Yousef Haj-Ahmad

Potential Urinary miRNA Biomarker Candidates for the Accurate Detection of Prostate Cancer among Benign Prostatic Hyperplasia Patients

MicroRNAs (miRNAs) are a class of short (~22nt), single stranded RNA molecules that function as post-transcriptional regulators of gene expression. MiRNAs can regulate a variety of important biological pathways, including: cellular proliferation, differentiation and apoptosis. Profiling of miRNA expression patterns was shown to be more useful than the equivalent mRNA profiles for characterizing poorly differentiated tumours. As such, miRNA expression “signatures” are expected to offer serious potential for diagnosing and prognosing cancers of any provenance. The aim of this study was to investigate the potential of using deregulation of urinary miRNAs in order to detect Prostate Cancer (PCa) among Benign Prostatic Hyperplasia (BPH). To identify the miRNA signatures specific for PCa, miRNA expression profiling of 8 PCa patients, 12 BPH patients and 10 healthy males was carried out using whole genome expression profiling. Differential expression of two individual miRNAs between healthy males and BPH patients was detected and found to possibly target genes related to PCa development and progression. The sensitivity and specificity of miR-1825 for detecting PCa among BPH individuals was found to be 60% and 69%, respectively. Whereas, the sensitivity and specificity of miR-484 were 80% and 19%, respectively. Additionally, the sensitivity and specificity for miR-1825/484 in tandem were 45% and 75%, respectively. The proposed PCa miRNA signatures may therefore be of great value for the accurate diagnosis of PCa and BPH. This exploratory study has identified several possible targets that merit further investigation towards the development and validation of diagnostically useful, non-invasive, urine-based tests that might not only help diagnose PCa but also possibly help differentiate it from BPH.

Sequence and Sequence Analysis of E1 and pIX Regions of the BAV3 Genome

Bovine adenovirus type 3 (BAV3) is a DNA virus that causes respiratory and gastrointestinal disorders in cattle. We have sequenced the extreme left end of BAV3 genome (0-11.7 map units). Partial analysis of the nucleotide sequence revealed 19 potential open reading frames (ORFs) that could encode for polypeptides of 50 or more amino acids. Four of these ORFs show homology to known adenovirus polypeptides. The four BAV3 ORFs are located in approximately the same area as the Ad5 E1a, E1b, and pIX ORFs. ORF 1 has the potential to code for a 208 amino acid long polypeptide that is 75.5% homologous to the E1a conserved region III of Ad5. ORFs 2 and 3 encode 157 and 420 amino acid long polypeptide, respectively. The 157 amino acid polypeptides exhibits 69.3% homology to the Ad5 small T antigen, and the 420 amino acid polypeptide exhibits 73% homology to the large T antigen of Ad5. ORF4 has the potential to code for a 125 amino acid long polypeptide that has 73% homology to the hexon-associated pIX of Ad7.

Differences in the quantity of DNA found in the urine and saliva of smokers versus nonsmokers: implications for the timing of epigenetic events

TP53 is a tumor-suppressor gene coding for p53, a protein responsible for cell-cycle arrest and DNA repair. Smoking has been demonstrated to lead to the methylation of tumor-suppressor genes in noncancerous lung biopsy tissues of smokers, and in bodily fluids, promoter hypermethylation occurs very early in the progression of cancer. Thus, DNA methylation changes may be initiated long before cells become cancerous. As this association has never been explored in young, healthy individuals, we decided to look at DNA isolated from urine and saliva samples taken from young male and female smoking and nonsmoking participants. While p53 methylation was not found in any of the samples tested, differences in DNA concentration between the two groups may shed light on the timing of epigenetic alterations, as well as better explain why the negative impact of smoking is not often found in young, healthy adults.

Promising Urinary Protein Biomarkers for the Early Detection of Hepatocellular Carcinoma among High-Risk Hepatitis C Virus Egyptian Patients

Hepatocellular Carcinoma is a major healthcare problem, representing the third most common cause of cancer-related mortality worldwide. There are 130 million Hepatitis C virus infected patients worldwide who are at a high-risk for developing Hepatocellular Carcinoma. Due to the fact that reliable parameters and/or tools for the early detection of Hepatocellular Carcinoma among high-risk individuals are severely lacking, Hepatocellular Carcinoma patients are always diagnosed at a late stage where surgical solutions or effective treatment are not possible. Urine was collected from 106 Hepatitis C infected patients patients, 32 of whom had already developed Hepatocellular Carcinoma and 74 patients who were diagnosed as Hepatocellular Carcinoma -free at the time of initial sample collection. In addition to these patients, urine samples were also collected from 12 healthy control individuals. Total urinary proteins were isolated from the urine samples and LC-MS/MS was used to identify potential protein HCC biomarker candidates. This was followed by validating relative expression levels of proteins present in urine among all the patients using quantitative real time-PCR. This approach revealed that significant over-expression of three proteins: DJ-1, Chromatin Assembly Factor-1 (CAF-1) and Heat Shock Protein 60 (HSP60), was a characteristic event among Hepatocellular Carcinoma - post Hepatitis C virus infected patients. As a single-based Hepatocellular Carcinoma biomarker, CAF-1 over-expression identified Hepatocellular Carcinoma among Hepatitis C virus infected patients with a specificity of 90%, sensitivity of 66% and with an overall diagnostic accuracy of 78%. Moreover, the CAF-1/HSP60 tandem identified Hepatocellular Carcinoma among Hepatitis C virus infected patients with a specificity of 92%, sensitivity of 61% and with an overall diagnostic accuracy of 77%.

Sequence Analysis of the Putative E3 Region of Bovine Adenovirus Type 2

Adenoviruses are nonenveloped icosahedral-shaped particles. The double-stranded viral DNA genome contains four major early transcription units, designated E1 (a and b), E2 (a and b), E3 and E4, which are expressed in a regulated manner soon after infection. The gene products of the region E3, shown to be nonessential for viral replication in vitro, are believed to be involved in counteracting host immunosurveillance. Human adenovirus type 5 DNA sequences of transcription units L4 and L5 adjacent to E3 were used to localize E3 within the bovine adenovirus type 2. The DNA sequences between 74.8 and 84.4 mu containing E3 and the fiber gene were determined. The E3 region was found to consist of about 2.3 kb pairs and to encode four proteins longer than 60 amino acids. However, these four open reading frames did not show significant homology to any other known adenovirus DNA or protein sequence.

Cloning and sequencing of the bovine adenovirus type 2 early region 4.

Bovine adenovirus type 2 (BAV2) is a medium size double-stranded DNA virus which infects both bovine and ovine species, resulting in mild respiratory and gastrointestinal disorders. To better understand the virus and its growth characteristics in Madin-Darby bovine kidney (MDBK) cells, we have cloned and sequenced the extreme right-end segment of the BAV2 genome (90.5-100 map units). Analysis of the nucleotide sequence revealed 40 potential open reading frames (ORFs) with coding capacity for polypeptides that are 25 or more amino acid (aa) residues long. Six of these ORFs encode polypeptides that show homology to well-characterized early region 4 (E4) proteins of human adenovirus type 2 (Ad2) and Ad12. ORF1 has the potential to encode a 114 aa long polypeptide that is 54% homologous to the E4 14kDa protein of Ad2. ORF2 encodes a 78 aa long polypeptide that exhibits 40% homology to the E4 13 kDa protein of Ad2. ORFs 3-6 encode polypeptides that have homology to the E4 34 kDa protein encoded by ORF6 of Ad2 and Ad12. ORFs 3, 4 and 5 encode 128, 96 and 31 aa long polypeptides, respectively. The 128-aa polypeptide exhibits 59% homology, while the 96 and 31 aa long polypeptides exhibit 61% and 70% homology to the E4 34 kDa protein, respectively. ORF6 has the potential to encode a 57 aa long polypeptide that has 67% homology to the E4 34 kDa protein of Ad2 and 50% homology to the E4 34 kDa protein of Ad12.

Subcloning and restriction enzyme mapping of bovine adenovirus type 2.

Bovine adenoviruses (BAVs), causing both respiratory and/or enteral diseases in cattle, have been isolated in many countries all over the world. In this study we report on the molecular cloning of the internal EcoRI fragments spanning 20.6-90.5%, and the internal SalI fragments spanning 3.1-65.2% of the BAV type 2 (BAV2; strain No. 19) genome into the plasmid vector pUC19. Moreover, the subcloning of the BAV2 genome facilitated the construction of a detailed physical restriction endonuclease map for BamHI, ClaI, EcoRI, HindIII, KpnI, NotI, NspV, PstI, PvuI, SalI, XbaI and XhoI.

Sequence Analysis of the Terminal Protein Precursor Coding Regions from Bovine Adenovirus Serotypes 2 and 3

As part of our efforts to develop bovine adenoviruses into a vector system, we sequenced the region predicted to encode the terminal protein precursor of either bovine adenoviruses type 2 and bovine adenoviruses type 3. We examined the regions containing the terminal protein precursors and showed that the bovine adenovirus early region 2B had an identical organization to all adenoviruses so far examined. The bovine adenovirus terminal protein precursors and those of other adenoviruses were also compared in a sequence alignment analysis and several highly conserved structural domains were identified. Finally, we showed how the various terminal protein precursors were related in a sequence pair similarity analysis and concluded that the terminal protein precursors of bovine adenoviruses types 2 and 3 are significantly different from each other at the protein sequence level.

A rapid simple procedure for direct measurement of excreted thiamine from yeast

A new rapid method for the detection of extracellular thiamine production in yeast is described. This method is based on a modified thiochrome assay where fluorescent zones surrounding thiamine excretor colonies can be directly visualized under UV light on agar plates. This new procedure is simple to perform on a large number of colonies simultaneously and results can be obtained within minutes.

Diversity and signature of small RNA in different bodily fluids using next generation sequencing

BackgroundSmall RNAs are critical components in regulating various cellular pathways. These molecules may be tissue-associated or circulating in bodily fluids and have been shown to associate with different tumors. Next generation sequencing (NGS) on small RNAs is a powerful tool for profiling and discovery of microRNAs (miRNAs).ResultsIn this study, we isolated total RNA from various bodily fluids: blood, leukocytes, serum, plasma, saliva, cell-free saliva, urine and cell-free urine. Next, we used Illumina’s NGS platform and intensive bioinformatics analysis to investigate the distribution and signature of small RNAs in the various fluids. Successful NGS was accomplished despite the variations in RNA concentrations among the different fluids. Among the fluids studied, blood and plasma were found to be the most promising fluids for small RNA profiling as well as novel miRNA prediction. Saliva and urine yielded lower numbers of identifiable molecules and therefore were less reliable in small RNA profiling and less useful in predicting novel molecules. In addition, all fluids shared many molecules, including 139 miRNAs, the most abundant tRNAs, and the most abundant piwi-interacting RNAs (piRNAs). Fluids of similar origin (blood, urine or saliva) displayed closer clustering, while each fluid still retains its own characteristic signature based on its unique molecules and its levels of the common molecules. Donor urine samples showed sex-dependent differential clustering, which may prove useful for future studies.ConclusionsThis study shows the successful clustering and unique signatures of bodily fluids based on their miRNA, tRNA and piRNA content. With this information, cohorts may be differentiated based on multiple molecules from each small RNA class by a multidimensional assessment of the overall molecular signature.