Hossam Murad

Algal sulfated carrageenan inhibits proliferation of MDA-MB-231 cells via apoptosis regulatory genes.

Marine algae are prolific sources of sulfated polysaccharides, which may explain the low incidence of certain cancers in countries that traditionally consume marine food. Breast cancer is one of the most common types of non‑skin cancer in females. In this study, extracted sulfated carrageenan (ESC), predominantly consisting of ι‑carrageenan extracted from the red alga Laurencia papillosa, was characterized using Fourier transform infrared spectrometry. The biological effects of the identified extract were investigated and its potential cytotoxic activity was tested against the MDA‑MB‑231 cancer cell line. The biological biometer of the inhibitory concentration of the polysaccharide‑treated MDA‑MB‑231 cells was determined as 50 µM. Treatment with 50 µM ESC inhibited cell proliferation and promptly induced cell death through nuclear condensation and DNA fragmentation. Characterization of polysaccharide‑treated MDA‑MB‑231 cell death revealed that induction of apoptosis occurred via the activation of the extrinsic apoptotic caspase‑8 gene. The apoptotic signaling pathway was regulated through caspase‑3, caspase‑9, p53, Bax and Bcl‑2 genes. These findings suggest that ESC may serve as a potential therapeutic agent to target breast cancer via prompting apoptosis.

Molecular Update of β-Thalassemia Mutations in the Syrian Population: Identification of Rare β-Thalassemia Mutations

Abstract β-Thalassemia (β-thal) is an autosomal recessive disorder characterized by variable degrees of anemia, bone marrow hyperplasia, splenomegaly, and complications related to the severity of the anemic state. The β-thalassemias result from mutations in and around the β-globin gene (HBB) located as a cluster on the short arm of chromosome 11. In Syria, β-thal is highly prevalent. The main aim of this study was to identify the frequency of HBB mutations in 189 Syrian β-thal patients and carriers of β-thal. Out of the 189 patients and carriers recruited in this study, 181 patients had at least one HBB mutation and eight patients did not show any mutation. The 10 most frequent ones constituted 77.5% of all HBB mutations. These mutations in order of frequency were: IVS-I-110 (G > A) (17.0%), IVS-I-1 (G > A) (14.7%), codon 39 (C > T) (14.4%), IVS-II-1 (G > A) (9.8%), codon 8 (–AA) (6.2%), IVS-I-6 (T > C) (5.2%), IVS-I-5 (G > C) (4.9%), codon 5 (–C) (3.2%), IVS-I-5 (G > A) (3.2%) and codon 37 (G > A) (2.2%). Another 21 mutations were less frequent or sporadic. These results provide important tools for adapting a prenatal molecular diagnostic test for the Syrian population.

Prokaryotic overexpression of TEV–rhGH and characterization of its polyclonal antibody

Recombinant protein technology represents one of the best solutions to achieve rapid, efficient, and cost-effective protein expression and purification of therapeutic proteins. Growth hormone (GH) is an excellent example of these proteins used in the therapy of hormone deficiencies. In this work, a plasmid, pRSET-TEV-rhGH, has been constructed to overexpress recombinant human GH (rhGH) by cloning its gene downstream of an N-terminal 6 × His-tagged polypeptide (43 aa) in the T7 promoter-plasmid pRSET. This polypeptide was cleavable by means of the integrated recognition site for the tobaccos etch virus (TEV) protease, resulting in an rhGH protein at an exact length and sequence. After IPTG induction, this plasmid effectively expressed TEV-rhGH protein (27 kDa) in the cytoplasm of Escherichia coli, which accumulated in the form of inclusion bodies. The 6 × His-tagged protein, with a yield of ~150 mg/L of culture, was purified from the cell extract using metal affinity chromatography, as shown after SDS-PAGE blue staining, and was confirmed by immunoblotting using specific commercial monoclonal antibodies. In order to detect TEV-rhGH, in ELISA and immunoblotting, specific polyclonal antibody, with high titer (~10⁻⁵ fold dilution), was produced in a rabbit and purified using affinity chromatography. Preliminary tests have proved that TEV-rhGH protein and its specific purified IgG antibody could provide valuable tools for rhGH productive and diagnostic purposes.

Mutation spectrum of phenylketonuria in Syrian population: genotype-phenotype correlation.

Characterization of the molecular basis of phenylketonuria (PKU) in Syria has been accomplished through the analysis of 78 unrelated chromosomes from 39 Syrian patients with PKU. Phenylalanine hydroxylase (PAH) gene mutations have been analyzed by using molecular detection methods based on the restriction fragment length polymorphism (RFLP), artificial constructed restriction sites (ACRS) PCR and direct DNA sequencing. 56.4% of the patients had cPKU. A mutation detection rate of 79.49% was achieved and sixteen different mutations were found: missense 56.25%, splice site 37.5%, and frameshift 6.25%. The predominant mutation in this population sample was p.R261Q G>A, p.F55>Lfs and p.R243Q G>A. No mutation in six PKU patients was observed. In 57.9% of patient genotypes, the metabolic phenotype could be predicted. The identification of the mutations in the PAH gene and the genotype-phenotype correlation should facilitate the evaluation of metabolic phenotypes, diagnosis, implementation of optimal dietary therapy, and determination of prognosis in the patients and genetic counseling for the patients relatives.

Frequency of HLA-DRB1 and HLA-DQB1 Alleles and Haplotype Association in Syrian Population

ABSTRACT The study of Human Leukocyte Antigen (HLA) system is very important in health and diseases. As the HLA loci are the most polymorphic in the human genome, it plays a very important role in the immune responses to self and nonself antigens. In the light of the growing importance of typing the HLA alleles in transplantation, autoimmune diseases, cancer, and many other diseases, we studied 225 unrelated healthy Syrian subjects for their HLA class II genotypes in an attempt to reveal the distribution of the HLA (DRB1-DQB1) alleles in the general Syrian population. Our results revealed that the most common alleles for the DRB1 locus were DRB1*11 (26.4%), DRB1*04 (14%), and DRB1*07 (12%). However, the most frequent alleles for the DQB1 locus were DQB1*03 (40.9%) and DQB1*05 (25.1%). The frequent of two-locus haplotypes carry the most frequent alleles at these loci. The most frequently detected class II ‘‘haplotypes’’ are DRB1*11-DQB1*03 (8.9%), DRB1*01-DQB1*05 (3.6%), and DRB1*04-DQB1*03 (2.7%). Compared with other populations, our result, deduced from the analysis of genetic distances and the construction of neighbor-joining (NJ) dendrogram, and principal component analysis (PCA) indicates that Syrians are related to Middle Eastern populations. Our data about the Syrian population will aid researchers in studying the relation of HLA class II with different diseases in a Syrian population and will add to the available international literature associated with these loci.

Prenatal Molecular Diagnosis of β-Thalassemia and Sickle Cell Anemia in the Syrian Population

Abstract Our objective was to evaluate the prenatal diagnosis (PND) of β-thalassemia (β-thal) and sickle cell anemia in Syria. Mutations detected from blood of at-risk couples and 55 amniotic fluid samples collected at the second trimester of pregnancy (14–22 weeks’ gestation) were characterized. Molecular screening and direct DNA sequencing of the HBB gene was carried out. DNA analyses showed 14 affected fetuses (25.45%), 32 (58.18%) carriers and eight (14.54%) normal fetuses. It appears that 20.0% of individuals carried the sickle cell anemia mutation and 80.0% carried the β-thal mutation. Thirteen different known mutations were detected in the fetuses. The most common mutations were: IVS-II-1 (G > A), codon 39 (C > T)], IVS-I-110 (G > A), IVS-I-1 (G > A) and IVS-I-5 (G > C). The Hb S [β6(A3)Glu → Val; HBB: c.20A > T] mutation was the only abnormal hemoglobin (Hb) that was found. The results point to a successful future for PND of β-thal and sickle cell anemia in Syria, using a rapid and accurate molecular method. We hope that this method will be used as a common application approach to decrease the incidence of β-thal major (β-TM).

Characterization of Annexin V Fusion with the Superfolder GFP in Liposomes Binding and Apoptosis Detection

Programed cell death is a critical and unavoidable part of life. One of the most widely used markers for dying cells, by apoptosis or pyroptosis, is the redistribution of phosphatidylserine (PS) from the inner to the outer plasma membrane leaflet. Annexin V protein is a sensitive and specific probe to mark this event because of its high affinity to the exposed PS. Beyond that, annexin V can bind to any PS-containing phospholipid bilayer of almost all tiny forms of membranous vesicles like blood platelets, exosomes, or even nanostructured liposomes. In this work, recombinant human annexin V was produced as a fusion with a highly fluorescent superfolder derivative of the green fluorescent protein (sfGFP) in Escherichia coli. The fusion protein(sfGFP-ANXV, 64 kDa), annexin V (ANXV, 40 kDa), and sfGFP (27 kDa) were separately produced after cloning their encoding genes in pRSET plasmid, and all proteins were expressed in a soluble form, then purified in high yields because of their N-terminal 6× His tag (~150 mg of pure protein per 1 L culture). Superiority of this fluorescent fusion protein over fluorescein-conjugated annexin V was demonstrated in binding to phospholipids (and their liposomes), prepared from natural sources (soya bean and egg yolk) that have different content of PS, by using different methods including ELISA, dot-blotting, surface plasmon resonance, and flow cytometry. We also applied fluorescent annexin V in the detection of apoptotic cells by flow cytometry and fluorescent microscopy. Interestingly, sfGFP-ANXV fusion was more sensitive to early apoptotic stressed HeLa cells than fluorescein-conjugated-ANXV. This highly expressed and functional sfGFP-ANXV fusion protein provides a promising ready-to-use molecular tool for quantifying liposomes (or similarly exosomes) and detecting apoptosis in cells.

Effects of γ-radiation on cell growth, cell cycle and promoter methylation of 22 cell cycle genes in the 1321NI astrocytoma cell line

PURPOSE DNA damage caused by radiation initiates biological responses affecting cell fate. DNA methylation regulates gene expression and modulates DNA damage pathways. Alterations in the methylation profiles of cell cycle regulating genes may control cell response to radiation. In this study we investigated the effect of ionizing radiation on the methylation levels of 22 cell cycle regulating genes in correlation with gene expression in 1321NI astrocytoma cell line. METHODS 1321NI cells were irradiated with 2, 5 or 10Gy doses then analyzed after 24, 48 and 72h for cell viability using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliu bromide) assay. Flow cytometry were used to study the effect of 10Gy irradiation on cell cycle. EpiTect Methyl II PCR Array was used to identify differentially methylated genes in irradiated cells. Changes in gene expression was determined by qPCR. Azacytidine treatment was used to determine whether DNA methylation affectes gene expression. RESULTS Our results showed that irradiation decreased cell viability and caused cell cycle arrest at G2/M. Out of 22 genes tested, only CCNF and RAD9A showed some increase in DNA methylation (3.59% and 3.62%, respectively) after 10Gy irradiation, and this increase coincided with downregulation of both genes (by 4 and 2 fold, respectively). TREATMENT with azacytidine confirmed that expression of CCNF and RAD9A genes was regulated by methylation. CONCLUSIONS 1321NI cell line is highly radioresistant and that irradiation of these cells with a 10Gy dose increases DNA methylation of CCNF and RAD9A genes. This dose down-regulates these genes, favoring G2/M arrest.

Effects of fenofibrate on Semaphorin 6B gene expression in rat skeletal muscle

Semaphorin 6B is a member of the semaphorin family of signaling proteins, which is implicated in a variety of biological processes, such as axon guidance and muscle regeneration. PPARα is known to be a regulator of semaphorin 6B in human cancer cell lines. In this study, we examined the effect of fenofibrate as a PPARα activator on the expression of the Sema6B gene in rat skeletal muscle. A total of 18 rats were divided into two groups: one fed with standard chow (control group), and the other with fenofibrate chow (treatment group) for 4 weeks by gavage with 30 mg/kg per day. The expression of rSema6B mRNA was analyzed by qPCR. rSema6B protein content in rat skeletal muscle was measured by Western blotting. A significant down-regulation of rSema6B was observed at both the mRNA and protein levels. Using the ChIP assay, a putative PPARα binding region (PPRE) was identified in the rSema6B promoter. We conclude that the expression of Sema6B is down-regulated by fenofibarte in rat skeletal muscle.

Geographical distribution of β-globin gene mutations in Syria

ABSTRACT Objectives: β-Thalassemia disease is caused by mutations in the β-globin gene. This is considered as one of the common genetic disorders in Syria. The aim of this study was to identify the geographical distribution of the β-thalassemia mutations in Syria. Methods: β-Globin gene mutations were characterized in 636 affected patients and 94 unrelated carriers using the amplification refractory mutations system-polymerase chain reaction technique and DNA sequencing. Results: The study has revealed the presence of 38 β-globin gene mutations responsible for β-thalassemia in Syria. Important differences in regional distribution were observed. IVS-I.110 [G > A] (22.2%), IVS-I.1 [G > A] (17.8%), Cd 39 [C > T] (8.2%), IVS-II.1 [G > A] (7.6%), IVS-I.6 [T > C] (7.1%), Cd 8 [−AA] (6%), Cd 5 [−CT] (5.6%) and IVS-I.5 [G > C] (4.1%) were the eight predominant mutations found in our study. The coastal region had higher relative frequencies (37.9 and 22%) than other regions. A clear drift in the distribution of the third common Cd 39 [C > T] mutation in the northeast region (34.8%) to the northwest region (2.5%) was noted, while the IVS-I.5 [G > C] mutation has the highest prevalence in north regions. The IVS-I.6 [T > C] mutation had a distinct frequency in the middle region. Ten mutations −86 [C > G], −31 [A > G], −29 [A > G], 5′UTR; +22 [G > A], CAP + 1 [A > C], Codon 5/6 [−TG], IVS-I (−3) or codon 29 [C > T], IVS-I.2 [T > A], IVS-I.128 [T > G] and IVS-II.705 [T > G] were found in Syria for the first time. Conclusions: These data will significantly facilitate the population screening, genetic counseling and prenatal diagnosis in Syrian population.