Future Nanotechnology-Based Strategies for Improved Management of Helicobacter pylori Infection.

Helicobacter pylori (H. pylori) is a recalcitrant pathogen, which can cause gastric disorders. During the past decades, polypharmacy-based regimens, such as triple and quadruple therapies have been widely used against H. pylori. However, polyantibiotic therapies can disturb the host gastric/gut microbiota and lead to antibiotic resistance. Thus, simpler but more effective approaches should be developed. Here, some recent advances in nanostructured drug delivery systems to treat H. pylori infection are summarized. Also, for the first time, a drug release paradigm is proposed to prevent H. pylori antibiotic resistance along with an IVIVC model in order to connect the drug release profile with a reduction in bacterial colony counts. Then, local delivery systems including mucoadhesive, mucopenetrating, and cytoadhesive nanobiomaterials are discussed in the battle against H. pylori infection. Afterward, engineered delivery platforms including polymer-coated nanoemulsions and polymer-coated nanoliposomes are poposed. These bioinspired platforms can contain an antimicrobial agent enclosed within smart multifunctional nanoformulations. These bioplatforms can prevent the development of antibiotic resistance, as well as specifically killing H. pylori with no or only slight negative effects on the host gastrointestinal microbiota. Finally, the essential checkpoints that should be passed to confirm the potential effectiveness of anti-H. pylori nanosystems are discussed.

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.

The inhibitory effects of live and UV-killed Akkermansia muciniphila and its derivatives on cytotoxicity and inflammatory response induced by Clostridioides difficile RT001 in vitro.

Clostridioides difficile infection (CDI) is the leading cause of healthcare-acquired infections worldwide. Probiotics are widely recommended to prevent CDI and its recurrences. Akkermansia muciniphila, as a therapeutic symbiont colonizing the intestinal mucosal layer, is considered to be a promising next-generation probiotic. In this work, we assessed the inhibitory effects of A. muciniphila MucT and its derivatives on cytotoxicity and inflammatory response induced by C. difficile RT001 in Caco-2 cells. The results obtained from SEM revealed that the morphology of UV-killed A. muciniphila remained unchanged after UV inactivation. TEM analysis showed that A. muciniphila-isolated extracellular vesicles (EVs) were spherical and ranged from 50 to 200 nm in size. Toxigenic supernatant (Tox-S) of C. difficile RT001 (500 µg/ml) significantly (P <0.01) reduced the cell viability of Caco-2 cells. Caco-2 cells treated with live (MOI 10), UV-killed (MOI 10), cell-free supernatant (CFS, 106 cfu/ml), and EVs (20 µg/ml) of A. muciniphila exhibited over 90% viability in comparison to untreated control. The neutralized CFS preparation using A. muciniphila and its derivatives could notably reduce the expression level of inflammatory markers. Additionally, A. muciniphila and its derivatives modulated the production of IL-1ß, TNF-α, and IL-10 in Tox-S stimulated Caco-2 cells. We demonstrated that A. muciniphila and its derivatives can modulate changes in the gut barrier-related genes and inflammatory response caused by C. difficile Tox-S in Caco-2 cells.

Alterations in the gut microbiota and their metabolites in human intestinal epithelial cells of patients with colorectal cancer.

BACKGROUND: The gut microbiota has become one of the main risk factors for the formation and development of colorectal cancer (CRC). CRC intensification may be due to the microbial pathogens' colonization and their released metabolites. Here, we analyzed Bacteroidetes and Clostridia bacteria in CRC patients and studied bacterial metabolome in cancerous tissues compared to their adjacent normal tissues. METHODS AND RESULTS: The population of selected bacteria in biopsy specimens of 30 patients with CRC was studied by RT-qPCR. The mutagenicity and cytotoxicity effects of microbiota metabolites were evaluated by Ames test and MTT Assay, respectively. Moreover, gene expression in carcinogenic pathways was studied by RT-qPCR, and genes with different expressions in tumor and non-tumor tissues were diagnosed. Based on microbiota analysis, the relative abundance of Clostridia and C. difficile was significantly higher in CRC tissue, whereas C. perfringens showed higher relative abundance in normal tissue. AIMES test confirmed the proliferation and mutagenicity effects of the bacterial metabolites in CRC patients. Significant upregulation of C-Myc, GRB2, IL-8, EGFR, PI3K, and AKT and downregulation of ATM were observed in CRC samples compared to the control. CONCLUSIONS: The influence of bacterial metabolites on inflammation and altered expression of genes in the cell signaling pathways was observed. The findings confirm the role gut microbiota composition and bacterial metabolites as key players in CRC onset and development.

Outer Membrane Vesicles Derived from Adherent-Invasive Escherichia coli Induce Inflammatory Response and Alter the Gene Expression of Junction-Associated Proteins in Human Intestinal Epithelial Cells.

Adherent-invasive Escherichia coli (AIEC) pathobionts, which are characterized by their ability to adhere to and invade intestinal epithelial cells, are associated with the etiopathogenesis of inflammatory bowel diseases (IBDs). Outer membrane vesicles (OMVs) released by AIEC strains can facilitate the interaction of these bacteria with host cells through delivering bacterial effectors. The aim of this study was to determine the ability of OMVs derived from AIEC strain LF82 to induce the host immune response, leading to production of proinflammatory cytokines and also altering the gene expression of junction-associated proteins in the human epithelial colorectal adenocarcinoma Caco-2 cell line. OMVs were extracted from AIEC strain LF82, and the cell viability of Caco-2 cells treated with these vesicles was assessed by MTT assay. The morphology and size distribution of vesicles were analyzed using transmission electron microscopy and dynamic light scattering, respectively. Gene expression of occludin, ZO-1, claudin-2, E-cadherin, TLR-2, and TLR-4 in response to OMVs was assessed in Caco-2 cells by RT-qPCR. Moreover, the secretion of IL-8 and TNF-α into the supernatant of Caco-2 cells upon treatment with OMVs was measured using ELISA. Our results demonstrated that OMVs upregulated the gene expression level of TLRs and also altered the gene expression level of junction-associated proteins. OMVs derived from AIEC may play a major role in the promotion of intestinal inflammation and epithelial barrier dysfunction. However, further investigations are needed to elucidate the putative role of OMVs in the pathogenesis of AIEC and IBD.

Characterisation of extracellular vesicles isolated from hydatid cyst fluid and evaluation of immunomodulatory effects on human monocytes.

Hydatidosis is a disease caused by the larval stage of Echinococcus granulosus, which involves several organs of intermediate hosts. Evidence suggests a communication between hydatid cyst (HC) and hosts via extracellular vesicles. However, a little is known about the communication between EVs derived from HC fluid (HCF) and host cells. In the current study, EVs were isolated using differential centrifugation from sheep HCF and characterized by western blot, electron microscope and size distribution analysis. The uptake of EVs by human monocyte cell line (THP-1) was evaluated. The effects of EVs on the expression levels of pro- and anti-inflammatory cytokines were investigated using quantitative real-time PCR (RT-PCR), 3 and 24 h after incubation. Moreover, the cytokine level of IL-10 was evaluated in supernatant of THP-1 cell line at 3 and 24 h. EVs were successfully isolated and showed spherical shape with size distribution at 130.6 nm. After 3 h, the expression levels of pro-inflammatory cytokine genes (IL1Β, IL15 and IL8) were upregulated, while after 24 h, the expression levels of pro-inflammatory cytokines were decreased and IL13 gene expression showed upregulation. A statistically significant increase was seen in the levels of IL-10 after 24 h. The main mechanism of the communication between EVs derived from HCF and their host remains unclear; however, time-dependent anti-inflammatory effects in our study suggest that HC may modulate the immune responses via EVs.

Toxoplasma gondii profilin induces NLRP3 activation and IL-1ß production/secretion in THP-1 cells.

Toxoplasma gondii is a highly prevalent protozoan that infects a broad spectrum of warm-blooded animals. Profilin is a critical protein that plays a role in the movement and invasion of T. gondii. In the current study, we assessed how profilin stimulates inflammasomes and how it induces transcription and secretion of IL-1ß. For this purpose, we assessed the level of TLR 2, 4, 5, and 9 expressions in a THP-1 cell line treated with profilin from T. gondii (TgP). In addition, we analyzed the expression levels of various inflammasomes, as well as IL-1ß, and IL-18 in THP-1 cells treated with the NLRP3 inhibitor MCC950. TgP significantly increased the expression of TLR5 but the expression of TLR2, 4, and 9 was not significantly increased. In addition, TgP did not significantly increase the level of inflammasomes after 5 h. Treatment with MCC950 significantly reduced NLRP3 and IL-1ß on both transcription and protein levels. Although the transcription level of NLRP3 was reduced 5 h after treatment with TgP, western blot analysis showed an increase in NLRP3. The western blot and ELISA analysis also showed that TgP increased both pro- and mature IL-1ß. In summary, our study showed that NLRP3 most probably plays a pivotal role in the expression and production levels of IL-1ß during the interaction between TgP and macrophages.

Clostridioides difficile PCR ribotypes 001 and 084 can trigger autophagy process in human intestinal Caco-2 cells.

Autophagy is a homeostatic process that can promote cell survival or death. However, the exact role of autophagy in Clostridioides difficile infection (CDI) is still not precisely elucidated. Here, we investigate the role of distinct C. difficile ribotypes (RTs) in autophagy induction using Caco-2 cells. The expression analysis of autophagy-associated genes and related miRNAs were examined following treatment of Caco-2 cells with C. difficile after 4 and 8 h using RT-qPCR. Toxin production was assessed using enzyme-linked immunosorbent assay (ELISA). Immunofluorescence analysis was performed to detect MAP1LC3B/LC3B, followed by an autophagic flux analysis. C. difficile significantly reduced the viability of Caco-2 cells in comparison with untreated cells. Elevated levels of LC3-II and SQSTM1/p62 by C. difficile RT001 and RT084 in the presence of E64d/leupeptin confirmed the induction of autophagy activity. Similarly, the immunofluorescence analysis demonstrated that C. difficile RT001 and RT084 significantly increased the amount of LC3-positive structures in Caco-2 cells. The induction of autophagy was further demonstrated by increased levels of LC3B, ULK1, ATG12, PIK3C3/VPS34, BECN1 (beclin 1), ATG5, and ATG16L1 transcripts and reduced levels of AKT and MTOR gene expression. The expression levels of MIR21 and MIR30B, microRNAs that suppress autophagy, were differentially affected by C. difficile. In conclusion, the present work revealed that C. difficile bacteria can induce autophagy through both toxin-dependent and -independent mechanisms. Also, our results suggest the potential role of other C. difficile virulence factors in autophagy modulation using intestinal cells in vitro.

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.

Evaluation of gut microbiota of iranian patients with celiac disease, non-celiac wheat sensitivity, and irritable bowel syndrome: are there any similarities?

BACKGROUND AND AIMS: Individuals with celiac disease (CD), non-celiac wheat sensitivity (NCWS), and irritable bowel syndrome (IBS), show overlapping clinical symptoms and experience gut dysbiosis. A limited number of studies so far compared the gut microbiota among these intestinal conditions. This study aimed to investigate the similarities in the gut microbiota among patients with CD, NCWS, and IBS in comparison to healthy controls (HC). MATERIALS AND METHODS: In this prospective study, in total 72 adult subjects, including CD (n = 15), NCWS (n = 12), IBS (n = 30), and HC (n = 15) were recruited. Fecal samples were collected from each individual. A quantitative real-time PCR (qPCR) test using 16S ribosomal RNA was conducted on stool samples to assess the relative abundance of Firmicutes, Bacteroidetes, Bifidobacterium spp., and Lactobacillus spp. RESULTS: In all groups, Firmicutes and Lactobacillus spp. had the highest and lowest relative abundance respectively. The phylum Firmicutes had a higher relative abundance in CD patients than other groups. On the other hand, the phylum Bacteroidetes had the highest relative abundance among healthy subjects but the lowest in patients with NCWS. The relative abundance of Bifidobacterium spp. was lower in subjects with CD (P = 0.035) and IBS (P = 0.001) compared to the HCs. Also, the alteration of Firmicutes to Bacteroidetes ratio (F/B ratio) was statistically significant in NCWS and CD patients compared to the HCs (P = 0.05). CONCLUSION: The principal coordinate analysis (PCoA), as a powerful multivariate analysis, suggested that the investigated gut microbial profile of patients with IBS and NCWS share more similarities to the HCs. In contrast, patients with CD had the most dissimilarity compared to the other groups in the context of the studied gut microbiota.

Characterization of the mucosal microbiota in patients with nodular lymphoid hyperplasia with concurrent irritable bowel syndrome compared to healthy controls.

BACKGROUND: Nodular lymphoid hyperplasia (NLH) is known as a lymphoproliferative lesion in which multiple small nodules appear on the intestinal wall. It has been documented that patients who struggle with irritable bowel syndrome (IBS) are at greater risk of developing NLH. Here, we aimed to investigate the previously reported pathogens and the abundance of a selection of mucosal microbiota in IBS + NLH patients compared to IBS, and healthy controls. METHODS AND RESULTS: Terminal ileum biopsies were collected from 37 IBS + NLH, 37 IBS, and 29 healthy controls. Bacterial culture and PCR was performed to detect the presence of pathogens in biopsies. A qPCR assay was applied to assess the abundance of a selection of bacterial taxa. Totally, five bacterial isolates including two enteropathogenic and one enteroaggregative Escherichia coli (EPEC, EAEC), one enterotoxigenic Staphylococcus aureus (SEA), and one Yersinia enterocolitica strains were detected among the IBS + NLH cases. The relative abundance of Bacteroidetes and Streptococcus spp. in IBS + NLH patients was significantly less than IBS and healthy controls. Firmicutes, Pseudomonas spp., Haemophilus spp., and Campylobacter spp. were notably more abundant in IBS + NLH than in IBS patients. The abundance of Verrucomicrobia was higher in NLH + IBS than in healthy controls. Actinobacteria was also significantly more abundant among NLH + IBS patients than the controls. CONCLUSION: Our results demonstrated that mucosal microbiota composition in NLH + IBS patients slightly differs from that of IBS patients and healthy controls. Further research using large-scale cohorts are needed to enhance current understanding of the contribution of the mucosal microbiota to NLH pathogenesis with concurrent IBS.

The role of mir-197-3p in regulating the tight junction permeability of celiac disease patients under gluten free diet.

BACKGROUND: Celiac disease (CD) is a hereditary immune-mediated disorder, which is along with the enormous production of pro-inflammatory cytokines and the reduced level of tight junction proteins. The aim of this study was to determine the expression of TNF-α, IFN-γ, IL-18, Occludin, miR-122-5p and miR-197-3p genes in duodenal biopsies of treated CD patients in comparison to the controls. METHODS AND RESULTS: Biopsy specimens were taken from the duodenum of 50 treated CD patients (36 (72%) females and 14 (28%) males with mean age of 37.06 ± 7.02 years) and 50 healthy controls (17 (34%) females and 33 (66%) males with mean age of 34.12 ± 4.9). Total RNA was isolated, cDNA was synthesized and mRNA expression of TNF-α, IFN-γ, IL-18, Occludin, miR-122-5p and miR-197-3p were quantified by relative qPCR using B2M and U6 as internal control genes. All data were evaluated using SPSS (V.21) and GraphPad Prism (V.5). Our results showed that there was no significant difference between patients and controls for intestinal mRNA expression of TNF-α, IFN-γ, IL-18, Occludin, and miR-122-5p (p > 0.05) and the expression of miR-197-3p was significantly increased in CD patients relative to control subjects (p = 0.049). CONCLUSION: This study suggests that adherence to GFD may have a positive effect on the tight junction (TJ) permeability and in this process, miR-197-3p plays an important role. Increased expression of miR-197-3p with a final protective effect on Occludin expression can be further studied as a complement therapeutic target for Celiac disease.

Regulation of the Th17/Treg balance by human umbilical cord mesenchymal stem cell-derived exosomes protects against acute experimental colitis.

Increasing evidence suggests that mesenchymal stem cells (MSCs) have immunosuppressive properties mediated by MSC-derived small extracellular vesicles (sEV). Exosomes are small extracellular vesicles that contain components that regulate immune cell function. We investigated the immunomodulatory effects of MSC-derived Exosome (MSC-Exo) on the severity of colitis using the dextran sulfate sodium (DSS)-induced colitis model. Exosomes were administrated intraperitoneally. Daily changes in body weight, stool consistency, and bleeding were assessed to determine the impact of MSC-Exos on colitis. Several measurements were taken, including the colon weight, length, and histological analysis of the colon tissues. The percentage of regulatory T cells and IL-10, TGF-ß, IL-17, TNF-α, and IFN-γ levels were calculated in the mesenteric lymph node (MLN) and spleen. The results showed MSC-Exos improved clinical manifestations of colitis. Colon macroscopic and histological observations also showed improvement in tissue destruction. The results illustrated that MSC-Exos might attenuate colitis by regulating Treg/Th17 balance, increasing anti-inflammatory, and decreasing pro-inflammatory cytokines expression. As a result, MSC-Exos could be used as an immunomodulatory approach to treating bowel inflammation.

Identification of antisense and sense RNAs of intracrine fibroblast growth factor components as novel biomarkers in colorectal cancer and in silico studies for drug and nanodrug repurposing.

INTRODUCTION: Colorectal cancer (CRC) is one of the most malignant tumors and in which various efforts for screening is inconclusive.The intracrine FGF panel, the non-tyrosine kinase receptors (NTKR) FGFs and affiliated antisenses play a pivotal role in FGF signaling.The expression levels of coding and non-coding intracrine FGFs were assessed in CRC donors.Also, substantial costs and slow pace of drug discovery give high attraction to repurpose of previously discovered drugs to new opportunities. OBJECTIVES: The aim of present study was to evaluate the potential role of the coding and non-coding intracrine FGFs as a new biomarkers for CRC cases and defining drug repurposing to alleviate FGF down regulation. METHODS: RNA-seq data of colon adenocarcinomas (COAD) was downloaded using TCGA biolinks package in R.The DrugBank database (https://go.drugbank.com/) was used to extract interactions between drugs and candidate genes. A total of 200 CRC patients with detailed criteria were enrolled.RNAs were extracted with TRIzol-based protocol and amplified via LightCycler® instrument.FGF11 and FGF13 proteins validation was performed by used of immunohistochemistry technique in tumor and non-tumoral samples.Pearson's correlation analysis and ROC curve plotted by Prism 8.0 software. RESULTS: RNA-seq data from TCGA was analyzed by normalizing with edgeR.Differentially expressed gene (DEG) analysis was generated. WCC algorithm extracted the most significant genes with a total of 47 genes. Expression elevation of iFGF antisenses (12AS,13As,14AS) compared with the normal colon tissue were observed (P = 0.0003,P = 0.042,P = 0.026, respectively). Moreover,a significant decrease in expression of the corresponding sense iFGF genes was detected (P < 0.0001).Plotted receiver operating characteristic (ROC) curves for iFGF components' expression showed an area of over 0.70 (FGF11-13: 0.71% and FGF12-14: 0.78%, P < 0.001) for sense mRNA expression, with the highest sensitivity for FGF12 (92.8%) and lowest for FGF11 (61.41%).The artificial intelligence (AI) revealed the valproic acid as a repurposing drug to relief the down regulation of FGF12 and 13 in CRC patients. CONCLUSION: Intracrine FGFs panel was down regulated versus up regulation of dependent antisenses. Thus, developing novel biomarkers based on iFGF can be considered as a promising strategy for CRC screening.In advanced, valporic acid detected by AI as a repurposing drug which may be applied in clinical trials for CRC treatment.

The PI3K/Akt/mTOR axis in colorectal cancer: Oncogenic alterations, non-coding RNAs, therapeutic opportunities, and the emerging role of nanoparticles.

Colorectal cancer (CRC) is one of the deadliest human malignancies worldwide. Several molecular pathways have been demonstrated to be involved in the initiation and development of CRC which among them, the overactivation of the phosphatidyl-inositol 3-kinase (PI3K)/Akt/mTOR axis is of importance. The current review aims to unravel the mechanisms by which the PI3K/Akt/mTOR pathway affects CRC progression; and also, to summarize the original data obtained from international research laboratories on the oncogenic alterations and polymorphisms affecting this pathway in CRC. Besides, we provide a special focus on the regulatory role of noncoding RNAs targeting the PI3K/Akt/mTOR pathway in this malignancy. Questions on how this axis is involved in the inhibition of apoptosis, in the induction of drug resistance, and the angiogenesis, epithelial to mesenchymal transition, and metastasis are also responded. We also discussed the PI3K/Akt pathway-associated prognostic and predictive biomarkers in CRC. In addition, we provide a general overview of PI3K/Akt/mTOR pathway inhibition whether by chemical-based drugs or by natural-based medications in the context of CRC, either as monotherapy or in combination with other therapeutic agents; however, those treatments might have life-threatening side effects and toxicities. To the best of our knowledge, the current review is one of the first ones highlighting the emerging roles of nanotechnology to overcome challenges related to CRC therapy in the hope that providing a promising platform for the treatment of CRC.

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.

Modulation of microRNAs and claudin-7 in Caco-2 cell line treated with Blastocystis sp., subtype 3 soluble total antigen.

BACKGROUND: Blastocystis sp., is a eukaryote of the large intestine, which is reported from almost all countries. The pathogenesis of this protist is not clear. The current study aimed to analyze the effects of Blastocystis sp., ST3 soluble total antigen (B3STA) on the microRNAs (miRNAs) involved in the gut permeability and also pro-inflammatory cytokines, occludin, and claudin-7. METHODS: Blastocystis sp., ST3 isolated from stool sample was purified, and its soluble total antigen was extracted using freeze and thawing. The Caco-2 cell line was treated with B3STA for 24 h and the expression levels of mir-16, mir-21, mir-29a, mir-223, and mir-874 were analyzed. In addition, the expression levels of il-8, il-15, occludin, and claudin-7 genes were assessed. RESULTS: B3STA significantly upregulated the expression of mir-223, and mir-874, and downregulated mir-29a. The expression of mir-16 and mir-21 was not significant. In addition, the expression of il-8 and il-15 was not significant. B3STA significantly decreased the expression level of claudin-7 (P-value < 0.0001), but the expression of occludin was not significant. Our results showed significant correlation between all studied miRNAs, except mir-29a, with downregulation of claudin-7. CONCLUSIONS: This is the first study investigating the effects of Blastocystis sp., ST3 isolated from symptomatic subjects on the expression levels of miRNAs involved in the gut permeability. Our results demonstrated that B3STA may change miRNA expression, which are involved in the gut barrier integrity, and downregulates claudin-7, which is known as sealing factor.

Surface layer protein A from hypervirulent Clostridioides difficile ribotypes induce significant changes in the gene expression of tight junctions and inflammatory response in human intestinal epithelial cells.

BACKGROUND: Surface layer protein A (SlpA), the primary outermost structure of Clostridioides difficile, plays an essential role in C. difficile pathogenesis, although its interaction with host intestinal cells are yet to be understood. The aim of this study was to investigate the effects of SlpA extracted from C. difficile on tight junction (TJ) proteins expression and induction of pro-inflammatory cytokines in human colon carcinoma cell line HT-29. SlpA was extracted from three toxigenic C. difficile clinical strains including RT126, RT001, RT084 as well as C. difficile ATCC 700057 as non-toxigenic strain. Cell viability was performed by MTT assay, and the mRNA expression of TJ proteins and inflammation-associated genes was determined using quantitative RT-PCR. Additionally, the secretion of IL-8, IL-1ß and TNF-α cytokines was measured by ELISA. RESULTS: C. difficile SlpA from selected RTs variably downregulated the expression level of TJs-assassinated genes and increased the expression level of TLR-4 and pro-inflammatory cytokines in HT-29 treated cells. SlpA from RT126 significantly (padj<0.05) decreased the gene expression level of claudins family and JAM-A and increased the secretion of IL-8, TNF-α and IL1-ß as compared to untreated cells. Moreover, only SlpA from RT001 could significantly induce the expression of IL-6 (padj<0.05). CONCLUSION: The results of the present study highlighted the importance of SlpA in the pathogenesis of CDI and C. difficile-induced inflammatory response in the gut. Further studies are required to unravel the significance of the observed results in promoting the intestinal inflammation and immune response induced by C. difficile SlpA from different RTs.

Surface layer protein A from hypervirulent Clostridioides difficile ribotype 001 can induce autophagy process in human intestinal epithelial cells.

Clostridioides difficile is the leading cause of nosocomial diarrhea with high morbidity and mortality worldwide. C. difficile strains produce a crystalline surface layer protein A (SlpA), which is an absolute necessity for its pathogenesis. However, its pathogenic mechanisms and its pro-inflammatory behavior are not yet fully elucidated. Herein, we report for the first time that SlpA extracted from C. difficile can induce autophagy process in Caco-2 cells. SlpA protein was purified from two C. difficile strains (RT001 and ATCC 700075). The cell viability of Caco-2 cells after exposure with different concentrations (15, 20, 25 µg/mL) of SlpA at various time points (3, 6, 12, 24 h) was measured by MTT assay. Acridine orange staining was used to visualize the hypothetical acidic vesicular organelles. The gene expression of autophagy mediators including LC3B, Atg5, Atg16L, and Beclin-1 was determined by quantitative real-time PCR assay. Western blotting assay was used to detect the expression of LC3B protein. MTT assay showed that different concentrations of SlpA did not induce significant changes in the viability of Caco-2 cells. SlpA at concentration of 20 µg/mL enhanced the formation of acidic vesicular organelles in Caco-2 cells after 12 h of exposure. Moreover, SlpA treatment significantly increased the expression of autophagy-associated genes, and increased the expression of LC3B protein in Caco-2 cells. In conclusion, our study demonstrated that SlpA is capable to induce autophagy in intestinal epithelial cells. These findings reveal a novel mechanism for the pathogenesis of C. difficile mediated by its SLPs.

Dietary and Lifestyle Inflammation Scores Are Inversely Associated with Metabolic-Associated Fatty Liver Disease among Iranian Adults: A Nested Case-Control Study.

BACKGROUND: Diet and lifestyle may affect risk for metabolic-associated fatty liver disease (MAFLD) by chronically elevating systemic inflammation. OBJECTIVES: In this study we investigated the separate and joint associations of dietary and lifestyle inflammation scores (DIS and LIS, respectively) with MAFLD risk. METHODS: For this nested case-control study we identified and recruited 968 patients with MAFLD (defined as having a fatty liver index ≥60 plus ≥1 of the following conditions: overweight or obese, type II diabetes mellitus, evidence of metabolic dysregulation) and 964 controls from among 35-70-y-old men and women in the baseline phase of the Sabzevar Persian Cohort Study. We collected demographic, lifestyle, anthropometric, biochemical, and dietary intake information (via a validated FFQ) from which we calculated a circulating inflammation biomarker-weighted, predominantly whole foods and beverages-based, 19-component DIS and a 3-component LIS. We estimated DIS- and LIS-MAFLD associations using multivariable unconditional logistic regression. We also calculated equal-weight DIS and LIS to capture all potential mechanisms (inflammation plus other mechanisms) for associations of diet and lifestyle with MAFLD risk. RESULTS: Among those in the highest relative to the lowest DIS and LIS tertiles, the multivariable-adjusted ORs and their 95% CIs were OR: 1.84; 95% CI: 1.61, 2.07; Ptrend < 0.001, and OR: 1.96; 95% CI: 1.69, 2.21; Ptrend < 0.001, respectively. For those in the highest relative to the lowest joint DIS and LIS tertile, the values were OR: 2.56; 95% CI: 2.19, 2.93; Pinteraction < 0.001. The findings were similar by sex. The third tertile values for the equal-weight DIS- and LIS-MAFLD associations were OR: 1.87; 95% CI: 1.41, 2.34; and OR: 2.16; 95% CI: 1.85, 2.46, respectively. CONCLUSIONS: Our results suggest that higher balances of pro- relative to anti-inflammatory dietary and lifestyle exposures, separately and especially jointly, may be associated with higher MAFLD risk among adults. Also, inflammation may be the primary mechanism through which diet affects MAFLD risk.