Molecular Design, Spectroscopic, DFT, Pharmacological, and Molecular Docking Studies of Novel Ruthenium(III)-Schiff Base Complex: An Inhibitor of Progression in HepG2 Cells.

A novel ruthenium(III)-pyrimidine Schiff base was synthesized and characterized using different analytical and spectroscopic techniques. Molecular geometries of the ligand and ruthenium complex were investigated using the DFT-B3LYP level of theory. The quantum global reactivity descriptors were also calculated. Various biological and molecular docking studies of the complex are reported to explore its potential application as a therapeutic drug. Cytotoxicity of the complex was screened against cancer colorectal (HCT116), breast (MCF-7 and T47D), and hepatocellular (HepG2) cell lines as well as a human normal cell line (HSF). The complex effectively inhibited the tested cancer cells with variable degree with higher activity towards HepG2 (IC50 values were 29 µM for HepG2, 38.5 µM for T47D, 39.7 µM for HCT, and 46.7 µM for MCF-7 cells). The complex induced apoptosis and cell cycle arrest in the S phase of HepG2 cells. The complex significantly induced the expression of H2AX and caspase 3 and caspase 7 gene and the protein level of caspase 3, as well as inhibited VEGF-A and mTOR/AKT, SND1, and NF-kB gene expression. The molecular docking studies supported the increased total apoptosis of treated HepG2 cells due to strong interaction of the complex with DNA. Additionally, the possible binding interaction of the complex with caspase 3 could be responsible for the elevated activity of caspase 3-treated cells. The score values for the two receptors were -3.25 and -3.91 kcal/mol.

Next generation sequencing of BRCA genes in glioblastoma multiform Egyptian patients: a pilot study.

BACKGROUND: Germ line mutations of BRCA1 and BRCA2 were correlated with a variety of cancer Authors aimed to use next-generation sequencing (NGS) to detect BRCA1 and BRCA2 germ line mutations in glioblastoma multiform (GBM) Egyptian patients. MATERIALS AND METHODS: Genomic DNA was extracted from six GBM cases, amplified using Ion AmpliSeq BRCA1 and BRCA2 panel. DNA libraries were pooled, barcoded and finally sequenced using Ion Torrent Personal Genome Machine sequencer. RESULTS: BRCA1 the previously reported rs1799966, rs1799950, rs16941 were found in five cases and they are in a linkage disequilibrium forming two distinct haplotypes, which might support their role in cancer predisposition. Out of the 18 reported variants in BRCA2, three denovo mutations were detected which leads to frame shift. CONCLUSION: Further studies on large number of GBM patients and control cases to determine BRCA1 and BRCA2 germline mutations and haplotypes; diagnostic and prognostic role are encouraged.

Upregulation of long noncoding RNA Lnc-IRF2-3 and Lnc-ZNF667-AS1 is associated with poor survival in B-chronic lymphocytic leukemia.

BACKGROUND: Lnc-IRF2-3 and Lnc-ZNF667-AS1 were recently studied as a positive biomarker for many tumor cells. However, experimental studies found that they are associated with worse outcomes in B-CLL. METHODS: A prospective case study was conducted on 135 B-CLL patients that were compared to thirty healthy controls. The patients were followed up for 40 months and quantitative measurements of Lnc-IRF2-3 and Lnc-ZNF667-AS1 were measured and compared between the two groups as well as high-risk and low low-risk B-CLL. RESULTS: Lnc-IRF2-3 and Lnc-ZNF667-AS1 had a high specificity (94% and 85%) and sensitivity (85%, 87%), respectively, to differentiate B-CLL from healthy controls. Furthermore, they showed high expression levels in high-risk CLL groups. For survival analysis, there was a negative correlation between overall survival (OS) and progression-free survival (PFS) and both biomarkers. However, it was not evident in multivariate Cox regression analysis; in patients with Lnc-IRF2-3 expression level, >67 had a significant decrease in OS and PFS. However, there is no significant effect for high expression levels of Lnc-ZNF667-AS1 on OS (P = .16) or PFS (P = .48). CONCLUSION: The Lnc-IRF2-3 and Lnc-ZNF667-AS1 are promising prognostic biomarkers in B-CLL.

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.

Genotype-phenotype correlation in 18 Egyptian patients with glutaric acidemia type I.

Glutaric acidemia I (GAI) is an autosomal recessive metabolic disease caused by a deficiency of glutaryl-CoA dehydrogenase enzyme (GCDH). Patients with GAI are characterized by macrocephaly, acute encephalitis-like crises, dystonia and frontotemporal atrophy. In this study, we investigated 18 Egyptian patients that were diagnosed with GAI based on their clinical, neuroradiological, and biochemical profiles. Of the 18 patients, 16 had developmental delay and/or regression, dystonia was prominent in 75% of the cases, and three patients died. Molecular genetics analysis identified 14 different mutations in the GCDH gene in the 18 patients, of the 14 mutations, nine were missense, three were in the 3'-Untranslated Region (3'-UTR), one was nonsense, and one was a silent mutation. Four novel mutations were identified (c.148 T > A; p.Trp50Arg, c.158C > A; p.Pro53Gln, c.1284C > G; p.Ile428Met, and c.1189G > T; p.Glu397*) that were all absent in 300 normal chromosomes. The 3'-UTR mutation (c.*165A > G; rs8012), was the most frequent mutation observed (0.5; 18/36), followed by the most common mutation among Caucasian patients (p.Arg402Trp; rs121434369) with allele frequency of 0.36 (13/36), and the 3'-UTR mutation (c.*288G > T; rs9384, 0.22; 8/16). The p.Arg257Gln mutation was found with allele frequency of ~0.17 (6/36). The marked homozygosity observed in our patients is probably due to the high level of consanguinity that is observed in 100% of the cases. We used nine in silico prediction tools to predict the pathogenicity (SIFT, PhD-SNP, SNAP, Meta-SNP, PolyPhen2, and Align GVGD) and protein stability (I-Mutant2.0, Mupro, and istable) of the nine missense mutants. The mutant p.Arg402Trp was predicted to be most deleterious by all the six pathogenicity prediction tools and destabilizing by all the three-stability prediction tools, and highly conserved by the ConSurf server. Using the clinical, biochemical, family history of the 18 patients, and the in silico analysis of the missense mutations, our study showed a mix of conclusive and inconclusive genotype-phenotype correlations among our patient's cohort and suggests the usefulness of using various sophisticated computational analysis to be utilized for future variant classifications in the genetic clinics.

Molecular diagnosis of Entamoeba spp. versus microscopy in the Great Cairo.

Amoebiasis is a human disease produced by Entamoeba histolytica which causes widespread mortality and morbidity worldwide through diarrheal disease and abscess establishment in parenchymal tissues such as liver, lung, and brain. The true prevalence of infection is unknown for most areas of the world due to the difficulty to characterise Entamoeba histolytica versus other non-pathogenic amoebas with identical morphology, as Entamoeba dispar, and Entamoeba moshkovskii. To overcome microscopy misidentification issues, we tested a nested multiplex polymerase chain reaction (PCR) and a real-time PCR on 194 stool samples collected from incoming dysentery patients in Cairo hospitals diagnosed with E. histolytica by microscopy. Nested PCR showed only 20 (10.3%) samples positive to E. histolytica and 17 (8.7%) to E. dispar. The real-time PCR detected only 19 and 11 samples positive to E. histolytica and E. dispar respectively, showing less sensitivity than the nested PCR. The data show that prevalence of E. histolytica in Cairo is lower when specific diagnosis methods are used instead of traditional microscopy, allowing to differentiate between morphologically identical human amoebas species.

Astaxanthin and Docosahexaenoic Acid Reverse the Toxicity of the Maxi-K (BK) Channel Antagonist Mycotoxin Penitrem A.

Penitrem A (PA) is a food mycotoxin produced by several terrestrial and few marine Penicillium species. PA is a potent tremorgen through selective antagonism of the calcium-dependent potassium BK (Maxi-K) channels. Discovery of natural products that can prevent the toxic effects of PA is important for food safety. Astaxanthin (AST) is a marine natural xanthophyll carotenoid with documented antioxidant activity. Unlike other common antioxidants, AST can cross blood brain barriers (BBBs), inducing neuroprotective effects. Docosahexaenoic acid (DHA) is polyunsaturated ω-3 fatty acid naturally occurring in fish and algae. DHA is essential for normal neurological and cellular development. This study evaluated the protective activity of AST and DHA against PA-induced toxicity, in vitro on Schwann cells CRL-2765 and in vivo in the worm Caenorhbitidis elegans and Sprague Dawley rat models. PA inhibited the viability of Schwann cells, with an IC50 of 22.6 µM. Dose-dependent treatments with 10-100 µM DHA significantly reversed the PA toxicity at its IC50 dose, and improved the survival of Schwann cells to 70.5%-98.8%. Similarly, dose-dependent treatments with 10-20 µM AST reversed the PA toxicity at its IC50 dose and raised these cells' survival to 61.7%-70.5%. BK channel inhibition in the nematode C. elegans is associated with abnormal reversal locomotion. DHA and AST counteracted the in vivo PA BK channel antagonistic activity in the C. elegans model. Rats fed a PA-contaminated diet showed high levels of glutamate (GLU), aspartate (ASP), and gamma amino butyric acid (GABA), with observed necrosis or absence of Purkinjie neurons, typical of PA-induced neurotoxicity. Dopamine (DA), serotonin (5-HT), and norepinephrine (NE) levels were abnormal, Nitric Oxide (NO) and Malondialdehyde (MDA) levels were significantly increased, and total antioxidant capacity (TAC) level in serum and brain homogenates was significantly decreased in PA-treated rats. DHA and AST treatments effectively counteracted the toxic effects of PA and normalized most biochemical parameters in rats. DHA and AST can be useful food additives to prevent and reverse PA food-induced toxicity.

Severe liver disease is caused by HBV rather than HCV in children with hematological malignancies.

UNLABELLED: Patients receiving chemotherapy experience exacerbations of chronic hepatitis B (HBV) or hepatitis C (HCV) viral infections. We examined the pattern of liver disease induced by these infections in 92 children and adolescents with elevated transaminases (median age: 9 years). This included 76 with hematological malignancies (55 ALL, 15 NHL and six Hodgkin's disease) and 16 with thalassemia major. Liver disease was graded: A--occasional hypertransaminemia, B--persistent hypertransaminemia, C--severe hepatitis without encephalopathy, D--fulminant hepatic failure (FHF) and death. Screening included HBsAg, anti-HCV antibody, HBV-DNA and HCV-RNA: 26 had liver biopsies. A total of 60 (79%) patients with malignancies were HBsAg and/or HBV-DNA(+)(genotype D-E) and 47 (62%) were anti-HCV and/or HCV-RNA(+); 33 were coinfected with HBV and HCV. Grade A (n=24) included 16 with HCV and 12 with HBV (six coinfected); 18 with HBV and 11 with HCV (10 coinfected) were graded B (n=22). All grade C (n=25) had HBV with 16 HCV coinfected. FHF and death occurred in five HBV-DNA(+) patients, in four within a month of i.v. methotrexate. Patterns C and D were associated with HBsAg and HBV-DNA (P=0.001 and P<0.001, respectively). In all, 70% of HBV-infected children suffered chemotherapy-associated flares. None of the thalassemics had severe hepatitis exacerbations; 94% had HCV markers with none HBV-DNA(+). One died of progressive cirrhosis. CONCLUSIONS: Children with hematological malignancies have worse liver disease when associated with chronic HBV. FHF occurred in HBsAg/HBV-DNA(+) children following i.v. methotrexate. Early recognition of hepatic dysfunction in HBV carriers is essential in order to reduce incidence of life-threatening complications.

Analysis of Schistosoma mansoni genes using the expressed sequence tag approach.

Expressed sequence tags (ESTs) are partial cDNA sequences read from both ends of random expressed gene fragments used for discovering new genes. DNA libraries from four different developmental stages of Schistosoma mansoni used in this study generated 141 ESTs representing about 2.5% of S. mansoni sequences in dbEST. Sequencing was done by the dideoxy chain termination method. The sequences were submitted to GenBank for homology searching in nonredundant databases using Basic Local Alignment Search Tool for DNA (BLASTN) alignment and for protein (BLASTX) alignment at the National Center for Biotechnology Information (NCBI). Among submitted ESTs, 29 were derived from lambdagt11 sporocyst library, 70 from lambdaZap adult worm library, 31 from lambdaZap cercarial library, and 11 from lambdaZap female B worm library. Homology search revealed that eight (5.6%) ESTs shared homology to previously identified S.mansoni genes in dbEST, 15 (10.6%) are homologous to known genes in other organisms, 116 (81.7%) showed no significant sequence homology in the databases, and the remaining sequences (2.1%) showed low homologies to rRNA or mitochondrial DNA sequences. Thus, among the 141 ESTs studied, 116 sequences are derived from noval, uncharactarized S. mansoni genes. Those 116 ESTs are important for identification of coding regions in the sequences, helping in mapping of schistosome genome, and identifying genes of immunological and pharmacological significance.

Cloning and characterization of a Schistosoma mansoni 1H and 30S clones as two tegumental vaccine candidate antigens.

Two Schistosoma mansoni cDNA clones 30S and 1H were identified by immunoscreening of sporocyst lambdagt11 library and by random sequencing of clones from lambdaZap libraries, respectively. Clone 30S was one of 30 clones identified by an antibody raised against tegument of 3-h schistosomules. The clone was found to encode an 81 amino-acid protein fragment. It was expressed in Escherichia coli as a fusion protein of calculated molecular mass of about 35 kDa with C-terminus of Schistosoma japonicum glutathione-S-transferase (Sj26; about 26 kDa). The recombinant fusion protein was specifically recognized by serum of rabbits immunized with irradiated cercariae. Clone 1H is one of 76 expressed sequence tags derived from an adult worm library. It encodes the complete sequence of a tegumental membrane protein, Sm13. The 104 amino-acid open reading frame encodes a protein with a calculated molecular mass of about 11.9 kDa. Clone 1H was expressed in E. coli as an insoluble fusion protein with Sj26 of about 40 kDa. In Western blots, the fusion protein was recognized by serum from rabbits vaccinated with irradiated cercariae but not by preimmune rabbit sera. The cloning, characterization and expression of those proteins are therefore potentially usefull for vaccine development.