoms, endoscopic, histologic, radiological, and serological findings. Extracellular vesicles (EVs) are now considered an additional mechanism for intercellular communication, allowing cells to exchange biomolecules. Long noncoding RNAs (lncRNAs) that are enriched in EVs have been defined as an ideal diagnostic biomarker for diseases. In this study, we investigated the expression differences of 5 lncRNAs in tissue and plasma EVs of active IBD patients compared with patients in the remission phase and healthy controls to introduce an EV-lncRNA as a noninvasive IBD diagnostic biomarker. METHODS: Twenty-two active IBD patients, 14 patients in the remission phase, 10 active rheumatoid arthritis (RA) patients, 14 irritable bowel syndrome (IBS) patients, and 22 healthy individuals were recruited in the discovery cohort. In addition, 16 patients with active IBD, 16 healthy controls, 10 inactive IBD patients, 12 active RA patients, and 14 IBS patients were also included in the validation cohort. The expression levels of 5 lncRNAs in tissue and EV-plasma were evaluated by quantitative real-time polymerase chain reaction (qRT-PCR) . Machine learning and receiver operating characteristic (ROC) curve analysis were performed to investigate the distinguishing ability of the candidate biomarkers. RESULTS: While the expression levels of lncRNAs CDKN2B-AS1, GAS5, and TUG1 were significantly downregulated, lncRNAs H19 and CRNDE were overexpressed in active IBD lesions. Expression of H19 was detected in plasma EVs whose isolation had been confirmed via dynamic light scattering, microscopy images, and western blotting. The classification results demonstrated the excellent ability of H19 in distinguishing IBD/active from IBD/remission, healthy control, RA, and IBS (area under the ROC curve = 0.95, 0.97,1, and 0.97 respectively). CONCLUSIONS: Our study suggests that circulating EV-lncRNA H19 exhibited promising potential for the diagnosis of active IBD.

The upregulation of lncRNA H19 in active IBD tissues and plasma extracellular vesicles indicated the possible association of H19 with the disease activity. In addition, the high sensitivity and specificity of this marker proposed it as a potential biomarker for the diagnosis of IBD patients.

Intestinal organoids: A versatile platform for modeling gastrointestinal diseases and monitoring epigenetic alterations.

Considering the significant limitations of conventional 2D cell cultures and tissue in vitro models, creating intestinal organoids has burgeoned as an ideal option to recapitulate the heterogeneity of the native intestinal epithelium. Intestinal organoids can be developed from either tissue-resident adult stem cells (ADSs) or pluripotent stem cells (PSCs) in both forms induced PSCs and embryonic stem cells. Here, we review current advances in the development of intestinal organoids that have led to a better recapitulation of the complexity, physiology, morphology, function, and microenvironment of the intestine. We discuss current applications of intestinal organoids with an emphasis on disease modeling. In particular, we point out recent studies on SARS-CoV-2 infection in human intestinal organoids. We also discuss the less explored application of intestinal organoids in epigenetics by highlighting the role of epigenetic modifications in intestinal development, homeostasis, and diseases, and subsequently the power of organoids in mirroring the regulatory role of epigenetic mechanisms in these conditions and introducing novel predictive/diagnostic biomarkers. Finally, we propose 3D organoid models to evaluate the effects of novel epigenetic drugs (epi-drugs) on the treatment of GI diseases where epigenetic mechanisms play a key role in disease development and progression, particularly in colorectal cancer treatment and epigenetically acquired drug resistance.

Extracellular vesicles as reconfigurable therapeutics for eye diseases: Promises and hurdles.

A large number of people worldwide suffer from visual impairment. However, most available therapies rely on impeding the development of a particular eye disorder. Therefore, there is an increasing demand for effective alternative treatments, specifically regenerative therapies. Extracellular vesicles, including exosomes, ectosomes, or microvesicles, are released by cells and play a potential role in regeneration. Following an introduction to EV biogenesis and isolation methods, this integrative review provides an overview of our current knowledge about EVs as a communication paradigm in the eye. Then, we focused on the therapeutic applications of EVs derived from conditioned medium, biological fluid, or tissue and highlighted some recent developments in strategies to boost the innate therapeutic potential of EVs by loading various kinds of drugs or being engineered at the level of producing cells or EVs. Challenges faced in the development of safe and effective translation of EV-based therapy into clinical settings for eye diseases are also discussed to pave the road toward reaching feasible regenerative therapies required for eye-related complications.

Evaluation of the mTORC activity in the presence of Toxoplasma gondii and azathioprine in human monocyte cell line.

BACKGROUND: Autophagy is an important part of pathogenesis of IBD. Thiopurines such as azathioprine (AZA) are approved drugs for clinical practices in IBD patients. Besides, as an escape strategy, Toxoplasma gondii can use the mTORC1 complex to inactivate autophagy. METHODS: In this study, we investigated whether T. gondii tachyzoites may modulate autophagy and interfere the effects of azathioprine in IBD treatment. PMA-activated human monocyte cell line (THP-1) was infected with fresh T. gondii RH tachyzoites. After 5 h of infection, the cells were treated with AZA for 6 h. The expression of atg5, atg7, atg12, lc3b, and ß-actin (BACT) genes was evaluated using quantitative real-time PCR. To analyze the phosphorylation of ribosomal protein S6 (rpS6), western blot using specific primary antibodies was performed. RESULTS: The results of real-time PCR revealed that AZA, T. gondii tachyzoites, and a combination of AZA and T. gondii tachyzoites upregulated atg5 gene for 4.297-fold (P-value = 0.014), 2.49-fold (P-value = 0.006), and 4.76-fold (P-value = 0.001), respectively. The atg7 gene showed significant upregulation (2.272-fold; P-value = 0.014) and (1.51-fold; P-value = 0.020) in AZA and AZA / T. gondii, respectively. The expression of atg12 gene was significantly downregulated in AZA and T. gondii tachyzoites for (8.85-fold; P-value = 0.004) and (2.005-fold; P-value = 0.038), respectively, but upregulated in T. gondii/AZA (1.52-fold; P-value = 0.037). In addition, the lc3b gene was only significantly changed in AZA / T. gondii (3.028-fold; P-value = 0.001). Western blot analysis showed that T. gondii tachyzoites significantly phosphorylated rpS6, and tachyzoites did not interfere the effects of AZA to phosphorylate the rpS6. CONCLUSION: Taken together, although AZA and T. gondii similarly affects the expression levels of atg5, atg7, and atg12, but T. gondii does not seem to modulate the effects of AZA via mTORC functions.

Clinical perspective: Antibody-drug conjugates for the treatment of HER2-positive breast cancer.

Antibody-drug conjugates (ADCs) are a promising class of cancer biopharmaceuticals that exploit the specificity of a monoclonal antibody (mAb) to selectively deliver highly cytotoxic small molecules to targeted cancer cells, leading to an enhanced therapeutic index through increased antitumor activity and decreased off-target toxicity. ADCs hold great promise for the treatment of patients with human epidermal growth factor receptor 2 (HER2)-positive breast cancer after the approval and tremendous success of trastuzumab emtansine and trastuzumab deruxtecan, representing a turning point in both HER2-positive breast cancer treatment and ADC technology. Additionally and importantly, a total of 29 ADC candidates are now being investigated in different stages of clinical development for the treatment of HER2-positive breast cancer. The purpose of this review is to provide an insight into the ADC field in cancer treatment and present a comprehensive overview of ADCs approved or under clinical investigation for the treatment of HER2-positive breast cancer.

TGF-ß receptor I inhibitor may restrict the induction of EMT in inflamed intestinal epithelial cells.

Despite the extensive body of research, understanding the exact molecular mechanisms governing inflammatory bowel diseases (IBDs) still demands further investigation. Transforming growth factor-ß1 (TGF-ß1) signaling possesses a multifacial effect on a broad range of context-dependent cellular responses. However, long-term TGF-ß1 activity may trigger epithelial-mesenchymal transition (EMT), followed by fibrosis. This study aimed to determine the role of epithelial TGF-ß1 signaling in inflammatory bowel disease (IBD) pathogenesis. The expression of TGF-ß1 signaling components and EMT-related and epithelial tight junction markers was examined in IBD patients (n = 60) as well as LPS-induced Caco-2/RAW264.7 co-culture model using quantitative real-time polymerase chain reaction (qRT-PCR), Western blotting, and immunofluorescence staining. Furthermore, the effect of A83-01, as a TGF-ß receptor I (TßRI) inhibitor, on the inflamed epithelial cells was evaluated in vitro. To evaluate the cytotoxic effects of the TßRI inhibitor, a cell viability assay was performed by the MTS method. Considering the activation of canonical and non-canonical TGF-ß1 signaling pathways in IBD patients, expression results indicated that administering A83-01 in inflamed Caco-2 cells substantially blocked the expression level of TGF-ß1, SMAD4, and PI3K and the phosphorylation of p-SMAD2/3, p-AKT, and p-RPS6 as well as prevented downregulation of LncGAS5 and LncCDKN2B. Further analysis revealed that the inhibition of TGF-ß1 signaling in inflamed epithelial cells by the small molecule could suppress the EMT-related markers as well as improve the expression of epithelial adherens and tight junctions. Collectively, these findings indicated that the inhibition of the TGF-ß1 signaling could suppress the induction of EMT in inflamed epithelial cells as well as exert a protective effect on preserving tight junction integrity. There is a pressing need to determine the exact cellular mechanisms by which TGF-ß1 exerts its effect on IBD pathogenesis.

Imatinib suppresses activation of hepatic stellate cells by targeting STAT3/IL-6 pathway through miR-124.

The activation of hepatic stellate cells is the primary function of facilitating liver fibrosis. Interfering with the coordinators of different signaling pathways in activated hepatic stellate cells (aHSCs) could be a potential approach in ameliorating liver fibrosis. Regarding the illustrated anti-fibrotic effect of imatinib in liver fibrosis, we investigated the imatinib's potential role in inhibiting HSC activation through miR-124 and its interference with the STAT3/hepatic leukemia factor (HLF)/IL-6 circuit. The anti-fibrotic effect of imatinib was investigated in the LX-2 cell line and carbon tetrachloride (CCl4 )-induced Sprague-Dawley rat. The expression of IL-6, STAT3, HLF, miR-124, and α-smooth muscle actin (α-SMA) were quantified by quantitative real-time PCR (qRT-PCR) and the protein level of α-SMA and STAT3 was measured by western blot analysis both in vitro and in vivo. The LX-2 cells were subjected to immunocytochemistry (ICC) for α-SMA expression. After administering imatinib in the liver fibrosis model, histopathological examinations were done, and hepatic function serum markers were checked. Imatinib administration alleviated mentioned liver fibrosis markers. The expression of miR-124 was downregulated, while IL-6/HLF/STAT3 circuit agents were upregulated in vitro and in vivo. Notably, imatinib intervention decreased the expression of IL-6, STAT3, and HLF. Elevated expression of miR-124 suppressed the expression of STAT3 and further inhibited HSCs activation. Our results demonstrated that imatinib not only ameliorated hepatic fibrosis through tyrosine kinase inhibitor (TKI) activity but also interfered with the miR-124 and STAT3/HLF/IL-6 pathway. Considering the important role of miR-124 in regulating liver fibrosis and HSCs activation, imatinib may exert its anti-fibrotic activity through miR-124.

Effective role of Curcumin on expression regulation of EZH2 histone methyltransferase as a dynamic epigenetic factor in osteogenic differentiation of human mesenchymal stem cells.

BACKGROUND: Efficient differentiation of mesenchymal stem cells (MSCs) into a desired cell lineage remains challenging in cell-based therapy and regenerative medicine. Numerous efforts have been made to efficiently promote differentiation of MSCs into osteoblast lineage. Accordingly, epigenetic signatures emerge as a key conductor of cell differentiation. Among them, Enhancer of Zeste Homolog 2 (EZH2), a histone methyltransferase appears to suppress osteogenesis. Curcumin is an osteoinductive natural polyphenol compound which supposedly modulates epigenetic mechanisms. Hence, the current study aims to address the role of the EZH2 epigenetic factor in osteogenic activity of MSCs after Curcumin treatment. METHODS: The effect of Curcumin on viability and osteogenic differentiation was evaluated at different time points in vitro. The expression level of EZH2 was assessed using quantitative real-time polymerase chain reaction (qRT-PCR) after 14 and 21 days. RESULTS: MTT results showed no cytotoxic effects at concentrations of 10 and 15 µM of Curcumin and cells survived up to 70 % at all time-points. qRT-PCR results demonstrated that Curcumin significantly enhanced the expression levels of osteogenic markers that included Runx2, Osterix, Collagen type I, Osteopontin and Osteocalcin at day 21. CONCLUSIONS: Interestingly, we observed that the expression level of the EZH2 gene was downregulated in the presence of Curcumin compared to the control group during osteogenesis. This study confirmed that Curcumin acts as an epigenetic switch to regulate osteoblast differentiation specifically through the EZH2 suppression.

New insights into extracellular and intracellular redox status in COVID-19 patients.

BACKGROUND: The imbalance of redox homeostasis induces hyper-inflammation in viral infections. In this study, we explored the redox system signature in response to SARS-COV-2 infection and examined the status of these extracellular and intracellular signatures in COVID-19 patients. METHOD: The multi-level network was constructed using multi-level data of oxidative stress-related biological processes, protein-protein interactions, transcription factors, and co-expression coefficients obtained from GSE164805, which included gene expression profiles of peripheral blood mononuclear cells (PBMCs) from COVID-19 patients and healthy controls. Top genes were designated based on the degree and closeness centralities. The expression of high-ranked genes was evaluated in PBMCs and nasopharyngeal (NP) samples of 30 COVID-19 patients and 30 healthy controls. The intracellular levels of GSH and ROS/O2• - and extracellular oxidative stress markers were assayed in PBMCs and plasma samples by flow cytometry and ELISA. ELISA results were applied to construct a classification model using logistic regression to differentiate COVID-19 patients from healthy controls. RESULTS: CAT, NFE2L2, SOD1, SOD2 and CYBB were 5 top genes in the network analysis. The expression of these genes and intracellular levels of ROS/O2• - were increased in PBMCs of COVID-19 patients while the GSH level decreased. The expression of high-ranked genes was lower in NP samples of COVID-19 patients compared to control group. The activity of extracellular enzymes CAT and SOD, and the total oxidant status (TOS) level were increased in plasma samples of COVID-19 patients. Also, the 2-marker panel of CAT and TOS and 3-marker panel showed the best performance. CONCLUSION: SARS-COV-2 disrupts the redox equilibrium in immune cells and the upper respiratory tract, leading to exacerbated inflammation and increased replication and entrance of SARS-COV-2 into host cells. Furthermore, utilizing markers of oxidative stress as a complementary validation to discriminate COVID-19 from healthy controls, seems promising.

Identifying novel host-based diagnostic biomarker panels for COVID-19: a whole-blood/nasopharyngeal transcriptome meta-analysis.

BACKGROUND: Regardless of improvements in controlling the COVID-19 pandemic, the lack of comprehensive insight into SARS-COV-2 pathogenesis is still a sophisticated challenge. In order to deal with this challenge, we utilized advanced bioinformatics and machine learning algorithms to reveal more characteristics of SARS-COV-2 pathogenesis and introduce novel host response-based diagnostic biomarker panels. METHODS: In the present study, eight published RNA-Seq datasets related to whole-blood (WB) and nasopharyngeal (NP) swab samples of patients with COVID-19, other viral and non-viral acute respiratory illnesses (ARIs), and healthy controls (HCs) were integrated. To define COVID-19 meta-signatures, Gene Ontology and pathway enrichment analyses were applied to compare COVID-19 with other similar diseases. Additionally, CIBERSORTx was executed in WB samples to detect the immune cell landscape. Furthermore, the optimum WB- and NP-based diagnostic biomarkers were identified via all the combinations of 3 to 9 selected features and the 2-phases machine learning (ML) method which implemented k-fold cross validation and independent test set validation. RESULTS: The host gene meta-signatures obtained for SARS-COV-2 infection were different in the WB and NP samples. The gene ontology and enrichment results of the WB dataset represented the enhancement in inflammatory host response, cell cycle, and interferon signature in COVID-19 patients. Furthermore, NP samples of COVID-19 in comparison with HC and non-viral ARIs showed the significant upregulation of genes associated with cytokine production and defense response to the virus. In contrast, these pathways in COVID-19 compared to other viral ARIs were strikingly attenuated. Notably, immune cell proportions of WB samples altered in COVID-19 versus HC. Moreover, the optimum WB- and NP-based diagnostic panels after two phases of ML-based validation included 6 and 8 markers with an accuracy of 97% and 88%, respectively. CONCLUSIONS: Based on the distinct gene expression profiles of WB and NP, our results indicated that SARS-COV-2 function is body-site-specific, although according to the common signature in WB and NP COVID-19 samples versus controls, this virus also induces a global and systematic host response to some extent. We also introduced and validated WB- and NP-based diagnostic biomarkers using ML methods which can be applied as a complementary tool to diagnose the COVID-19 infection from non-COVID cases.

Inhibition of epithelial SHH signaling exerts a dual protective effect against inflammation and epithelial-mesenchymal transition in inflammatory bowel disease.

Inflammatory bowel disease (IBD) is a debilitating and incurable inflammatory disorder. Despite its increasing prevalence, the underlying pathogenic mechanisms of IBD have not been fully clarified. In addition to the regulatory role of Sonic Hedgehog (SHH) signaling in the maintenance of gut homeostasis, its involvement in development of inflammatory disorders and organ fibrosis has also been reported. Here, we investigated the role of SHH signaling in IBD and examined the molecular mechanisms targeted by the SHH signaling blockade. In addition to increased inflammatory responses and induced Epithelial-mesenchymal transition (EMT) process, SHH signaling activity also increased in active lesions of IBD patients. These findings were similar to what was observed in the LPS-induced Caco2-RAW264.7 co-culture model. Inhibition of SHH signaling in the intestinal epithelial cells using SHH inhibitors influenced inflammatory responses through decreased expression of inflammatory cytokines. Moreover, treatment of differentiated Caco2 cells with SHH signaling inhibitors prevented the overexpression of EMT markers and downregulation of epithelial adherens and tight junctions in inflammatory conditions. This study demonstrated that the inhibition of SHH signaling by small molecules might have therapeutic benefit in IBD, and provided compelling experimental evidence that SHH signaling inhibitors can impose anti-inflammatory effects in intestinal epithelial cells while preserving their epithelial characteristics by restricting the induction of EMT.