miRNAs: possible regulators of toll like receptors and inflammatory tumor microenvironment in colorectal cancer.

BACKGROUND: Colorectal cancer (CRC) is ranked as the third most commonly diagnosed cancer and the third cause of cancer related deaths. CRC is greatly attributed to genetic and epigenetic mutations and immune dysregulation. Tumor aberrant expression of Toll-like Receptors (TLRs) can contribute to tumorigenesis. Recent studies suggested that microRNAs act as direct ligands of TLRs altering their expression and signaling pathways. AIM: To prove our concept that specific miRNA mimics may act as antagonists of their specific toll like receptors inhibiting their expression that could limit the release of pro-inflammatory and pro-tumorigenic cytokines leading to apoptosis of tumor cells. METHODS: From public microarray databases, we retrieved TLRs and miRNAs related to CRC followed by in silico docking of the selected miRNA ligands into the TLRs. Clinical validation after co-immunoprecipitation of TLRs and their interacting miRNA ligands was done. Expression of TLRs 1, 7,8 was determined by ELISA while miRNAs was measured by RT-qPCR. In addition, microRNA mimics of the down regulated miRNAs were transfected into human CRC cell lines. RESULTS: Our data demonstrate that TLRs 1, 7, 8 are up regulated in CRC compared to controls. Further, three miRNAs (-122, -29b and -15b) are relatively downregulated, while 4 miRNAs (-202, miRNA-98, -21 and -let7i) are upregulated in CRC patients compared to those with benign tumor and healthy controls. Transfection of down regulated miRNA mimics into CRC cell lines resulted in a significant reduction of the number and viability of cells as well as down regulating the expression of TLRs 1, 7 and 8 with ultimate reduction of downstream effector IL6 protein, suggesting that these miRNAs are negative regulators of carcinogenesis. CONCLUSION: MicroRNAs could act as antagonistic ligands of TLRs limiting the inflammatory tumor microenvironment.

Virtual versus paper-based PBL in a pulmonology course for medical undergraduates.

BACKGROUND: Problem-based learning (PBL) remains a valid and effective tool for small-group medical education. Using Virtual patients (VP) case simulation in PBL is a recognizable educational method that has successfully prepared students to focus learning on core information that uses realistic patient-based cases relating to everyday clinical scenarios. Using other modalities as the virtual patient in PBL instead of the paper-based methods remains debatable. This study aimed to evaluate the effectiveness of using VP case simulation mannequin in PBL versus the PBL in paper-based cases in improving the cognitive skills by comparing the grades of a multiple-choice question test and assess its ability to reach students' satisfaction using questionnaire with Likert survey instrument. METHODS: The study was conducted on 459 fourth-year medical students studying in the pulmonology module of the internal medicine course, Faculty of Medicine, October 6 University. All students were divided into 16 PBL classes and randomly divided into groups A and B by simple manual randomization. The groups were parallel with a controlled cross-over study between paper-based and virtual patient PBL. RESULTS: The pre-test showed no significant difference between both, while post-test scores were significantly higher in both VP PBL cases 1 discussing COPD (6.25 ± 0.875) and case 2 discussing pneumonia (6.56 ± 1.396) compared to paper-based PBL (5.29 ± 1.166, 5.57 ± SD1.388, respectively) at p < 0.1 When students in Group A experienced PBL using VP in case 2 after paper-based PBL in case 1, their post-test score improved significantly. (from 5.26 to 6.56, p < .01). Meanwhile, there was a significant regression in the post-test score of the students in Group B when they experienced the paper-based PBL session in case 2 after using PBL using VP in case 1, (from 6.26 to 5.57, p < .01). Most of the students recommended using VP in PBL as they found VP was more engaging and inducing concentration in gathering the information needed to characterize the patient's problem than in a classroom- paper-based cases session. They also enjoyed the teaching of the instructor and found it a suitable learning style for them. CONCLUSION: Implementing virtual patients in PBL increased knowledge acquisition and understanding in medical students and was more motivating for students than paper based PBL to gather the needed information.

Modulation of hepatic stellate cells by Mutaflor® probiotic in non-alcoholic fatty liver disease management.

BACKGROUND: NAFLD and NASH are emerging as primary causes of chronic liver disease, indicating a need for an effective treatment. Mutaflor® probiotic, a microbial treatment of interest, was effective in sustaining remission in ulcerative colitis patients. OBJECTIVE: To construct a genetic-epigenetic network linked to HSC signaling as a modulator of NAFLD/NASH pathogenesis, then assess the effects of Mutaflor® on this network. METHODS: First, in silico analysis was used to construct a genetic-epigenetic network linked to HSC signaling. Second, an investigation using rats, including HFHSD induced NASH and Mutaflor® treated animals, was designed. Experimental procedures included biochemical and histopathologic analysis of rat blood and liver samples. At the molecular level, the expression of genetic (FOXA2, TEAD2, and LATS2 mRNAs) and epigenetic (miR-650, RPARP AS-1 LncRNA) network was measured by real-time PCR. PCR results were validated with immunohistochemistry (α-SMA and LATS2). Target effector proteins, IL-6 and TGF-ß, were estimated by ELISA. RESULTS: Mutaflor® administration minimized biochemical and histopathologic alterations caused by NAFLD/NASH. HSC activation and expression of profibrogenic IL-6 and TGF-ß effector proteins were reduced via inhibition of hedgehog and hippo pathways. Pathways may have been inhibited through upregulation of RPARP AS-1 LncRNA which in turn downregulated the expression of miR-650, FOXA2 mRNA and TEAD2 mRNA and upregulated LATS2 mRNA expression. CONCLUSION: Mutaflor® may slow the progression of NAFLD/NASH by modulating a genetic-epigenetic network linked to HSC signaling. The probiotic may be a useful modality for the prevention and treatment of NAFLD/NASH.

mRNA-miRNA-lncRNA Regulatory Network in Nonalcoholic Fatty Liver Disease.

AIM: we aimed to construct a bioinformatics-based co-regulatory network of mRNAs and non coding RNAs (ncRNAs), which is implicated in the pathogenesis of non-alcoholic fatty liver disease (NAFLD), followed by its validation in a NAFLD animal model. MATERIALS AND METHODS: The mRNAs-miRNAs-lncRNAs regulatory network involved in NAFLD was retrieved and constructed utilizing bioinformatics tools. Then, we validated this network using an NAFLD animal model, high sucrose and high fat diet (HSHF)-fed rats. Finally, the expression level of the network players was assessed in the liver tissues using reverse transcriptase real-time polymerase chain reaction. RESULTS: in-silico constructed network revealed six mRNAs (YAP1, FOXA2, AMOTL2, TEAD2, SMAD4 and NF2), two miRNAs (miR-650 and miR-1205), and two lncRNAs (RPARP-AS1 and SRD5A3-AS1) that play important roles as a co-regulatory network in NAFLD pathogenesis. Moreover, the expression level of these constructed network-players was significantly different between NAFLD and normal control. Conclusion and future perspectives: this study provides new insight into the molecular mechanism of NAFLD pathogenesis and valuable clues for the potential use of the constructed RNA network in effective diagnostic or management strategies of NAFLD.

Identification of Novel Insulin Resistance Related ceRNA Network in T2DM and Its Potential Editing by CRISPR/Cas9.

BACKGROUND: Type 2 diabetes mellitus is one of the leading causes of morbidity and mortality worldwide and is derived from an accumulation of genetic and epigenetic changes. In this study, we aimed to construct Insilco, a competing endogenous RNA (ceRNA) network linked to the pathogenesis of insulin resistance followed by its experimental validation in patients', matched control and cell line samples, as well as to evaluate the efficacy of CRISPR/Cas9 as a potential therapeutic strategy to modulate the expression of this deregulated network. By applying bioinformatics tools through a two-step process, we identified and verified a ceRNA network panel of mRNAs, miRNAs and lncRNA related to insulin resistance, Then validated the expression in clinical samples (123 patients and 106 controls) and some of matched cell line samples using real time PCR. Next, two guide RNAs were designed to target the sequence flanking LncRNA/miRNAs interaction by CRISPER/Cas9 in cell culture. Gene editing tool efficacy was assessed by measuring the network downstream proteins GLUT4 and mTOR via immunofluorescence. RESULTS: LncRNA-RP11-773H22.4, together with RET, IGF1R and mTOR mRNAs, showed significant upregulation in T2DM compared with matched controls, while miRNA (i.e., miR-3163 and miR-1) and mRNA (i.e., GLUT4 and AKT2) expression displayed marked downregulation in diabetic samples. CRISPR/Cas9 successfully knocked out LncRNA-RP11-773H22.4, as evidenced by the reversal of the gene expression of the identified network at RNA and protein levels to the normal expression pattern after gene editing. CONCLUSIONS: The present study provides the significance of this ceRNA based network and its related target genes panel both in the pathogenesis of insulin resistance and as a therapeutic target for gene editing in T2DM.

Determination of certain urinary microRNAs as promising biomarkers in diabetic nephropathy patients using gold nanoparticles.

Diabetic nephropathy (DN) is a major leading cause of kidney failure. So, early detection of DN by assessing urinary microRNAs (miRNAs) expression may be of clinical value. In this study, the diagnostic value of two urinary miRNAs (miR-210 & miR-34a) as biomarkers for diagnosis of DN was assessed using a simple colorimetric gold nanoparticle (AuNP) assay and real-time PCR. MiR-(210 & 34a) were markedly up-regulated in DN groups (micro-albuminuric and macro-albuminuric groups) compared to the non-albuminuric group and healthy controls. The sensitivity and specificity for the qualitative detection of urinary miR-(210 & 34a) using the AuNP assay were (78% and 72%) & (81% and 69%), respectively, which were consistent with the results of real-time PCR. There was a highly significant correlation between urinary miR-(210 & 34a) detected by either qRT-PCR or qualitative AuNP assay. Accordingly, this simple AuNP assay may be considered a valid test for the detection of these two urinary miRNAs as potential biomarkers that can aid in the noninvasive diagnosis of DN.

Diabetic Nephropathy Assessment: Microtubule-Associated Protein 1 Light-Chain 3B a New Promising Biomarker.

Autophagy is a highly conserved pathway. Impairment of autophagy is implicated in the pathogenesis of diabetic nephropathy. The current study applied a bioinformatics analysis to retrieve promising autophagy biomarker relevant diabetic nephropathy. Urinary expression of Microtubule-associated protein 1 light-chain 3B (LC3B) RNA was assessed. Urine samples of 86 type II diabetic kidney disease Egyptian patients (albuminuria group) were provided to quantify urinary expression of LC3B. A group of 30 healthy volunteers were also enrolled in addition to non-albuminuria group including 44 patients. Our study revealed a cut-off value for urinary LC3B expression level that was calculated by receiver-operating characteristic curve as 0.866. Sensitivity and specificity of LC3B were 83.7 and 78.4% respectively. The positivity rate of urinary LC3B expression level was significantly lower in diabetic nephropathy patients than control group. LC3B has great clinical value as promising biomarker in diabetic nephropathy assessment.

Caffeic Acid Modulates miR-636 Expression in Diabetic Nephropathy Rats.

We investigated the action of caffeic acid in regulating miR-636 expression level in kidney of streptozotocin-induced diabetic rats. Streptozotocin-induced diabetic rats were orally treated with caffeic acid at 40 mg/kg/day for 8 weeks. At the end of the treatment, body and kidney weight and blood glucose levels were determined, blood, urine, and kidneys were collected for biochemical and histological examination. Expression levels of miR-636 were determined in liver by qRT-PCR. Induction of diabetic nephropathy by streptozotocin was evidenced by displayed elevated levels of serum creatinine, blood urea nitrogen, microalbuminuria and urinary albumin/creatinine ratio in addition to renal hypotrophy. Caffeic acid (CA) can ameliorate renal damage and significantly decreased the fasting blood glucose, cholesterol and triglyceride in diabetic rats. CA treatment improved histological architecture in the diabetic kidney. CA significantly down regulate miR-636 expression level in the kidney of diabetic rats in comparison to healthy group. Overall, caffeic acid down regulates miR-636 expression level which is involved in development of diabetic nephropathy and might therefore be potential attractive therapeutic agent to pursue in DN.

Dual approach for the colorimetric determination of unamplified microRNAs by using citrate capped gold nanoparticles.

The authors describe a method for the colorimetric determination of unamplified microRNA. It is based on the use of citrate-capped gold nanoparticles (AuNPs) and, alternatively, a microRNA-probe hybrid or a magnetically extracted microRNA that serve as stabilizers against the salt-induced aggregation of AuNPs. The absorbance ratios A525/A625 of the reacted AuNP solutions were used to quantify the amount of microRNA. The assay works in the range of 5-25 pmol microRNA. The lower limit of detection (LOD) is 10 pmol. The performance of the method was tested by detection of microRNA-210-3p in totally extracted urinary microRNA from normal, benign, and bladder cancer subjects. The sensitivity and specificity for qualitative detection of urinary microRNA-210-3p using the assay are 74% and 88% respectively, which is consistent with real time PCR based assays. The assay was applied to the determination of specific microRNA by using its specific oligo targeter or following magnetic isolation of the desired microRNA. The method is simple, cost-efficient, has a short turn-around time and requires minimal equipment and personnel. Graphical abstract Schematic of the two detection schemes: In the first approach, matched microRNA hybridizes with its specific probe to stabilize gold nanoparticles (AuNPs) against salt induced aggregation and to leave the red color of the AuNPs unchanged. In the second one, microRNA extracted via magnetic nanoparticles (MNP) stabilizes AuNPs against aggregation.

Identification and validation of a novel autophagy gene expression signature for human bladder cancer patients.

We sought to identify and validate a novel urinary autophagy transcript signature in patients with bladder cancer and evaluate its clinical utility. We performed an initial screening for seven autophagy transcript-based panel (autophagy-related protein 12 (ATG12); WD repeat domain, phosphoinositide interacting 2 (WIPI2); FYVE and coiled-coil domain-containing protein 1 (FYCO1); microtubule-associated protein light chain (MAPLC3); RB1-inducible coiled-coil 1 (RB1CC1); tachylectin-II-like beta-propeller domain 1 (TECPR1); and Unc-51-like kinase (ULK1)) that was identified based on bioinformatics analysis followed by SYBR Green-based polymerase chain reaction array validation in paired tissue and urine samples. Afterward, we evaluated the expression of differentially expressed autophagy transcripts in an independent validation set with reverse transcription quantitative real-time polymerase chain reaction in urine sediments of 140 patients with bladder cancer, 68 patients with benign urological lesions, and 74 healthy controls (age and sex matched). The expression levels of ATG12, FYCO1, TECPR1, and ULK1 in paired bladder tissue and urine samples were significantly lower in bladder cancer than in control group (p < 0.001). In the validation set, the receiver-operating characteristic curve analyses demonstrated that each urinary autophagy transcripts showed high sensitivity and specificity for distinguishing bladder cancer from non-bladder cancer patients (ATG12, 75.4% and 86.1%; FYCO1, 87% and 75.7%; ULK1, 85.5% and 75.6%; and TECPR1, 90% and 81.9%). We document and validate a novel autophagy transcript signature for human bladder cancer diagnosis: bilharzial and non-bilharzial types.

MicroRNA-10b and minichromosome maintenance complex component 5 gene as prognostic biomarkers in breast cancer.

The aim of this study is to identify micro-ribonucleic acid (microRNA) and its target, in addition to their relationship to the outcome in breast cancer (BC). To achieve this aim, we investigated microRNA-10b (miR-10b) and minichromosome maintenance complex component 5 (MCM5 mRNA) expression in 230 breast tissue samples by real-time PCR and semiquantitative conventional RT-PCR, respectively. Relapse-free survival (RFS) associated with miRNA-10b and MCM5 mRNA were tested by Kaplan-Meier survival analysis. The impact of miRNA-10b andMCM5 mRNA expression on the survival was evaluated by Cox proportional hazard regression model. The expression of miRNA-10b and MCM5 mRNA was positive in 86.4 and 79.7 % breast cancer patients, respectively. The overall concordance rate between miRNA-10b and MCM5 RNA was 90.4 %. The median follow-up period was 50 months. The survival analysis showed that high levels of both miR-10b and MCM5 were associated with short relapse free survival of BC. We identified MCM5 mRNA expression changes consistent with the miRNA-10b target regulation. Thus, we could consider miRNA-10b and MCM5 mRNA as prognostic markers and potential therapeutic targets in breast cancer to be applied to other patient data sets.

Integrative functional genetic-epigenetic approach for selecting genes as urine biomarkers for bladder cancer diagnosis.

Early screening for bladder cancer (BC) holds the key to combat and control the increasing global burden of BC mortality. We presented a simple approach to characterize, analyze, and validate a panel of biomarkers in BC and their relationship to bilharziasis. We investigated voided urine and blood samples from patients with bladder cancer (n = 94), benign bladder lesions (n = 60), and age-matched normal controls (n = 56). This study was divided into the following phases. (1) We analyzed the expression of urinary Hyaluronoglucosaminidase 1 (HYAL1) protein in BC and control samples by zymography. (2) We performed bioinformatics analysis to retrieve a set of epigenetic regulators of HYAL1. (3) This set of three selected genes [long non-coding RNA-urothelial cancer associated 1(lncRNA-UCA1), microRNA-210, and microRNA-96] was then analyzed in the same urine samples used in phase I by quantitative real-time PCR. (4) A high reproducibility of gene selection results was also determined from statistical validation. The urinary expression of HYAL1 protein and its epigenetic regulators were higher in BC patients (P < .001). The receiver-operating characteristic curve analyses demonstrated that each one had good sensitivity and specificity for distinguishing BC patients from non-BC ones (HYAL1, 89.4 and 91.2 %; miR-210, 76.6 and 93 %; miR-96, 76.6 and 89.4 %; and lncRNA-UCA1, 91.5 and 96.5 %). There was a significant positive correlation between HYAL1 and the selected epigenetic biomarkers. The performance of this urine biomarker panel reached 100 % sensitivity and 89.5 % specificity for bladder cancer diagnosis.

Rapid detection of urinary long non-coding RNA urothelial carcinoma associated one using a PCR-free nanoparticle-based assay.

We developed a specific hybridization assay for direct detection of long non-coding RNA urothelial carcinoma associated-1 (lncRNA-UCA1). Total RNA was extracted from urine pellet samples (bladder carcinoma patients and controls). Then, we compared the developed nanoassay with quantitative real time polymerase chain reaction (qRT-PCR) results in detection of urine UCA1 in bladder cancer and control samples. The sensitivity and the specificity of UCA1 nanoassay were 92.1% and 93.3%, respectively. The concordance of the two methods was 98%. Interestingly, all bilharzial benign cases showed negative lncRNA-UCA1 using both methods. UCA1-nanoassay is a valid test for direct detection of urine UCA1 for bladder cancer detection.

Evaluation of urinary human telomerase reverse transcriptase mRNA and scatter factor protein as urine markers for diagnosis of bladder cancer.

BACKGROUND: Expression of the human telomerase reverse transcriptase (hTERT) gene, which codes for the catalytic subunit of telomerase is considered an important tumor marker used for bladder cancer detection being found in the majority of cancer cells. Scatter Factor (SF) is a secretory protein produced by fibroblasts and smooth muscles and induces scattering of the epithelial cells. The aim of the current study was to evaluate the potential usefulness of hTERT and SF measurement as urinary markers for bladder cancer diagnosis. METHODS: Voided urine specimens were collected from patients with histologically confirmed bladder urothelial carcinoma (malignant group: n = 60), urological patients without urothelial carcinoma (benign group: n = 25), and healthy volunteers (control group: n = 20). All cases underwent urine cytology, serological schistosomiasis antibody assay and detection of urinary hTERT mRNA using RT-PCR and SF using ELISA. RESULTS: Positivity rate of hTERT mRNA was markedly higher in malignant versus benign or control cases (86.67%, 8%, and 0%, respectively, p-value < 0.001). Combining hTERT and cytology increased the sensitivity of cytology to 95%. According to a cut-off value of urinary SF (> or = 410 ng/mg protein), 57 (95%) of the patients with bladder carcinoma, 10 (40%) with benign lesions, and non of the control individuals were positive and the difference between the 3 groups was statistically significant (p < 0.001). The sensitivity of cytology was increased to 98.33% when combined with the SF assay. When associating the two urinary markers with different clinicopathological factors of the bladder cancer group, only SF exerted a significantly higher positivity rate at the invasive stage (100%) than the superficial stage (88.46%) as well as in transitional cell carcinoma (100% thansquamous cell carcinoma type (87.5%). CONCLUSIONS: hTERT and SF can be considered potential useful markers for detection of bladder cancer.

The diagnostic efficacy of urinary survivin and hyaluronidase mRNA as urine markers in patients with bladder cancer.

BACKGROUND: A new, sensitive, noninvasive method for the detection of urothelial carcinomas of the bladder would open new possibilities in both the diagnosis and follow up of patients. METHODS: Voided urine specimens were collected from patients with histologically confirmed bladder urothelial carcinoma (Group 1: n = 60), urological patients without urothelial carcinoma (Group 2: n = 20), and healthy volunteers (Group 3: n = 20). All underwent serological assessment of schistosomiasis antibody, quantitative measurement of survivin by ELISA in urine supernatant, urine cytology, and detection of hyaluronidase (HYAL-1) by RT-PCR in urothelial cells of voided urine samples. RESULTS: Urinary survivin mean rank was higher in malignant and benign groups than in the healthy group (p < 0.001). Urinary survivin best-cutoff was determined using receiver operating characteristic curve to discriminate between malignant and nonmalignant groups (2537.25 pg/mg protein) at 78.33% sensitivity and 82.5% specificity. HAase mRNA showed superior sensitivity (86.67%) over cytology (38.33%) and urinary survivin (78.33%) with specificity of 97.5%, 100%, and 82.5%, respectively. The sensitivity of urine cytology was increased on combination with either survivin (83.33%) or HAase (90%). Also, the combination of both markers increased overall sensitivity (95%). CONCLUSIONS: Survivin can be reliably and quantitatively measured in urine of bladder cancer patients, improving the sensitivity and specificity of urine cytology for the diagnosis of bladder cancer. Combined use of cytology with survivin and HAase was the best recommended combination for bladder cancer detection.

Evaluation of the role of kefir in management of non-alcoholic steatohepatitis rat model via modulation of NASH linked mRNA-miRNA panel.

Non-alcoholic steatohepatitis (NASH) is the clinically aggressive variant of non-alcoholic fatty liver disease. Hippo pathway dysregulation can contribute to NASH development and progression. The use of probiotics is effective in NASH management. Our aim is to investigate the efficacy of kefir Milk in NASH management via modulation of hepatic mRNA-miRNA based panel linked to NAFLD/NASH Hippo signaling and gut microbita regulated genes which was identified using bioinformatics tools. Firstly, we analyzed mRNAs (SOX11, SMAD4 and AMOTL2), and their epigenetic regulator (miR-6807) followed by validation of target effector proteins (TGFB1, IL6 and HepPar1). Molecular, biochemical, and histopathological, analyses were used to evaluate the effects of kefir on high sucrose high fat (HSHF) diet -induced NASH in rats. We found that administration of Kefir proved to prevent steatosis and development of the inflammatory component of NASH. Moreover, Kefir improved liver function and lipid panel. At the molecular level, kefir down-regulated the expression of miR 6807-5p with subsequent increase in the expression of SOX 11, AMOTL2 associated with downregulated SMAD4, resulting in reduction in the expression of the inflammatory and fibrotic markers, IL6 and TGF-ß1 in the treated and prophylactic groups compared to the untreated rats. In conclusion, Kefir suppressed NASH progression and improved both fibrosis and hepatic inflammation. The produced effect was correlated with modulation of SOX11, SMAD4 and AMOTL2 mRNAs) - (miR-6807-5p) - (TGFB, IL6 and, HepPar1) expression.

Epigenetic modulation of autophagy genes linked to diabetic nephropathy by administration of isorhamnetin in Type 2 diabetes mellitus rats.

Aim: To assess isorhamnetin efficacy for diabetic kidney disease in a Type 2 diabetes mellitus rat model, through investigating its effect at the epigenetic, mRNA and protein levels. Materials & methods: Type 2 diabetes mellitus was induced in rats by streptozotocin and high-fat diet. Rats were treated with isorhamnetin (50 mg/kg/d) for 4 or 8 weeks. Fasting blood glucose, renal and lipid profiles were evaluated. Renal tissues were examined by light and electron microscopy. Autophagy genes (FYCO1, ULK, TECPR1 and WIPI2) and miR-15b, miR-34a and miR-633 were assessed by qRT-PCR, and LC3A/B by immunoblotting. Results: Isorhamnetin improved fasting blood glucose, renal and lipid profiles with increased autophagosomes in renal tissues. It suppressed miRNA regulation of autophagy genes. Conclusion: We propose a molecular mechanism for the isorhamnetin renoprotective effect by modulation of autophagy epigenetic regulators.

New insight into the role of isorhamnetin as a regulator of insulin signaling pathway in type 2 diabetes mellitus rat model: Molecular and computational approach.

We intended to examine the molecular mechanism of action of isorhamnetin (IHN) to regulate the pathway of insulin signaling. Molecular analysis, immunofluorescence, and histopathological examination were used to assess the anti-hyperglycemic and insulin resistance lowering effects of IHN in streptozotocin /high fat diet-induced type 2 diabetes using Wistar rats. At the microscopic level, treatment with IHN resulted in the restoration of myofibrils uniform arrangement and adipose tissue normal architecture. At the molecular level, treatment with IHN at three different doses showed a significant decrease in m-TOR, IGF1-R & LncRNA-RP11-773H22.4. expression and it up-regulated the expression of AKT2 mRNA, miR-1, and miR-3163 in both skeletal muscle and adipose tissue. At the protein level, IHN treated group showed a discrete spread with a moderate faint expression of m-TOR in skeletal muscles as well as adipose tissues. We concluded that IHN could be used in the in ameliorating insulin resistance associated with type 2 diabetes mellitus.

Probiotic-prebiotic-synbiotic modulation of (YAP1, LATS1 and NF2 mRNAs/miR-1205/lncRNA SRD5A3-AS1) panel in NASH animal model.

AIM: To investigate the prophylactic efficacy of gut microbiota-based treatments on nonalcoholic steatohepatitis (NASH) management via modulation of Hippo signaling pathway-related genes (YAP1, LATS1 and NF2), and their epigenetic regulators (miR-1205 and lncRNA SRD5A3-AS1) retrieved from in-silico data analysis. MATERIALS & METHODS: Histopathological, biochemical, molecular and immunohistochemistry analyses were used to assess the effects of multistrain probiotic mixture and prebiotic inulin fiber on high sucrose high fat (HSHF) diet-induced NASH in rats. These treatments were administered orally either alone or in combination, along with HSHF diet. RESULTS: Both probiotic mixture and prebiotic inulin fiber attenuated steatosis, inflammation and fibrosis grades in HSHF diet-induced NASH rats. Moreover, the applied treatments significantly prevented the elevation of serum liver enzymes and improved lipid panel. At the molecular level, both treatments down-regulated hepatic YAP1 mRNA and miR-1205 expressions, and concomitantly up-regulated the expression of hepatic LATS1& NF2 mRNAs and the lncRNA SRD5A3-AS1. At the protein level, both treatments decreased the hepatic content of the inflammatory marker IL6 and the fibrotic marker TGFß1. Moreover, an observable reduction in α-SMA together with noticeable elevation in LATS1/2 protein expression levels were detected in liver sections compared to the untreated rats. CONCLUSION: Probiotic mixture and prebiotic inulin fiber, either alone or in combination, attenuated NASH progression and ameliorated both fibrosis and hepatic inflammation in the applied animal model. The produced effect was correlated with modulation of the retrieved (YAP1, LATS1 and NF2) - (miR-1205) - (lncRNA SRD5A3-AS1) RNA panel.

Diagnostic evaluation of apoptosis inhibitory gene and tissue inhibitor matrix metalloproteinase-2 in patients with bladder cancer.

Bladder carcinoma is an important worldwide health problem. Both cystoscopy and urine cytology used in detecting bladder cancer suffer from drawbacks where cystoscopy is an invasive method and urine cytology shows low sensitivity in low-grade tumors. This study validates easier and less time-consuming techniques for the estimation of survivin and TIMP-2 in urine of bladder cancer patients to evaluate them in comparison with cytology. This study includes malignant (bladder cancer patients, n = 42), benign (patients with bilharzial cystitis, n = 22) and healthy (n = 21) groups. The studied groups were subjected to cystoscopic examination, detection of bilharzial antibodies, urine cytology, and estimation of urinary survivin by qualitative RT-nested PCR and TIMP-2 by ELISA. Significantly higher positivity rates of urinary survivin and TIMP-2 were observed in the malignant group compared with benign and healthy groups. On associating the two urinary markers with different clinicopathological factors, only TIMP-2 exerted significantly higher positivity rate in invasive stage (100%) than superficial stage (82.3%). Survivin showed 78.6% sensitivity, 95.3% specificity, 94.3% PPV, 82% NPV, and 87% accuracy. When combined with urine cytology, the sensitivity increased to 83.3%. While on applying the cutoff value of urinary TIMP-2 (< or =639.5 pg/mg protein), it showed 93% sensitivity, 83.7% specificity, 85% PPV, 92.3% NPV, and 88.2% accuracy. When combined with urine cytology, the TIMP-2 sensitivity remained 93%. On combining cytology with both urinary survivin and TIMP-2, the highest sensitivity was reached (98%). Survivin and TIMP-2 can be considered as potentially useful urine markers in early detection of bladder cancer.