Rhamnetin ameliorates non-alcoholic steatosis and hepatocellular carcinoma in vitro.

Non-alcoholic fatty liver (NAFLD) is a widespread disease with various complications including Non-alcoholic steatohepatitis (NASH) that could lead to cirrhosis and ultimately hepatocellular carcinoma (HCC). Up till now there is no FDA approved drug for treatment of NAFLD. Flavonoids such as Rhamnetin (Rhm) have been ascribed effective anti-inflammatory and anti-oxidative properties. Thus, Rhm as a potent flavonoid could target multiple pathological cascades causing NAFLD to prevent its progression into HCC. NAFLD is a multifactorial disease and its pathophysiology is complex and is currently challenged by the 'Multiple-hit hypothesis' that includes wider range of comorbidities rather than previously established theory of 'Two-hit hypothesis'. Herein, we aimed at establishing reliable in vitro NASH models using different mixtures of variable ratios and concentrations of oleic acid (OA) and palmitic acid (PA) combinations using HepG2 cell lines. Moreover, we compared those models in the context of oil red staining, triglyceride levels and their altered downstream molecular signatures for genes involved in de novo lipogenesis, inflammation, oxidative stress and apoptotic machineries as well. Lastly, the effect of Rhm on NASH and HCC models was deeply investigated. Over the 10 NASH models tested, PA 500 µM concentration was the best model to mimic the molecular events of steatosis induced NAFLD. Rhm successfully ameliorated the dysregulated molecular events caused by the PA-induced NASH. Additionally, Rhm regulated inflammatory and oxidative machinery in the HepG2 cancerous cell lines. In conclusion, PA 500 µM concentration is considered an effective in vitro model to mimic NASH. Rhm could be used as a promising therapeutic modality against both NASH and HCC pathogenesis.

Genetics and meta-analysis of recessive non-syndromic hearing impairment and Usher syndrome in Maghreb population: lessons from the past, contemporary actualities and future challenges.

Hereditary hearing impairment (HI) is a heterogeneous condition with over 130 genes associated with genetic non-syndromic HI (NSHI) and Usher syndrome (USH). Approximately 80% of hereditary NSHI cases have autosomal recessive (AR) mode of inheritance. The high rate of consanguinity and endogamy in the Maghreb countries, including Tunisia, Algeria and Morocco, represents a major contributing factor to the development of ARHI. Since the 90s, those populations, with their particular large familiar structure, represented an effective key towards the discovery of the first HI loci and genes. In this study, we performed a deep literature database search to analyze the mutational spectrum and the distribution of pathogenic variants responsible of USH and the NSHI among those populations. To date, 124 pathogenic variants were identified in 32 genes of which over 70% represent population-specific variants. The particular variants' distribution is related to the high rate of consanguinity as well as the multiple shared features such as demographic history of migrations and social behavior that promoted the spreading of several founder mutations within those countries. This is the first study to report lessons from the past and current actualities of HI within the three Maghreb countries. However, despite the great impact placed by such population for the HI genetic studies, only a few next-generation sequencing platforms have so far been implemented with those countries. We, therefore, believe that those countries should be supported to implement this technology that would definitely be of great value in the discovery of additional novel HI genes/variants.

Novel pathogenic mutations and further evidence for clinical relevance of genes and variants causing hearing impairment in Tunisian population.

Introduction: Hearing impairment (HI) is characterized by complex genetic heterogeneity. The evolution of next generation sequencing, including targeted enrichment panels, has revolutionized HI diagnosis. Objectives: In this study, we investigated genetic causes in 22 individuals with non-GJB2 HI. Methods: We customized a HaloplexHS kit to include 30 genes known to be associated with autosomal recessive nonsyndromic HI (ARNSHI) and Usher syndrome in North Africa. Results: In accordance with the ACMG/AMP guidelines, we report 11 pathogenic variants; as follows; five novel variants including three missense (ESRRB-Tyr295Cys, MYO15A-Phe2089Leu and MYO7A-Tyr560Cys) and two nonsense (USH1C-Gln122Ter and CIB2-Arg104Ter) mutations; two previously reported mutations (OTOF-Glu57Ter and PNPT1-Glu475Gly), but first time identified among Tunisian families; and four other identified mutations namely WHRN-Gly808AspfsX11, SLC22A4-Cys113Tyr and two MYO7A compound heterozygous splice site variants that were previously described in Tunisia. Pathogenic variants in WHRN and CIB2 genes, in patients with convincing phenotype ruling out retinitis pigmentosa, provide strong evidence supporting their association with ARNSHI. Moreover, we shed lights on the pathogenic implication of mutations in PNPT1 gene in auditory function providing new evidence for its association with ARNSHI. Lack of segregation of a previously identified causal mutation OTOA-Val603Phe further supports its classification as variant of unknown significance. Our study reports absence of otoacoustic emission in subjects using bilateral hearing aids for several years indicating the importance of screening genetic alteration in OTOF gene for proper management of those patients. Conclusion: In conclusion, our findings do not only expand the spectrum of HI mutations in Tunisian patients, but also improve our knowledge about clinical relevance of HI causing genes and variants.

Investigation of the relationship between CTLA4 and the tumor suppressor RASSF1A and the possible mediating role of STAT4 in a cohort of Egyptian patients infected with hepatitis C virus with and without hepatocellular carcinoma.

The Ras association domain family 1 isoform A (RASSF1A), cytotoxic T lymphocyte antigen 4 (CTLA-4), and signal transducer and activator of transcription 4 (STAT4) genes play a role in regulating the cell cycle, apoptosis, and the autoimmune response against cancer. We investigated the genotype frequency and the possible association of the rs2073498 (RASSF1A), rs5742909 (CTLA-4) and rs7574865 (STAT4) genetic variants with hepatitis C virus (HCV)-G4-mediated hepatocellular carcinoma (HCC) progression in Egyptian patients. Fifty patients with HCV infection, 50 patients with HCV-mediated HCC, and 50 age- and sex-matched healthy controls were recruited. The investigated variants were genotyped based on polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP). The Ser133 mutant G4 variant of the rs2073498 SNP in RASSF1A exhibited a positive correlation with HCC incidence risk (OR = 0.571, 95% CI = 0.175-1.865, P < 0.001). The rs7574865 variant in STAT4 (G/T) occurred frequently in both HCV groups, with a significant incidence risk (OR = 1.583, 95% CI = 1.123-2.232, P = 0.005). The rs5742909 change in CTLA4 (C/T) did not show a significant difference between HCV-mediated HCC cases and the control group (OR = 4.5, 95% CI = 1.326-15.277, P > 0.001). Activation of the immune checkpoint gene CTLA4 or polymorphism in the encoded CTLA4 protein causes phosphorylation of kinases needed for RAS gene activation. This in turn downregulates the tumor suppressor RASSF1, inhibiting apoptosis and leading to HCC development, indicating a negative impact of CTLA4 gene polymorphism on HCV-mediated HCC cases. A major determinant of disease progression could be immune system genetic variants, together with the presence of costimulatory factors. The rs2073498 and rs7574865 variations in the RASSF1A and STAT4 genes, respectively, could be genetic susceptibility factors for Egyptian patients with HCV-mediated HCC.

Activation of FXR modulates SOCS3/Jak2/STAT3 signaling axis in a NASH-dependent hepatocellular carcinoma animal model.

Despite the recent substantial progress in the treatment of hepatocellular carcinoma (HCC) from viral etiology, non-alcoholic steatohepatitis (NASH) is on a trajectory to become the fastest growing indication for HCC-related liver transplantation. The Farnesoid X receptor (FXR) is a member of the nuclear receptor superfamily with multifaceted roles in several metabolic disorders, particularly NASH. Its role as a tumor suppressor was also highlighted. Herein, we investigated the effect of obeticholic acid (OCA), as an FXR agonist, on NASH-associated HCC (NASH-HCC) animal model induced by diethylnitrosamine and high fat choline-deficient diet, exploring the potential impact on the suppressor of cytokine signaling 3 (SOCS3)/Janus kinase 2 (Jak2)/signal transducer and activator of transcription 3 (STAT3) pathway. Results indicated that OCA treatment upregulated FXR and its key mediator, small heterodimer partner (SHP), with remarkable amelioration in the dysplastic foci observed in the NASH-HCC group. This was paralleled with noticeable downregulation of alpha fetoprotein along with reduction in interferon gamma and transforming growth factor beta-1 hepatic levels besides caspase-3 and p53 upregulation. Moreover, sirtuin-1 (SIRT-1), a key regulator of FXR that controls the regenerative response of the liver, was elevated following OCA treatment. Modulation in the SOCS3/Jak2/STAT3 signaling axis was also reported. In conclusion, OCA attenuated the development and progression of NASH-dependent HCC possibly by interfering with SOCS3/Jak2/STAT3 pathway suggesting the potential use of FXR activators in NASH-related disorders, even at later stages of the disease, to impede its progression to the more deteriorating condition of HCC.

Synthesis, Molecular Docking and Biological Evaluation of Novel Flavone Derivatives as Potential Anticancer Agents Targeting Akt.

BACKGROUND: Flavonoids are naturally occurring compounds with versatile healthpromoting effects against various diseases. OBJECTIVE: This aim of this paper is to synthesize and evaluate the biological activity of novel flavone derivatives against cancer. METHODS: A new series of 2-hydroxy-α,ß-unsaturated ketones 2a-h, was synthesized via the reaction of N-substituted-indole-3-carboxaldehyde 1a-h with 2-hydroxy acetophenone in the presence of piperidine. The oxidative cyclization of 2a-h using hydrogen peroxide/KOH and/or dimethyl sulfoxide/I2 produced the corresponding 2-(N-substituted-1H-indol-3-yl)-3-hydroxy-4H-chromen- 4-ones 3a-h and 2-(N-substituted-1H-indol-3-yl)-4H-chromen-4-ones 4a-h, respectively. Antiproliferative activities for synthesized series were investigated against HCT-116 colon and MCF- 7 breast cancer cell lines. Molecular downstream effects were evaluated using RT-PCR. Moreover, molecular docking was carried out to pinpoint the binding mode of the most active compounds into the active site of Akt enzyme (PDB ID: 3QKK). RESULTS: All compounds exhibited an anti-proliferative activity range of 52-97% and 67.2-99% against HCT-116 and MCF-7, respectively. Compounds 3b, 3h, 3g and 4h had a minimal inhibitory effect on normal BJ1 cells indicating their safety profile. Compounds 3b and 4h, in particular, exhibited the most potent antiproliferative activity against HCT116 and MCF7, meanwhile compounds 3g, 3h and 4g showed potent to moderate activity. Compound 3b had IC50 of 78.3 µM and 53.9 µM against HCT-116 and MCF-7 respectively with comparable IC50 for doxorubicin of 65.1 µM and 45.02 µM. Compound 3b exhibited significant down-regulation for Akt and significant up-regulation of CAS9 and CDKN1genes in all tested cell lines. CONCLUSION: The synthesized flavone derivatives and particularly compound 3b exhibited promising anticancer activity through Akt inhibition.

Circulatory miRNA-484, 524, 615 and 628 expression profiling in HCV mediated HCC among Egyptian patients; implications for diagnosis and staging of hepatic cirrhosis and fibrosis.

Circulatory microRNAs have recently emerged as non-invasive and effective biomarkers for diagnosis of various diseases. Currently there is no reliable biomarker for diagnosis, prognosis or even staging of fibrotic and cirrhotic complications arising from HCV infection. This study aimed at investigating plasma miR-484, miR-524, miR-615-5p and miR-628-3p expression signatures in Egyptian patients with HCV mediated cirrhosis, fibrosis and HCC. Plasma miRNAs expressions in 168 samples [(40 healthy controls, 47 with HCV liver fibrosis, 40 with HCV-cirrhosis and 41 with HCV-hepatocellular carcinoma (HCC)] were quantified using RT-PCR. The studied miRNAs were differentially expressed among all participating groups. Plasma miR-484 levels exhibited significant downregulation in advanced fibrosis as compared to mild fibrosis and HCC. Moreover, miR-484 showed significant upregulation in HCC versus cirrhosis. Both miR-524-5p and miR-615-5p were upregulated in cirrhotic group as compared to controls. Differential expression between HCC and controls was noticeable in miR-524-5p. Receiver operator characteristic curve analysis revealed promising diagnostic performance for miR-484 in discriminating late fibrosis from both mild fibrosis and HCC and also for miR-524 in distinguishing between cirrhosis and fibrosis. In conclusion, investigated miRNAs could serve as potential and sensitive biomarkers for staging, prognosis and early diagnosis of various HCV mediated hepatic disease progression.

Analysis of p.Gly12Valfs*2, p.Trp24* and p.Trp77Arg mutations in GJB2 and p.Arg81Gln variant in LRTOMT among non syndromic hearing loss Egyptian patients: implications for genetic diagnosis.

Hearing loss (HL) is a global sensory disorder that affects children and deprives them from their rights to enjoy standard social and educational levels. Although hundreds of genetic mutations across several genes have been linked to HL, very limited studies are available on Egyptian population which has high rate of consanguinity and HL. The frequency of the p.Gly12Valfs*2, p.Trp24* and p.Trp77Arg mutations in GJB2 along with the p.Arg81Gln variant in LRTOMT gene was investigated in Egyptian patients. 103 non-syndromic HL (NSHL) Egyptian patients and 100 control subjects were recruited in this study. PCR-RFLP and Direct sequencing were performed to screen and confirm presence/absence of those mutations in Egyptian population. The p.Gly12Valfs*2 mutation was found in eight patients (7.8%) (six homozygous and two heterozygous) with an allele frequency of 6.8%. The p.Trp24* and p.Trp77Arg were absent in both HL patients and controls. Another one patient had the heterozygous variant for p.Arg81Gln in LRTOMT gene. This study reports, for the first time, the presence of a heterozygous change for the p.Arg81Gln in LRTOMT gene in one Egyptian patient. The p.Gly12Valfs*2 mutation, but not the p.Trp24* nor the p.Trp77Arg, in GJB2 is the most frequent variant among Egyptian patients and would therefore be recommended for genetic counseling and diagnosis.

Encapsulation of cinnamon essential oil in whey protein enhances the protective effect against single or combined sub-chronic toxicity of fumonisin B1 and/or aflatoxin B1 in rats.

Fumonisin B1 (FB1) and aflatoxin B1 (AFB1) are fungal metabolites that frequently co-occur in foodstuffs and are responsible for mycotoxicosis and several primary cancers. Cinnamon essential oil (CEO) has a spacious range of benefit effects but also has some limitations owing to its strong taste or its interaction with some drugs. This study aimed to use the cinnamon oil emulsion droplets (COED) for the protection against oxidative stress, cytotoxicity, and reproductive toxicity in male Sprague-Dawley rats sub-chronically exposed to FB1 and/or AFB1. The composition of CEO was identified using GC-MS then was encapsulated using whey protein as wall material. Male rats were divided into eight groups and treated orally for 8 weeks as follows: control group, AFB1-trreated group (80 µg/kg b.w), FB1-treated group (100 mg/kg b.w), FB1 plus AFB1-treated group, and the groups treated with COED plus FB1 and/or AFB1. Blood and samples of the kidney, liver, and testis were collected for different analysis and histopathological examination. The GC-MS analysis revealed that cinnamaldehyde, α-copaene, trans-cinnamaldehyde, caryophyllene, and delta-cadinene were the main compounds in COE. The average size of COED was 235 ± 1.4 nm and the zeta potential was - 6.24 ± 0.56. Treatment with FB1 and/or AFB1 induced significant disturbances in the serum biochemical analysis, oxidative stress parameters, DNA fragmentation, gene expression, and testosterone and severe pathological changes in the tested organs. Moreover, treatment with both mycotoxins induced synergistic toxic effects. COED did not induce toxic effects and could normalize the majority of the tested parameters and improve the histological picture in rats treated with FB1 and/or AFB1. It could be concluded that COED induce potential protective effects against the single or combined exposure to FB1 and AFB1.

Advances in ligase chain reaction and ligation-based amplifications for genotyping assays: Detection and applications.

Genetic variants have been reported to cause several genetic diseases. Various genotyping assays have been developed for diagnostic and screening purposes but with certain limitations in sensitivity, specificity, cost effectiveness and/or time savings. Since the discovery of ligase chain reaction (LCR) in the late nineties, it became one of the most favored platforms for detecting these variants and also for genotyping low abundant contaminants. Recent and powerful modifications with the integration of various detection strategies such as electrochemical and magnetic biosensors, nanoparticles (NPs), quantum dots, quartz crystal and leaky surface acoustic surface biosensors, DNAzyme, rolling circle amplification (RCA), strand displacement amplification (SDA), surface enhanced raman scattering (SERS), chemiluminescence and fluorescence resonance energy transfer have been introduced to both LCR and ligation based amplifications to enable high-throughput and inexpensive multiplex genotyping with improved robustness, simplicity, sensitivity and specificity. In this article, classical and up to date modifications in LCR and ligation based amplifications are critically evaluated and compared with emphasis on points of strength and weakness, sensitivity, cost, running time, equipment needed, applications and multiplexing potential. Versatile genotyping applications such as genetic diseases detection, bacterial and viral pathogens detection are also detailed. Ligation based gold NPs biosensor, ligation based RCA and ligation mediated SDA assays enhanced detection limit tremendously with a discrimination power approaching 1.5aM, 2aM and 0.1fM respectively. MLPA (multiplexed ligation dependent probe amplification) and SNPlex assays have been commercialized for multiplex detection of at least 48 SNPs at a time. MOL-PCR (multiplex oligonucleotide ligation) has high-throughput capability with multiplex detection of 50 SNPs/well in a 96 well plate. Ligase detection reaction (LDR) is one of the most widely used LCR versions that have been successfully integrated with several detection strategies with improved sensitivity down to 0.4fM.

Evaluation of food products fortified with oyster shell for the prevention and treatment of osteoporosis.

Production and evaluation of different diet formulas fortified with oyster shell for the prevention and treatment of osteoporosis. Eighty-eight female albino rats were recruited and divided into 11 groups (8 rats each). Group 1 represented negative control while the remaining groups were ovariectomized. Group 2 acted as positive control. Groups 3-5 were fed on basal diet. Groups 6-8 were fed on lentil soup while groups 9-11 were fed on vegetable soup. Group 4, 7, 10 were fed on diets fortified with oyster shell. Groups 5, 8 and 11 were fed on diet formulas fortified with calcium citrate. All calcium fortified diet formulas, especially lentil soup, have minimized risk factors associated with osteoporosis as indicated from the significant increase in tibial weight, total protein, total calcium and phosphorus with noticeable reduction in ALP activity compared to positive group. Maximum recovery was observed for diet fortified with oyster shell. These data suggest that food products fortified with oyster shell as natural and inexpensive source could be beneficial for the prevention and treatment of osteoporosis.

The p.Arg86Gln change in GARP2 (glutamic acid-rich protein-2) is a common West African-related polymorphism.

BACKGROUND/AIMS: The aim of the present study is to probe the potential association between previously-reported GARP2 mutations and retinitis pigmentosa (RP) using Scottish RP patients and controls. METHODS: Exons 4, 5 and 8 in DNA from blood or buccal samples (130 autosomal recessive and simplex RP patients, 31 controls) were amplified and analysed for single-strand conformational polymorphism by capillary electrophoresis (CE-SSCP) and confirmed by sequencing. RESULTS: The p.Arg86Gln mutation in exon 4 was found in just one patient (out of 130), and in 10 of the 31 unaffected subjects. All of these occurrences were in people of West African origin (patient and controls). Two polymorphisms in exon 5, p.His100Arg and p.Gly109Gly, and a c.534+20A>G change in the intronic region flanking the 3' end of exon 8 were also found not to be associated with RP. CONCLUSIONS: The Scottish population examined here had no mutations in the GARP2 exons surveyed that could be associated with RP. The p.Arg86Gln mutation actually appears to be a polymorphism common in ethnic West Africans and not associated with RP. This change may provide a useful marker for West African ancestry.

Options available for labelling nucleic acid samples in DNA microarray-based detection methods.

DNA microarrays are considered by many researchers to be the platform of choice for the high-throughput analysis of nucleic acids. Since the past two decades, they have been used constantly as powerful tools in differential gene expression, SNP genotyping, DNA sequencing, gene discovery, disease diagnostic and pathways reconstruction. Several methods have been developed to enable samples of limited amounts of RNA to be quantified. Here we evaluate classical and up-to-date assays made available for labelling those samples. This review also sheds light on the recently developed strategies that ensure high sensitivity such as sample and signal amplification, quantum dot, surface plasmom resonance, nanoparticles and cationinc polythiophenes.