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.

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.

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.

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.

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.

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.

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.

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.

Measurement of Urinary Level of a Specific Competing endogenous RNA network (FOS and RCAN mRNA/ miR-324-5p, miR-4738-3p, /lncRNA miR-497-HG) Enables Diagnosis of Bladder Cancer.

OBJECTIVE: To assist in the diagnosis, treatment, and prognostic prediction of bladder cancer, the molecular patterns associated with it should be elucidated. Competing endogenous RNA network: MicroRNA (miRNA), long noncoding RNA (lncRNA), and their target autophagy genes have been strongly implicated in tumor development and metastasis. PATIENTS AND METHODS: Bioinformatics analysis was performed to retrieve a ceRNA: lncRNA-miRNA-mRNA network linked to autophagy and relevant to bladder cancer. Expression of selected noncoding human RNAs (miR-324-5p, miR-4738-3p, and lncRNA miR-497-HG) and their target genes (RCAN1 mRNA and FOSB mRNA) was examined by qPCR in bladder tissues and urine samples obtained from 196 individuals (98 patients with bladder cancer, 48 patients with benign lesions, and 50 healthy controls). RESULTS: Expression levels of the selected genes in urine samples in the bladder cancer group were significantly different from those in the control group (P < 0.001). Expression in bladder cancer tissue samples correlated with that in urine samples. Urinary expression levels of all biomarkers had high accuracy to distinguish patients with and without bladder cancer, with FOSB mRNA and RCAN1 mRNA having the highest accuracy (99% for RCAN1 mRNA or FOSB mRNA, 87.8% for miR-324-5p, 84.7% for miR-4738-3p, and 90.5% for lncRNA miR-497-HG). FOSB mRNA and RCAN1 mRNA expression showed also a higher accuracy than cytology (77.6%). CONCLUSION: The significant differential expression of the ceRNA network: lncRNA-miRNA-mRNA network in bladder cancer as compared to noncancerous controls has revealed the superior accuracy of the chosen biomarkers to cytology, especially FOSB mRNA and RCAN1 mRNA, suggesting their involvement in bladder cancer pathogenesis and promising role for future diagnosis, and targeted therapy.

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.

Evaluation of urinary autophagy transcripts expression in diabetic kidney disease.

BACKGROUND: We identified and validated novel urinary autophagy markers in diabetic kidney disease (DKD) based on bioinformatics analysis and clinical validation. PATIENTS & METHODS: We retrieved three novel autophagy genes related to DKD from public microarray databases, namely; microtubule-associated protein light chain (MAP1LC3A), WD Repeat Domain, Phosphoinositide Interacting 2 (WIPI2), and RB1-Inducible Coiled-Coil 1 (RB1CC1). Secondly we assessed the expression of the chosen autophagy transcript in urine sediment of 86 patients with DKD and 74 (age and sex matched) controls by reverse transcription quantitative real-time PCR. RESULTS: The urinary expression levels of MAP1LC3A, WIPI, RB1CC1 were significantly lower in DKD than control group (P<0.001).The receiver-operating characteristic curve (ROC) analyses that each urinary autophagy transcript showed high sensitivity and specificity for distinguishing DKD from control (MAP1LC3A, 81.4% and 81.1%; WIPI, 74.4% and 67.6%, and RB1CC1, 81.4%,70.3%, respectively). Notably, a negative correlation was found between these autophagy markers, serum creatinine and urinary albumin creatinine ratio. The sensitivity and specificity of this urinary autophagy based panel reached 90.6% and 60% in diagnosis of DKD. CONCLUSION: We identified and validated a novel diagnostic urinary autophagy based panel with high sensitivity and moderate specificity representing a vital player in the pathogenesis of DKD.

Caffeic Acid Attenuates Diabetic Kidney Disease via Modulation of Autophagy in a High-Fat Diet/Streptozotocin- Induced Diabetic Rat.

The aim of this study is to evaluate the anti-diabetic nephropathy effect of Caffeic acid and to prove our hypothesis for its mechanism of action that it may occur by reactivation of autophagy pathway via suppression of autophagy regulatory miRNAs. In vivo, high-fat diet and streptozotocin-induced (HFD-STZ) diabetic rats were treated with Caffeic acid once per day for 12 weeks before and after development of diabetic nephropathy. Blood and urine biochemical parameters, autophagy transcripts and their epigenetic regulators together with renal tissue morphology were investigated. In diabetic rats, Caffeic acid intake, caused improvement in albumin excretion,blood glucose, reduced renal mesangial matrix extension with increased vacuolation and reappearance of autophagosomes. Meanwhile, it resulted in autophagy genes up-regulation [RB 1-inducible coiled coil protein (RB1CC1), Microtubule-associated proteins 1A/1B light chain 3(MAP1LC3B), Autophagy related gene (ATG-12),] with simultaneous reduction in their epigenetic regulators; miRNA-133b, -342 and 30a, respectively. These above mentioned effects were more significant in the diabetic nephropathy Caffeic treated rats than in the prophylactic group. Based on our results we postulated that caffeic acid modulates autophagy pathway through inhibition of autophagy regulatory miRNAs, that could explain its curative properties against diabetic kidney disease.

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.

Gelatin-modified gold nanoparticles for direct detection of urinary total gelatinase activity: Diagnostic value in bladder cancer.

Matrix metalloproteinases (MMPs), in particularly gelatinases (MMP-2 and MMP-9) were reported as urinary markers of bladder cancer. In this work, we developed a simple colorimetric gold nanoparticle (AuNP) assay for rapid and sensitive detection of urinary total gelatinase activity based on the surface plasmon resonance (SPR) property of AuNPs. Gelatin-modified AuNPs were stably suspended in solution even upon addition of an aggregation inducer as 6-mercaptohexan-1-ol (6-MCH). Gelatinases digest gelatin capping. Subsequently, addition of 6-MCH leads to AuNPs aggregation with red to blue color shift. In a pilot study, results of the developed AuNP assay were consistent with zymography for qualitative detection of urinary total gelatinase activity. The sensitivity and specificity of both assays were 80% and 90.9% respectively. The absorption ratios, A625/A530 of the reacted AuNP solutions were used to quantify the total gelatinase concentration. The best cut off value was 0.01895ng/µg protein, at which the sensitivity was 87.5% and the specificity was 86.4%. The developed AuNP assay is simple, low-cost and can aid non-invasive diagnosis of bladder cancer.

Urinary exosomal microRNA panel unravels novel biomarkers for diagnosis of type 2 diabetic kidney disease.

BACKGROUND: A potential approach adopted in the current study is to design a panel based on in silico retrieval of novel miRNAs related to diabetic kidney disease and to evaluate its usefulness in disease diagnosis. PATIENT AND METHODS: In the current study, we measured the differential expression of a 6 miRNA panel in urine pellet and exosome in an initial screening group using syber green-based PCR array. Also, we performed pathway enrichment analysis of the key target genes of these miRNAs. Finally, we selected the most significantly up-regulated miRNAs in DKD, exosomal miR-15b, miR-34a and miR-636, that were measured by real-time PCR in a larger independent set of 180 participants to evaluate their usefulness as novel urine biomarkers for diagnosis diabetic kidney disease. RESULTS: PCR array analysis showed that miR-15b, miR-34a, and miR-636 were upregulated in both urine pellet and exosome of type 2DKD patients. qRT-PCR validation in the larger independent set of participants confirmed the significant up-regulation of these urinary exosomal miRs (P<0.001). Notably, a positive correlation was found between these miRs, serum creatinine and urinary protein creatinine ratio. The sensitivity of this miRs based panel in urine exosomes reached 100% in diagnosis of DKD. CONCLUSION: We identified urinary exosomal miR-15b, miR-34a, and miR-636 as a novel diagnostic panel and a major contributor in the pathogenesis of diabetic kidney disease.

Clinical verification of a novel urinary microRNA panal: 133b, -342 and -30 as biomarkers for diabetic nephropathy identified by bioinformatics analysis.

BACKGROUND: Because microvascular disease is one of the major drivers of diabetic complications, early detection of diabetic nephropathy (DN) by assessing the expression of exosomal microRNAs (miRNAs) in DN patients and healthy controls, may be of clinical value. The aim of this study wasto identify a novel miRNA panel of DN by combining bioinformatics analysis of miRNA databases and clinical verification to evaluate the significance of this panel as urine biomarkers for type 2 diabetic nephropathy (T2DN). PATIENTS AND METHODS: Public miRNA databases e.g miro-Ontology and miRWalk were analyzed and a novel panel of 3 microRNAs was retrieved. Meanwhile, combinatorial target prediction algorithms were applied. Multiple case-matched normal were examined by quantative RT-PCR for differential expression in urine exosomes from 210 participants, and the three identified miRNAs were validated as DN biomarkers. RESULTS: We found urinary exosomalmiR-133b, miR-342, and miR-30a were expressed at significantly elevated levels in T2DN patients (P<0.001) compared to normal. Furthermore, high-level expression of the 3 miRNAs was associated withHbA1c,systolic-diastolic blood pressure, LDL, serum creatinine, urinary albumin creatinine ratio and estimated glomerular filtration rate(eGFR). Moreover, 39.3%, 19.6% and 17.9% of patients with normo-albuminuria had positive (miR-133b, miR-342 and miR-30a, respectively); indicating the possibility of molecular changes in these patients before onset of albuminuria. CONCLUSION: We have identified novel urinary exosomal miRNA biomarkers of DN which were altered not only in micro and macroalbuminuric groups but also in some normoalbuminuria cases prior to albuminuria.

Prognostic and biological significance of microRNA-221 in breast cancer.

INTRODUCTION: Breast cancer (BC) is the most notorious cancer between females with high rates of morbidity and mortality. The aim of this study was to determine the differential expression of breast tissues microRNA-221 (miR-221) and assess its prognostic and biological significance in breast cancer (BC). METHODS: A quantitative reverse transcription PCR (qPCR) assay was performed to detect the expression of breast tissue miR-221 in different subtypes of BC (n=76) and controls (n=36) and its correlations with clinicopathological factors of patients. Univariate and multivariate analyses using the Cox proportional hazards model were performed to analyze the prognostic significance of miR-221 expression. RESULT: Our data indicated that the relative level of miR-221 expression in BC tissues was significantly higher than that in noncancerous breast tissues (p<0.01). Of 76 BC patients, 62 (81.6%) were positive cases. By statistical analyses, high miR-221 expression was observed to be closely correlated with advanced clinical stage (p<0.01). Moreover, patients with high miR-221 expression had worse 5-year relapse free survival (p=0.0124). Univariate and multivariate analyses indicated that high miR-221 expression was an independent poor prognostic factor for BC patients. CONCLUSION: miR-221 is a potential biomarker for predicting the survival of BC patients and may be a molecular therapeutic target for BC.