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Correlation between IL28B/TLR4 genetic variants and HCC development with/without DAAs treatment in chronic HCV patients

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Genes & Diseases 2020年第3期7卷 392-400页

作者： Ghada M.Salum Reham M.Dawood Mai Abd el-Meguid Noha E.Ibrahim Ashraf O.Abdel Aziz mostafa k.el awady Department of Microbial Biotechnology Genetic Engineering DivisionNational Research Centre33 EL Bohouth St.(former El Tahrir St.)DokkiGizaP.O.12622Egypt Department of Tropical Medicine Faculty of MedicineCairo UniversityGizaP.O.12622Egypt

In Egypt,Sofosbuvir(SOF)in combination with Dataclasvir(DCV)is the broadly used DAAs with excellent therapeutic *** study is designed to explore the relation between IL28B/TLR4 genetic variants and each of the followings;HCC development post SOF/DCV treatment,progression to HCC in naı¨ve patients and SOF/DCV therapy outcome.A total of 493 blood samples were collected(controls(n = 70);HCV patients treated with SOF/DCV(n = 252)of whom 65 patients developed HCC,187 patients didn’t develop HCC(125 responders,62 relapsers);naı¨ve HCV patients(n = 171)had early(n = 48),late liver fibrosis(n = 21)and HCC(n = 102)).Both SNPs were genotyped using a TaqMan 50 allelic discrimination *** IL28B rs12979860 SNP,the C allele was significantly correlating with the response rate more than T allele(OR 1.9,95%CI 1.29e2.9,p=0.004),while at TLR4 rs4986791 SNP,no association was found(OR 6.5,95%0.57e75.28,p = 0.09).Both SNPs couldn’t detect the probability for HCC emergence after *** naı¨ve patients,the protective alleles were detected in their lowest frequency in HCC patients(p = 0.1,for rs12979860 and,p = 0.001 for rs4986791).SOF/DCV combination improved SVR rates in HCV genotype 4a infected patients regardless of IL28B genotype,with the best rates in those lacking the T allele.

关键词： DAAs DCV HCV HCC IL28B SOF TLR4

Positional effect of mutations in 5'UTR of hepatitis C virus 4a on patients' response to therapy

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World Journal of Gastroenterology 2009年第12期15卷 1480-1486页

作者： mostafa k el awady Hassan M Azzazy Ahmed M Fahmy Sherif M Shawky Noha G Badreldin Samar S Yossef Moataza H Omran Abdel Rahman N Zekri Said A Goueli Department of Biomedical Technology National Research CenterDokkiCairo 12622Egypt Department of Chemistry and Yousef Jameel Science and Technology Research Center the American University in CairoCairo 11511Egypt Structural biology department Free University of AmsterdamDe Boelelaan 10851081 HV AmsterdamThe Netherlands National Cancer Institute Cairo 11796Egypt Department of Pathology and Laboratory Medicine University of WisconsinMadisonWI 53711United States

AIM： To investigate the effects of mutations in domain Ⅲ of the hepatitis C virus （HCV） internal ribosome entry sequences （IRES） on the response of chronic HCV genotype 4a patients to interferon therapy.METHODS： HCV RNA was extracted from 19 chronic HCV 4a patients receiving interferon/ribavirin therapy who showed dramatic differences in their response to combination therapy after initial viral clearance. IRES domain Ⅲ was cloned and 15 clones for each patient were sequenced. The obtained sequences were aligned with genotype 4a prototype using the ClustaIW program and mutations scored. Prediction of stem-loop secondary structure and thermodynamic stability of the major quasispecies in each patient was performed using the MFOLD 3.2 program with Turner energies and selected constraints on base pairing.RESULTS： Analysis of RNA secondary structure revealed that insertions in domain Ⅲ altered WatsonCrick base pairing of stems and reduced molecular stability of RNA, which may ultimately reduce binding affinity to ribosomal proteins. Insertion mutations in domain - were statistically more prevalent in sustained viral response patients （SVR, n = 14） as compared to breakthrough （BT, n = 5） patients.CONCLUSION： The influence of mutations within domain Ⅲ on the response of HCV patients to combination therapy depends primarily on the position, but not the frequency, of these mutations within IRES domain Ⅲ.

关键词： Hepatitis C virus Internal ribosome entrysequences Domain Ⅲ Genotype 4a Ribosomal sub-unit Interferon therapy RNA folding

Association between low molecular polypeptide 7 single nucleotide polymorphism and response to therapy in hepatitis C virus infection

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World Journal of Hepatology 2013年第3期5卷 97-103页

作者： Moataza H Omran Basma E Fotouh Samar S Youssef Noha E Ibrahim Wael Nabil el-Sayed M Mahdy Wafaa G Shosha mostafa k el-awady Microbial Biotechnology Department Genetic Engineering Division National Research Center Chemistry Department Faculty of Science Helwan University

AIM: To investigate the relationship between low molecular polypeptide-7 (LMP-7) gene polymorphism and response to interferon (IFN) therapy in chronic hepatitis C virus (HCV) patients. METHODS: LMP-7 polymorphism at codon 49 with nucleotide substitution from A to C was amplified in 104 chronic HCV patients of genotype 4. The amplicons were digested with restriction endonuclease Bsm I and the produced restriction fragment length polymorphism was analyzed. Patients received IFN + regional blood volume therapy for 48 wk and the frequency of thissingle nucleotide polymorphism (SNP) was statistically correlated with treatment response. The exclusion criteria for these patients were stated by the national health program for treating viral hepatitis. Main exclusion criteria included co-infection with hepatitis B virus or schistosomiasis, thyroid dysfunction, uncontrolled diabetes mellitus, history of long term drug or alcohol intake and autoimmune hepatitis. Multivariate analyses were done to correlate LMP-7 SNP plus several factors such as age, gender, weight, serum alpha-fetoprotein (AFP) and alanine aminotransferase levels, liver activity, fibrosis score and viral load with response to therapy. RESULTS: The data presented in this study clearly demonstrated statistically significant differences between sustained virological response (SVR) (defined as the absence of HCV RNA levels in the patient's sera at least 6 mo after discontinuation of treatment) and non-response (NR) (where HCV RNA levels in the patient's sera never become undetectable for 6 mo during or after treatment). Variables were described as odds ratio with 95%CI. The data were considered significant if P values were ≤ 0.05; highly significant if P ele were associated with SVR, while the other two genotypes C/A and A/A were associated with NR patients, 83.3% and 64.3% respectively, showing that g

关键词： Hepatitis C virus Interferon therapy Low molecular mass polypeptide Host gene Single nucleotide polymorphism

Antibody to el peptide of hepatitis C virus genotype 4 inhibits virus binding and entry to HepG2 cells in vitro

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World Journal of Gastroenterology 2006年第16期12卷 2530-2535页

作者： mostafa k el-awady Ashraf A Tabll khaled Atef Samar S Yousef Moataza H Omran Yasmin EI-Abd Noha G Bader-eldin Ahmad M Salem Samir F Zohny Wael T EI-Garf Department of Biomedical Technology National Research CenterCairoEgypt Department of Biochemistry Faculty of ScienceAin Shams UniversityCairoEgypt

AIM： To analyze the neutralizing activity of antibodies against E1 region of hepatitis C virus （HCV）. Specific polydonal antibody was raised via immunization of New Zealand rabbits with a synthetic peptide that had been derived from the E1 region of HCV and was shown to be highly conserved among HCV published genotypes. METHODS： Hyper-immune HCV E1 antibodies were incubated over night at 4 ℃ with serum samples positive for HCV RNA, with viral loads ranging from 615 to 3.2 million IU/mL. Treated sera were incubated with HepG2 cells for 90 min. Blocking of viral binding and entry into cells by anti E1 antibody were tested by means of RTPCR and flow cytometry. RESULTS： Direct immunostaining using FITC conjugated E1 antibody followed by Flow cytometric analysis showed reduced mean fluorescence intensity in samples pre-incubated with E1 antibody compared with untreated samples. Furthermore, 13 out of 18 positive sera （72%） showed complete inhibition of infectivity as detected by RT-PCR. CONCLUSION： In house produced E1 antibody, blocks binding and entry of HCV virion infection to target cells suggesting the involvement of this epitope in virus binding and entry. Isolation of these antibodies that block virus attachment to human cells are useful as therapeutic reagents.

关键词： Flow cytometry Hepatitis C virus E1 envelope Therapeutic antibodies Direct immuno-fluorescence HepG2 cells

Flow cytometric detection of hepatitis C virus antigens in infected peripheral blood leukocytes: Binding and entry

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World Journal of Gastroenterology 2005年第33期11卷 5203-5208页

作者： mostafa k el-awady Ashraf A Tabll el-Rashdy M Redwan Samar Youssef Moataza H Omran Fouad Thakeb Maha el-Demellawy Department of Biomedical Technology National Research Center Tahrir Street Dokki Cairo Egypt Genetic Engineering and Biotechnology Research Institute New Borg el-Arab City Alexandria Egypt Department of Gastroenterology-Hepatology Faculty of Medicine Cairo University Egypt Genetic Engineering and Biotechnology Research Institute New Borg el-Arab City Alexandria Egypt

AIM： We designed two synthetic-core-specific peptides core 1 （C1） and core 2 （C2）, and an E1-specific peptide （el）. We produced specific polyclonal antibodies against these peptides and used the antibodies for detection of HCV antigens on surface and within infected peripheral blood leukocytes. METHODS： Peripheral blood from a healthy individual who tested negative for HCV RNA was incubated with HCV type 4 infected serum for i h and 24 h at 37 ℃. Cells were stained by direct and indirect immunofluorescence and measured by flow cytometry. RESULTS： After 1 h of incubation, antibodies against C1, C2, and el detected HCV antigens on the surface of 27%, 26% and 73% of monocytes respectively, while 10%, 5% and 9% of lymphocytes were positive with anti-C1, anti-C2 and anti-E1 respectively. Only 1-3% of granulocytes showed positive staining with anti-C1, anti-C2 and anti E1 antibodies. After 24 h of incubation, we found no surface staining with anti-C1, anti-C2 or anti-E1. Direct immunostaining using anti-C2 could not detect intracellular HCV antigens, after 1 h of incubation with the virus, while after 24 h of incubation, 28% of infected cells showed positive staining. Only plus strand RNA was detectable intracellularly as early as 1 h after incubation, and remained detectable throughout 48 h post-infection. Interestingly, minus RNA strand could not be detected after 1 h, but became strongly detectable intracellularly after 24 h post-infection. CONCLUSION： Monocytes and lymphocytes are the preferred target cells for HCV infection in peripheral blood leukocytes. Our specific anti-core and anti-E1 antibodies are valuable reagents for demonstration of HCV cell cycle. Also, HCV is capable of infecting and replicating in peripheral blood mononuclear cells as confirmed by detection of minus strand HCV RNA as well as intracellular staining of core HCV antigen.

关键词： Flow cytometry Hepatitis C virus Envelope Core Antibodies Indirect immunofluorescence Minus and plus RNA strand Peripheral blood mononudear cells

HepG2 cells support viral replication and gene expression of hepatitis C virus genotype 4 in vitro

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World Journal of Gastroenterology 2006年第30期12卷 4836-4842页

作者： mostafa k el-awady Ashraf A Tabll Yasmine S el-Abd Mahmoud M Bahgat Hussein A Shoeb Samar S Youssef Noha G Bader el Din el-Rashdy M Redwan Maha el-Demellawy Moataza H Omran Wael T el-Garf Said A Goueli Department of Biomedical Technology National Research Center Tahrir Street PO 12622 Dokki Cairo Egypt Department of Medicinal Chemistry Na- tional Research Center Tahrir Street PO 12622 Dokki Cairo Egypt Department of Microbiology and Immunology Faculty of Pharmacy Cairo University Cairo Egypt Genetic Engineering and Biotechnology Research Institute New Borg El-Arab City Alexandria Egypt Department of Pathology School of Medicine University of Wisconsin Madison WI United States

AIM： TO establish a cell culture system with longterm replication of hepatitis C virus （HCV） genome and expression of viral antigens in vitro. METHODS： HepG2 cell line was tested for its susceptibility to HCV by incubation with a serum from a patient with chronic hepatitis C. Cells and supernatant were harvested at various time points during the culture. Culture supernatant was tested for its ability to infect na＇ive cells. The presence of minus （antisense） RNA strand, and the detection of core and E1 antigens in cells were examined by RT-PCR and immunological techniques （flow cytometry and Western blot） respectively. RESULTS： The intracellular HCV RNA was first detected on d 3 after infection and then could be consistently detected in both cells and supernatant over a period of at least three months. The fresh cells could be infected with supernatant from cultured infected cells. Flow cytometric analysis showed surface and intracellular HCV antigen expression using in house made polyclonal antibodies （anti-core, and anti-E1）. Western blot analysis showed the expression of a cluster of immunogenic peptides at molecular weights extended between 31 and 45 kDa in an one month old culture of infected cells whereas this cluster was undetectable in uninfected HepG2 cells. CONCLUSION： HepG2 cell line is not only susceptible to HCV infection but also supports its replication in vitro. Expression of HCV structural proteins can be detected in infected HepG2 cells. These cells are also capable of shedding viral particles into culture media which in turn become infectious to uninfected cells.

关键词： Hepatitis C virus In vitro propagation Genomic replication Gene expression HepG2 cells

Tumor necrosis factor-α-G308A polymorphism is associated with liver pathological changes in hepatitis C virus patients

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World Journal of Gastroenterology 2016年第34期22卷 7767-7777页

作者： Noha G Bader el Din Sally Farouk Reem el-Shenawy Marwa k Ibrahim Reham M Dawood mostafa M elhady Ahmed M Salem Naglaa Zayed Ahmed khairy mostafa k el awady Department of Microbial Biotechnology National Research Centre Department of Biochemistry Faculty of ScienceAin Shams University Endemic Medicine Department Faculty of MedicineCairo University

AIM To investigate the association of tumor necrosis factor alpha(TNFα)-G308 A polymorphism with different liver pathological changes in treatment-na?ve Egyptian patients infected with hepatitis C virus(HCV) genotype *** This study included 180 subjects,composed of 120 treatment-na?ve chronic HCV patients with different fibrosis grades(F0-F4) and 60 healthy controls. The TNFα-G308 A region was amplified by PCR and the different genotypes were detected by restriction fragment length polymorphism analysis. The TNFα protein was detected by enzyme-linked immunosorbent assay. The influence of different TNFα-G308 A genotypes on TNFα expression and liver disease progression were statistically analyzed. The OR and 95%CI were calculated to assess the relative risk *** Current data showed that the TNFα-G308 A SNP frequency was significantly different between controls and HCV infected patients(P = 0.001). Both the AA genotype and A allele were significantly higher in late fibrosis patients(F2-F4,n = 60) than in early fibrosis patients(F0-F1,n = 60)(P = 0.05,0.04 respectively). Moreover,the GA or AA genotypes increased the TNFα serum level greater than the GG genotype(P = 0.002). The results showed a clear association between severe liver pathological conditions(inflammation,steatosis and fibrosis) and(GA + AA) genotypes(P = 0.035,0.03,0.04 respectively). The stepwise logistic regression analysis showed that the TNFα genotypes(GA + AA) were significantly associated with liver inflammation(OR = 3.776,95%CI: 1.399-10.194,P = 0.009),severe steatosis(OR = 4.49,95%CI: 1.441-14.0,P = 0.010) and fibrosis progression(OR = 2.84,95%CI: 1.080-7.472,P = 0.034). Also,the A allele was an independent risk factor for liver inflammation(P = 0.003),steatosis(P = 0.003) and fibrosis(P = 0.014). CONCLUSION TNFα SNP at nucleotide-308 represents an important genetic marker that can be used for the prognosis of different liver pathological changes in HCV infected

关键词： Hepatitis C virus immune response Tumor necrosis factor alpha Single nucleotide polymorphisms Cytokine expression Liver disease progression

IL28B polymorphism and cytomegalovirus predict response to treatment in Egyptian HCV type 4 patients

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World Journal of Gastroenterology 2013年第2期19卷 290-298页

作者： mostafa k el awady Noha G Bader el Din Ashraf Tabll Yaser el Hosary Ashraf O Abdel Aziz Hesham el khayat Mohsen Salama Tawfeek H Abdelhafez Department of Microbial Biotechnology National Research CenterCairoGiza 12622Egypt Department of Internal Medicin National Research CenterCairoGiza 12622Egypt Department of Tropical Medicine Cairo UniversityGiza 12622Egypt Theodor Bilharz Research Institute Giza 12622Egypt National Liver Institute Shebeen El Kom 32512Egypt

AIM:To test whether the status of positive cytomegalovirus(CMV) DNA detection adds to the predictive value of IL28B and to further categorize C/T allele ***:This study included 166 chronic hepatitis C(CHC) patients who received combined interferon and ribavirin therapy for 48 wk,84 spontaneous hepatitis C virus(HCV) resolvers who were positive for IgG anti-HCV antibody and negative for HCV RNA,and 100 healthy subjects who were negative for both HCV antibodies and RNA as *** DNA from peripheral blood was used for IL28B rs.12979860 single nucleotide polymorphism(SNP) and CMV DNA detection.A 139 bp fragment containing IL28B SNP was amplified in all subjects by polymerase chain reaction using a specifically designed *** the IL28B rs.12979860 SNP was detected by restriction fragment length polymorphism(RFLP) *** presence of CMV DNA was tested by amplification of the gB1 gene using nested polymerase chain *** role of CMV and IL28B rs.12979860 SNP genotypes in determining the response rate to combined interferon therapy and clinical status of patients were statistically ***:Current data showed that 67% of patients carrying the IL28B 12979860 C/C allele had a sustained viral response(SVR) while the genotypes C/T and TT were associated with lower SVR rates,50% and 48%,*** rates for the C/C allele were lower than other HCV genotypes and/or other *** CC was associated with the response to interferon(P = 0.025).Genotype C/C was reduced from 48% in controls to 14% in CHC patients suggesting its protective role against progression to *** majority of spontaneously cleared subjects(86%) were C/C,confirming its protective *** C/T allele was present in 71% of CHC patients compared with 38% of controls,so the use of IL28B SNP genotyping only in these patients may be of little value as a predictor of *** reactivation occurred in 40% of CHC ***-infection with

关键词： Hepatitis C Interleukin 28B Genetic polymorphisms Human cytomegalovirus Spontaneous clearance

Enhancing sustainability in phosphate ore processing:Performance of frying oil as alternative flotation collector for carbonate removal

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International Journal of Mining Science and Technology 2024年第4期34卷 557-571页

作者： Asmae el-bahi Yassine Taha Yassine Ait-khouia Abdellatif elghali mostafa Benzaazoua Geology&Sustainable Mining Institute(GSMI) Mohammed VI Polytechnic UniversityBen Guerir 43150Morocco

Recycling waste frying oils for the synthesis of flotation reagents presents a promising avenue for sustainable waste ***,it offers a cost-effective solution for crafting a specialized collector designed to efficiently remove carbonates and enhance phosphate enrichment in froth flotation *** study focuses on the synthesis of an anionic collector using the saponification reaction of a frying oil sample,subsequently applied to the flotation of calcite and *** elucidate the adsorption mechanisms of the frying oil collector(FrOC)and sodium oleate,a reference collector,on fluorapatite,calcite,dolomite,and quartz surfaces,comprehensive experiments were conducted,including zeta potential measurements and Fourier transform infrared *** revealed diverse adsorption affinities of the molecules towards these *** assess the practical performance of the collector,flotation tests were conducted using a natural phosphate ore mixture,employing a BoxBehnken experimental ***,under optimized conditions(pH 9,1000 g/t of FrOC,3.5 min of conditioning,and 6 min of flotation),FrOC exhibited excellent performance,with calcite and dolomite recoveries exceeding 80%,while apatite recovery in the concentrate fraction remained below 10%.This work exemplifies both circular economy practices and the distinctive approach to sustainable mineral processing.

关键词： Frying oils recycling Phosphate beneficiation Flotation separation Green surfactants Sustainability