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Background: Alpha calcitonin gene-related peptide (aCGRP), neuropeptide Y (NPY), and substance P (SP) are neuropeptides that have emerged recently as potent immunomodulatory factors with potential as novel biomarkers and therapeutic targets in multiple sclerosis (MS). background: Alpha calcitonin gene related peptide (aCGRP), neuropeptide Y (NPY), and substance P (SP) are neuropeptides that have emerged recently as potent immunomodulatory factors with potential as novel biomarkers and therapeutic targets in multiple sclerosis (MS). Objective: The study aimed to detect serum levels of aCGRP, NPY, and SP in MS patients versus healthy controls and their association with disease activity and severity. Methods: Serum levels were measured in MS patients and age and sex-matched healthy controls using ELISA. method: Serum levels were measured in MS patients and age and sex-matched healthy controls using ELISA. Results: We included 67 MS patients: 61 relapsing-remitting MS (RR-MS) and 6 progressive MS (PR-MS), and 67 healthy controls. Serum NPY level was found to be lower in MS patients than in healthy controls (p Conclusion: Lower serum levels of NPY were revealed in MS patients compared to healthy controls. Since serum levels of aCGRP are significantly associated with disease activity and severity, it is a potential disease progression marker. conclusion: Lower serum levels of NPY were revealed in MS patients as compared to healthy controls. Since serum levels of aCGRP significantly associated with disease activity and severity, it might be a suitable disease progression marker. other: None

BACKGROUND Multiple sclerosis (MS) is a chronic inflammatory neurodegenerative disease of the central nervous system. Vasoactive and intestinal peptide (VIP) and pituitary adenylate cyclase-activating peptide (PACAP) are neuropeptides that play roles in anti-inflammation and neuroprotection in MS. In this study, we aimed to determine the serum levels of VIP and PACAP in MS patients versus healthy controls and to correlate them with demographics and clinical characteristics. METHODS Serum samples were collected from MS patients (n = 145) and healthy controls (n = 73) to measure serum levels VIP and PACAP. RESULTS VIP serum levels were lower in MS patients than healthy controls (p

Khaled Z Alawneh,1 Liqaa A Raffee,2 Musa Ahmed Mohammed Alshehabat,3 Hazem Haddad,4 Saied A Jaradat5 1Department of Diagnostic Radiology and Nuclear Medicine, Faculty of Medicine, Jordan University of Science and Technology, Irbid, Jordan; 2Department of Accident and Emergency Medicine, Faculty of Medicine, Jordan University of Science and Technology, Irbid, Jordan; 3Department of Clinical Veterinary Medical Sciences, Faculty of Veterinary Medicine, Jordan University of Science and Technology, Irbid, Jordan; 4Haya Biotechnology Center, Jordan University of Science and Technology, Irbid, 22110, Jordan; 5Princess Haya Biotechnology Center, Jordan University of Science and Technology, Irbid, 22110, JordanCorrespondence: Liqaa A RaffeeDepartment of Accident and Emergency Medicine, Faculty of Medicine, Jordan University of Science and Technology, Irbid, JordanEmail laraffee5@just.edu.joPurpose: Ischemic stroke of the brain is still considered one of the most common causes of disability and death in developed and developing countries in human beings despite advances in medicine and technology. This study was conducted to characterize and profile tens of induced biomarkers (microRNAs) after experimentally inducing regional ischemic stroke of the brain by occluding the middle cerebral artery under fluoroscopic guidance using an autologous blood clot.Patient and Methods: A total of six healthy dogs were recruited for this study. The microRNAs were profiled in the blood and urine before and after occluding the middle cerebral artery using genetic techniques.Results: The very highly expressed genes were comprised within cluster A, followed by cluster D in both 24 and 48-hour brain samples. Clusters B and C revealed down-regulated genes, while miRNAs remained up-regulated in the 24-hour samples merely in cluster F. Upregulated genes at 48 hours of reperfusion were included in cluster E. On the other hand, changes were observed after a day on the cluster G genes. Exclusive upregulation was notified after 2 days due to the changes in mIR-138. The normalized gene expression in the test sample is witnessed through Fold-Change, which divides the control sample’s normalized gene expression. Moreover, fold-change has emerged as a significant approach for representing fold-regulation.Conclusion: The microRNAs expression in blood and urine may have a potential role in the diagnosis, prognosis, and assessment of therapy associated with cerebral artery occlusion under fluoroscopic guidance.Keywords: dogs, fluoroscopic induced ischemic brain, microRNAs, middle cerebral artery stroke

BACKGROUND Horizontal gaze palsy and progressive scoliosis (HGPPS) is a rare autosomal recessive disorder due to mutations in ROBO3 gene. Patients have characteristic clinical and imaging findings. We report six patients from two families with this disorder with two novel mutations. MATERIALS AND METHODS One patient from a non-consanguineous family and five patients from extended consanguineous families were clinically and radiologically examined. Blood samples from the patients and their parents were obtained and all the coding exons and flanking intronic sequences of the ROBO3 gene were amplified and subjected to bidirectional DNA sequencing. RESULTS All six patients had the characteristic clinical and radiological findings of HGPPS. Genetic testing showed two novel mutations including frame-shift and nonsense. CONCLUSION Two novel mutations in the ROBO3 gene were identified in two Jordanian families with six affected individuals. To our knowledge, this is the first molecular study of HGPPS in Jordan.

Bladder cancer is the most common cancer of the urinary tract with an estimate of 401,000 new cases diagnosed annually worldwide. In Jordan, bladder cancer accounts for 4.3% of all newly diagnosed cancer cases. Several studies have linked p53 molecular changes to tumor initiation, invasion, metastasis, and chemoresistance. In the present study, we investigated a cohort of 121 bladder cancer patients with various grades and stages of the tumor for molecular changes in p53. Overexpression of p53 was shown in 22%, 46%, 71% and 25% pTa, pT1, pT2 and pT4 cases, respectively. Additionally, p53 was overexpressed in 19%, 38% and 74% of G1, G2 and G3 cases of bladder cancer, respectively. p53 expression is not significantly associated with the stage or grade of the disease. Mutational analysis of exons 4–8 of p53 identified the following previously reported point mutations R72P (58%), P67T (9%), A159P (0.7%), D185N (28%), G199 V (0.7%), G199E (0.7%), D208N (0.7%), R283P (1.4%). Furthermore, we were able to identify two new deletion mutations located in the DNA-binding domain in two patients. The first mutation was a result of one nucleotide deletion in exon 5 causing a frameshift at codon 115 resulting in a truncated 168 amino acids long p53 protein and the second is a result of a whole codon deletion in exon 6 resulting in a p53 protein missing a valine residue at codon 218. P53 mutations were found to be significantly associated with late stage (pT2-pT4) muscle-invasive bladder tumors. Survival analysis indicates significant differences of overall survival between the patients with the p53 overexpression and those with negative p53 staining ( p = 0.0041). Additionally, were able to observe a significant difference of overall survival between p53 mutants and p53 wild-type ( p = 0.0062). The present work points to the variable spectrum of TP53 mutations and p53 expression profile in Jordanian patients with bladder cancer and reflects the importance of p53 alterations in predicting poor prognosis of bladder cancer patients.

Maple syrup urine disease (MSUD) is an autosomal-recessive inborn error of amino acid metabolism characterized by the accumulation of three branched-chain amino acids (BCAAs) in patients' cells due to reduced activity of the branched-chain α-ketoacid dehydrogenase complex (BCKD). In this study, we aimed to sequence the coding exons of the BCKDHA, BCKDHB, and DBT genes in five Jordanian families with MSUD and one family from Iraq with MSUD. BCAA levels were measured in probands initially presenting with developmental delay and encephalopathy. All of the coding exons and flanking intronic sequences of the BCKDHA, BCKDHB, and DBT genes were amplified and subjected to direct DNA sequencing. Four different mutations in the BCKDHA gene were identified, including a novel frame-shift mutation, c.908_909delTG, in family 4. Two novel missense mutations at residues Met263 and Gly353 in the DBT gene were also found to be cosegregated with the MSUD phenotype in families 5 and 6, respectively. Structural analyses suggested that these two mutations may affect the assembly of the intermediate E2 trimer. No BCKDHB gene mutations were found in Jordanian patients. The mutation profiles described in this study provide a basis for the early detection of MSUD disease. To our knowledge, this is the first molecular study of MSUD in Jordan and Middle Eastern Arabic countries.

Hereditary angioedema due to C1-inhibitor deficiency (C1-INH-HAE) is an autosomal dominant disease caused by mutations in the SERPING1 gene. A Jordanian family, including 14 individuals with C1-INH-HAE clinical symptoms, was studied. In the propositus and his parents, SERPING1 had four mutations leading to amino acid substitutions. Two are known polymorphic variants (c.167T>C; p.Val34Ala and c.1438G>A; p.Val458Met), the others are newly described. One (c.203C>T; p.Thr46Ile) is located in the N-terminal domain of the C1-inhibitor protein and segregates with angioedema symptoms in the family. The other (c.800C>T; p.Ala245Val) belongs to the serpin domain, and derives from the unaffected father. DNA from additional 24 family members were screened for c.203C>T mutation in the target gene. All individuals heterozygous for the c.203C>T mutation had antigenic and functional plasma levels of C1-inhibitor below 50% of normal, confirming the diagnosis of type I C1-INH-HAE. Angioedema symptoms were present in 14 of 16 subjects carrier for the c.203T allele. Among these subjects, those carrying the c.800T variation had more severe and frequent symptoms than subjects without this mutation. This family-based study provides the first evidence that multiple amino acid substitutions in SERPING1 could influence C1-INH-HAE phenotype.

Maha S Al-Keilani,1 Karem H Alzoubi,1 Saied A Jaradat2,3 1Department of Clinical Pharmacy, College of Pharmacy, Jordan University of Science and Technology, Irbid 22110, Jordan; 2Department of Biotechnology and Genetic Engineering, Jordan University of Science and Technology, Irbid 22110, Jordan; 3Princess Haya Center for Biotechnology, Jordan University of Science and Technology, Irbid 22110, Jordan Background: Chemotherapy resistance is the main cause of the marginal clinical benefit of platinum-based chemotherapy and tyrosine kinase inhibitors in advanced non-small-cell lung cancer (NSCLC). Thus, the identification of new therapeutic agents that can enhance the sensitivity of these drugs is of clinical importance. Histone deacetylase inhibitors (HDACIs) are emerging as new promising agents with strong antiproliferative effects against different types of cancers. This study investigates the synergistic potential of sodium phenylbutyrate (NaPB) added on top of standard chemotherapy used against NSCLC. Objective: The objective of this study was to evaluate the ability of NaPB to overcome the resistance of NSCLC cell lines to cisplatin, gefitinib, and erlotinib. Methods: MTT cell proliferation assay was used to measure the anticancer effects of cisplatin, erlotinib, or gefitinib alone or combined with various concentrations of NaPB against A549, Calu1, and H1650 NSCLC cell lines. Synergism was estimated by measuring synergy value (R), which is equal to the ratio of IC50 of each primary drug alone divided by combination IC50s. Student’s t-test analysis was used to evaluate the potential differences between IC50 values. ANOVA followed by Tukey’s post hoc was used to evaluate the potential differences among monotherapy and combination treatment groups. Analyses were performed using R 3.3.2 software. P-value 1.6, P-value

To identify certain risk factors associated with childhood obesity related to lifestyle; dietary patterns, physical activity, and sedentary behavior.A cross-sectional study was conducted among 977 schoolchildren (473 boys and 449 girls) aged 7-18 years. Children were selected randomly from three main cities in Jordan: Amman, Irbid, and Mafraq by using multistage cluster sampling method. Sedentary behaviors, physical activity and child eating behaviors were measured by using validated questionnaires. Overweight and obesity were defined by International Obesity Task Force (IOTF) criteria.Sedentary activities increase the risk of overweight among schoolchildren by 2-fold [RR(Relative risk) = 2.0, 95% CI(Confidence interval) (1.1-3.6), p = 0.02]. Whereas, sedentary activities for less than 3 h increased the risk of overweight by 0.8-fold [RR = 0.8, 95%CI (0.6-1.3), P = 0.388], Schoolchildren who spent30 min/day in exercising decreased the risk of overweight by 0.5-fold [RR = 0.5, 95% CI (0.2-1.0), P = 0.06)]. Both Students who ate one meal daily and daily ate snacks from schools cafeterias had a higher tendency to be obese [(RR = 1.8, 95%CI (0.5-5.9), P = 0.368], and [RR = 1.5, 95%CI (0.9-2.5), P = 0.169] respectively.Physical activity, eating meals regularly and homemade food, all together play a significant role in decreasing obesity among Jordanian schoolchildren, thus a national policy that promoting active living and healthy eating among schoolchildren is warranted.

Objective/background: Myeloproliferative neoplasms (MPNs) are heterogeneous clonal bone marrow stem cell disorders and include polycythemia vera (PV), essential thrombocythemia (ET), and idiopathic myelofibrosis (IMF) neoplasia. In 2005, the JAK2V617F mutation was identified in Philadelphia chromosome-negative patients. The aim of this study was to sequence coding exons 12 and 14 of the JAK2 gene in Jordanian patients with MPN. Methods: Both exons 12 and 14 of the JAK2 gene were amplified using polymerase chain reaction from DNA extracted from 68 blood and bone marrow samples belonging to 57 MPN patients and subjected to DNA sequencing. Results: JAK2V617F mutations were detected in 26 of 57 Jordanian patients (45%) with different MPNs. JAK2V617F was identified in 70%, 31%, and 14% of PV, ET, and IMF cases, respectively. Five men diagnosed with PV were homozygous for JAK2V617F, whereas the other 21 patients were heterozygous for the mutation. Neither the JAK2V617F mutation nor any DNA polymorphism in exon 12 or exon 14 of the JAK2 gene was detected among the 40 leukemic patients. A rare single nucleotide polymorphism, c.1860C→T (rs375442615), was detected in one patient with ET. Conclusion: This study is the first molecular investigation of the JAK2 gene in Jordan. We successfully identified the JAK2V617F mutation in Jordanian patients with Philadelphia chromosome-negative MPNs. Our results provide a basis for the early detection of this mutation and simplify the diagnostic workup for these disorders at the molecular level. Keywords: JAK2 mutations, Jordan, Myeloproliferative neoplasms, Polycythemia vera, V617F

Epilepsy is a severe neurological disorder characterized by altered electrical activity in the brain. Important pathophysiological mechanisms include disturbed metabolism and homeostasis of major excitatory and inhibitory neurotransmitters, glutamate and GABA. Current drug treatments are largely aimed at decreasing neuronal excitability and thereby preventing the occurrence of seizures. However, many patients are refractory to treatment and side effects are frequent. Temporal lobe epilepsy (TLE) is the most common type of drug-resistant epilepsy in adults. In rodents, the pilocarpine-status epilepticus model reflects the pathology and chronic spontaneous seizures of TLE and the pentylenetetrazole kindling model exhibits chronic induced limbic seizures. Accumulating evidence from studies on TLE points to alterations in astrocytes and neurons as key metabolic changes. The present review describes interventions which alleviate these disturbances in astrocyte-neuronal interactions by supporting mitochondrial metabolism. The compounds discussed are the endogenous transport molecule acetyl-L-carnitine and the triglyceride of heptanoate, triheptanoin. Both provide acetyl moieties for oxidation in the tricarboxylic acid cycle whereas heptanoate is also provides propionyl-CoA, which after carboxylation can produce succinyl-CoA, resulting in anaplerosis-the refilling of the tricarboxylic acid cycle.

Pentylenetetrazol, kainic acid, or pilocarpine can be used to induce seizures in animal models of epilepsy. The present Review describes disturbances in astrocyte-neuron interactions in the acute, latent, and chronic phases analyzed by magnetic resonance spectroscopy of brain tissue extracts from rats injected with [1-(13)C]glucose and [1,2-(13)C]acetate. The most consistent change after onset of seizures was the decrease in (13)C labeling of glutamate (GLU) from [1-(13) C]glucose regardless of brain area, severity, or duration of the period with seizures and toxin used. In most cases this decrease was accompanied by a reduction in glutamine (GLN) labeling from [1-(13)C]glucose, presumably as a direct consequence of the reduction in labeling of GLU and the GLU-GLN cycle. Amounts of GLN were never changed. Reduction in the content of N-acetyl aspartate (NAA) was first detectable some time after status epilepticus but before the occurrence of spontaneous seizures. This decrease can be an indication of neuronal death and/or mitochondrial impairment and might indicate beginning gliosis. It is known that gliosis occurs in the chronic phase of temporal lobe epilepsy in hippocampus, but astrocyte metabolism appears normal in this phase, indicating that the gliotic astrocytes have a somewhat reduced metabolism per volume. A decrease in (13)C labeling of GLU from [1-(13)C]glucose is a very sensitive measure for the onset of epileptogenesis, whereas reduction of NAA is first detectable later. In the chronic phases of the hippocampal formation, astrocyte metabolism is upregulated given that the number of neurons is reduced.

Background: Viral cause of sporadic breast cancer (SBC) has been suggested based on the experimental murine model of mammary tumor caused by mouse mammary tumor virus (MMTV), Epstein-Barr virus (EBV), and human papillomavirus (HPV). While some studies have demonstrated the presence of viral sequences of MMTV, HPV, and EBV in breast cancer cells, others failed. These contradictions may be attributed to the geographical distribution of breast cancer incidence and/or technical variations. In the current study, we aimed to investigate the correlation of MMTV, HPV, and EBV infections with the development of breast cancer in Jordanian patients. Methods: One hundred SBC tissue samples were subjected to laser capture microdissection for the selection of tumor cells populations. Fluorescence polymerase chain reaction (PCR) was used to detect the presence of the MMTV env-like sequences. Real-time PCR was used for HPV and EBV detection, and EBV was further confirmed by chromogen in situ hybridization (CISH). Results: Mouse mammary tumor virus, HPV, and EBV were detected in SBC in 11%, 21%, and 23%, respectively. Only 3 of 52 (5.7%) positive cases demonstrated multiple virus infections. However, 49 of 52 (94%) of the positive cases revealed the presence of 1 type of viral sequences. Consequently, 52% of the studied breast cancer cases were infected with at least 1 type of the aforementioned viruses. Conclusions: The current cohort suggests that MMTV, HPV, and EBV have a potential role in the development of breast cancer and adding more reasons to proceed with the quest of a possible viral origin of breast cancer.

Result: The frequency of heterozygous genotype AG= 42%, homozygous for the wild type allele genotype AA= 50%. Homozygous genotype of the mutant allele GG=8%. The allele frequency for the wild allele A=71%, and for mutant allele is G=29%, with unknown clinical findings but the frequency of phenotype for wild allele A=92% and the phenotype frequency for mutant allele G= 50%. At the same time for 50 control subjects were investigated for the Cytotoxic T Lymphocyte Association-4 +49 A/G polymorphism respectively with patients on same Single Nucleotide Polymorphism, as following : AG=40%; AA=54%; GG=6%; A=74%; G=26%; A=94% and G=46%. The distribution of Cytotoxic T Lymphocyte Association -4 + 49 A/G genotype frequency did not differ significantly between patients and controls (P=0.885). The result on the other Single Nucleotide Polymorphism -318 C/T Showing the frequency of genotype; for heterozygous (CT=16%), homozygous wild allele (CC=84%), homozygous genotype for T allele =0%. Normal allele C=92%, but for mutant allele is (T=8%) with unknown clinical finding, where the phenotype for wild allele is (C=100%), phenotype mutant allele is T=16%. On the other hand for control subject investigated for Cytotoxic T Lymphocyte Association -4 -318 C/T polymorphism were (CT=22%, CC=74%, TT=4%, C=85%, T=15%, C=96%, T=26%) respectively. The result of distribution of Cytotoxic T Lymphocyte Association -4 -318 C/T genotype frequency did not differ significantly with type 1 diabetes mellitus patients and controls (P=0.248). Conclusion: This case-control study suggests that the +49 A/G Single Nucleotide Polymorphism of the Cytotoxic T Lymphocyte Association-4 gene is not strongly associated with type one diabetes mellitus in Jordanian population, the apparent discrepancies between the present study and other studies could be due to the genetic heterogeneity among the population studied. The Cytotoxic T Lymphocyte Association-4 49 A/G Single Nucleotide Polymorphism may not be the true disease associated variant but a marker in linkage disequilibrium in different population.

To examine the antitumor activity of a new derivative of ciprofloxacin called methoxyphenylcipro (CMPP). Cell viability was assessed using the MTT assay and apoptotic cells and reactive oxygen species were evaluated using flow cytometry. Results revealed that CMPP induces antiproliferative activity against breast cancer cells and melanoma and to a lesser extent against colorectal cancer cells. Interestingly, compared to ciprofloxacin, CMPP-induced a selective cytotoxicity against human cancer cells but not human normal fibroblasts. The potential of CMPP to inhibit cellular growth in MD-MB-486 breast cancer cells and MV3 melanoma cells was largely due to induction of caspase-dependent apoptosis, as confirmed by caspase-3 activation and cleavage of its substrate PARP. In addition, results indicated that CMPP-induced apoptosis is mediated by generation of reactive oxygen species. These findings revealed that CMPP has a selective antitumor activity against cancer cells and warrants further clinical evaluation.

Drug dependence is a pattern of repeated self-administration of a drug, which can result in tolerance, withdrawal and compulsive drug-taking behaviour. It has been recently suggested that 5-HTTLPR ( LL/ LS/ SS) variants and rs25531 (A/G) polymorphism in the serotonin transporter gene ( SLC6A4) may play a role in drug dependence. The current study aimed to (1) identify allelic, haplotypic and genotypic frequencies of the 5-HTTLPR variants and rs25531 polymorphisms of SLC6A4 gene in drug and nondrug-dependent Jordanian Arab population and (2) determine whether there is an association of these variants in a drug-dependent population from the same area. Jordanian drug male addicts of Arab descent ( n = 192) meeting the Diagnostic and Statistical Manual of Mental Disorders – Fourth edition criteria for drug dependence and 230 healthy male controls from an ethnically homogenous Jordanian Arab population were examined. Genotyping was performed using the restriction fragment length polymorphism–polymerase chain reaction-based method to genotype the 5-HTTLPR variants and detect the A/G polymorphism at position rs25531. The biallelic analysis revealed that the frequency of 5-HTTLPR ( LL/ LS/ SS) genotypes was statistically significant different between drug-dependent individuals and controls ( χ2 (2, N = 422), p = 0.04). Drug-dependent subjects had a higher frequency of ‘ L’ allele. However, using the triallelic approach, the estimated frequency of haplotypes ( SA, SG, LA and LG) and phased genotypes ( LA/ LA, LA/ SA, LA/ LG, SA/ SA and SA/ SG) did not show significant association with drug dependence ( χ2 (3, N = 422), p = 0.53 and χ2 (4, N = 422), p = 0.06, respectively). This study suggests a putative role of the 5-HTTLPR for drug dependence in the Jordanian Nationals of Arab ancestry.

Inhalation of olive pollen is a major cause of allergic respiratory diseases in Mediterranean region. Using bags attached to the branches, pollens were collected from 36 olive cultivars grown in Jordan just before bloom. Proteins were extracted from pollens and analysed by SDS-PAGE and immunoblotting using antisera collected from patients with history of olive pollen-allergy. Immunoblot results showed that 25 cultivars contained one or two allergenic proteins in the range of 15–72 kDa with majority of the proteins corresponding to 15–20 kDa, while remainders did not exhibit any allergenic protein. Seven cultivars exhibited three allergenic proteins and only two cultivars exhibited four allergenic proteins and thus, were considered potentially hyperallergenic cultivars. This study highlights the great variability in the antigenic and allergenic composition of Olea europaea pollen extracts which may serve as guidance for selecting the olive tree cultivars that could be planted in urban areas.

Oral Diseases (2012) 18, 271–279 Objective: Interleukin-10 gene promoter polymorphisms have been associated with interleukin-10 decreased production, thereby playing a role in the pathogenesis of periodontitis. This study aimed to investigate whether interleukin-10 single nucleotide polymorphisms at positions -1087(G/A) and -597(C/A) are associated with generalised chronic periodontitis and localised aggressive periodontitis. Methods: Genomic DNA samples were isolated from 276 unrelated Jordanian participants. Subjects were categorised into 86 periodontally healthy controls, 105 chronic periodontitis patients and 85 localised aggressive periodontitis patients. Genotype frequencies were calculated, and differences were determined using Pearson chi-squared test, and odds ratio and 95% confidence intervals were included. Results: The frequencies of the -1087A and -597A alleles were significantly more common in chronic periodontitis patients than controls. The A-positive allele genotypes (GA, AA) at position -1087 and A-positive allele genotypes (CA, AA) at position -597 appeared to increase the risk of having chronic periodontitis. No significant differences were observed in the genotype frequencies between localised aggressive periodontitis patients and controls. Conclusions: These findings indicate the possible use of interleukin-10 single nucleotide polymorphisms as genetic markers in chronic periodontitis patients and further emphasise the molecular differences between chronic periodontitis and aggressive periodontitis.

Paratuberculosis (Johne’s disease) is an infectious, incurable, chronically progressive granulomatous enteritis which affects domestic and exotic ruminants. The causative agent is Mycobacterium avium paratuberculosis (Mycobacterium johnei), a slow growing mycobactin-dependent acid-fast bacillus. We investigated the occurrence of Johne’s disease in apparently healthy cattle, using 263 ileum and corresponding mesenteric lymph nodes, by histopathological examination, and 170 ileum and 120 mesenteric lymph nodes by immunohistochemical examination. The occurrence of the disease was 65% and 66% using immunohistochemistry and histopathology techniques, respectively. When Ziehl-Neelsen (ZN) and ELISA techniques were implemented, the occurrence was 1% (4/120) and 3% (8/278), respectively. Grading from I–IV of histopathological lesions based on type of cellular infiltrate and severity of lesions revealed that most of the positive cases were in grade I and II. Furthermore, staging I–III of immunohistochemistry results, has presented a high number of positive cases in stage I. ZN stain showed a very low occurrence; however, it is still used as a confirmatory test for clinical cases. On the other hand, ELISA technique showed a low occurrence of the disease (3%) in this study reflecting the low sensitivity of the technique in diagnosis of subclinical Johne’s disease (JD). These results showed that histopathology is a very good diagnostic method for subclinical paratuberculosis in cattle. From the present study, we conclude that the occurrence of JD in cattle is high in Jordan. It is interesting to note that this is the first study of JD in cattle in Jordan, and the results strongly suggest alarming fears of severity of the disease at the national level. The urgent need for national control strategies are well founded due to the economical importance of the disease.

Paratuberculosis is an endemic disease and induces high economical losses in Jordan. There is no information available on genotypic variation of Mycobacterium avium subsp. paratuberculosis (MAP) isolated from animals in Jordan. In this study, we investigated 150 fecal samples from sheep, goats, and cattle for the presence of paratuberculosis using bacterial culture and polymerase chain reaction (PCR)-RFLP analysis of insertion sequence IS1311. Analysis of the results revealed that genotypic information from sheep, goat, and cattle could classify them into cattle or sheep strains. All culture isolates from cattle, 12.5% of the isolates from sheep, and 50% of the isolates from goats were cattle strain, while 87.5% of the isolates from sheep and 50% of the isolates from goats were sheep strain. Sequencing of the IS1311 268 bp PCR product from the three animal species confirmed the different MAP patterns.

Here we report that paclitaxel induces variable degrees of apoptosis in human colorectal cancer cells. Paclitaxel induces multiple arms of the endoplasmic reticulum stress response, including upregulation of the 78-kDa glucose-regulatory protein (GRP78) and eukaryotic initiation factor alpha phosphorylation. Inhibition of the MEK/ERK pathway sensitized colorectal cancer cells to paclitaxel-induced apoptosis. A similar result was obtained by the inhibition of GRP78 using small interfering RNA molecules. Knockdown of MEK resulted in a significant downregulation of paclitaxel-induced upregulation of GRP78 indicating that activation of GRP78 is a downstream event of MEK/ERK pathway activation. These results indicate that GRP78 might be a novel mechanism underlying the resistance of colorectal cancer cells to microtubule-targeting drugs. A combination of compounds capable of suppressing GRP78 might be a golden approach for improving the effectiveness of taxanes.

Marfan syndrome is an autosomal dominant inheritance disorder with a 1/5000-live-birth prevalence. More than 3000 mutations have been characterized thus far in the FBN1 gene. The goal of this study is to facilitate Marfan syndrome diagnosis in Jordanian patients using a molecular genetic testing. All of the 65 coding exons and flanking intronic sequences of the FBN1 gene were amplified using polymerase chain reaction and were subjected to sequencing in five unrelated Jordanian patients suspected of having Marfan syndrome. Four different mutations were identified, including two novel mutations: the c.1553dupG frame-shift (p.Tyr519Ilefs*14) and the c.6650G>A (p.Cys2217Tyr) missense mutations. Two other missense mutations, c.2243G>A (p.Cys748Tyr) and c.2432G>A (p.Cys811Tyr), have been previously detected. Patient number five was heterozygous for the synonymous substitution variant c.1875T>C (p.Asn625Asn; rs#25458). Additionally, eight variants in the intronic sequence of the FBN1 gene were identified, of which the c.2168-46A>G mutation was a new variant. The data provide molecular-based evidence linking Marfan syndrome to pathogenic mutations in the FBN1 gene among Jordanians for the first time. Thus, our results will contribute to the better management of the disease using molecular tools and will help in genetic counseling of the patients’ families.

Recessive mutations in LPIN1, which encodes a phosphatidate phosphatase enzyme, are a frequent cause of severe rhabdomyolysis in childhood. Hence, we sequenced the 19 coding exons of the gene in eight patients with recurrent hereditary myoglobinuria from four unrelated families in Jordan. The long-term goal is to facilitate molecular genetic diagnosis without the need for invasive procedures such as muscle biopsies. Three different mutations were detected, including the novel missense mutation c.2395G>C (Gly799Arg), which was found in two families. The two other mutations, c.2174G>A (Arg725His) and c.1162C>T (Arg388X), have been previously identified, and were found to cosegregate with the disease phenotype in the other two families. Intriguingly, patients homozygous for Arg725His were also homozygous for the c.1828C>T (Pro610Ser) polymorphism, and were exercise-intolerant between myoglobinuria episodes. Notably, patients homozygous for Arg388X were also homozygous for the c.2250G>C silent variant (Gly750Gly). Taken together, the data provide family-based evidence linking hereditary myoglobinuria to pathogenic variations in the C-terminal lipin domain of the enzyme. This finding highlights the functional significance of this domain in the absence of structural information. This is the first analysis of LPIN1 in myoglobinuria patients of Jordanian origin, and the fourth such analysis worldwide., Highlights • LPIN1 mutations were cataloged in families with hereditary myoglobinuria. • A novel missense Gly799Arg mutation was identified. • Arg725His, the only other known missense mutation, was confirmed to be pathogenic. • Arg388X, a known nonsense mutation, was the most common among Arabic patients. • Patients exercise-intolerant between myoglobinuria episodes have a second mutation.

Sperm motility is highly dependent on aerobic energy metabolism, of which the apparent rate-limiting step of the mitochondrial respiratory chain is catalyzed by cytochrome c oxidase (COX). COX is the only electron transport chain complex to display isoforms, consistent with its suggested rate-limiting role. Isoforms were previously described for four of the 13 subunits. We now report the discovery that COX subunit VIb displays a testes-specific isoform in human, bull, rat, and mouse (COX VIb-2). Analysis of a variety of rat and mouse tissues, including ovaries, demonstrates exclusive expression of VIb-2 in testes, whereas VIb-1 transcripts are absent in rodent testes, even at early developmental stages. In contrast, both isoforms are transcribed in human testes. In situ hybridizations with human, rat, and mouse testes sections reveal VIb-2 transcripts in all testicular cell types. Within the seminiferous tubules, VIb-1 shows stronger signals in the periphery than in the lumen. Previously, cytochrome c was the only component of the mitochondrial respiratory chain known to express a testes-specific isoform in mammals. COX subunit VIb connects the two COX monomers into the physiological dimeric form, and is the only COX subunit that, like cytochrome c, is solely located in the inter-membrane space. Significant differences between the isoform sequences, in particular changes in charged amino acids, suggest interactions with cytochrome c and sperm-specific energy requirements. Mol. Reprod. Dev. 66: 8–16, 2003. © 2003 Wiley-Liss, Inc.

Large-scale gene expression profiling was performed on embryo-derived stem cell lines to identify molecular signatures of pluripotency and lineage specificity. Analysis of pluripotent embryonic stem (ES) cells, extraembryonic-restricted trophoblast stem (TS) cells, and terminally-differentiated mouse embryo fibroblast (MEF) cells identified expression profiles unique to each cell type, as well as genes common only to ES and TS cells. Whereas most of the MEF-specific genes had been characterized previously, the majority (67%) of the ES-specific genes were novel and did not include known differentiated cell markers. Comparison with microarray data from embryonic material demonstrated that ES-specific genes were underrepresented in all stages sampled, whereas TS-specific genes included known placental markers. Investigation of four novel TS-specific genes showed trophoblast-restricted expression in cell lines and in vivo, whereas one uncharacterized ES-specific gene, Esg-1, was found to be exclusively associated with pluripotency. We suggest that pluripotency requires a set of genes not expressed in other cell types, whereas lineage-restricted stem cells, like TS cells, express genes predictive of their differentiated lineage.[Supplemental material is available online at www.genome.org andhttp://lgsun.grc.nia.nih.gov/microarray/data.html]

The cytochrome c oxidase (COX) holoenzyme is a 13-subunit complex that carries out the terminal step in the electron transport chain. Three of the subunits, which contain the electron transfer function, are coded by mitochondrial DNA and the other ten subunits by nuclear DNA. Since the holoenzyme contains equivalent amounts of each subunit, we and others have examined transcriptional regulation of COX nuclear subunits to explore whether there is a common basis for co-regulation. Each gene is seen to have a unique pattern of recognition by regulatory factors; although some factors bind to more than one gene, not all COX genes seem to be regulated by the same set of factors. Current information about the COX promoters that have been examined is summarized, and the relation of promoter regulation to coordinate gene expression is discussed.

Cytogenetic and Morphological Analysis of De Novo Acute Myeloid Leukemia in Adults: A Single Center Study in JordanAcute myeloid leukemia (AML) in adults is known to be a heterogeneous disease with diverse chromosomal abnormalities. Some of these abnormalities are found with a high incidence in specific ethnic groups and in certain geographical areas. We report the results of cytogenetic studies of 35 adult Jordanian Arab patients withde novoAML diagnosed according to the French-American-British (FAB) criteria. Four patients did not have metaphases secondary to hypocellular bone marrow. The most common morphological subtype was M5 (55%) followed by M3 (19%). Cytogenetic abnormalities were present in 20 patients (65%); t(15;17) translocation in six patients (19%), inv(16) in four patients (13%), t(11;17) in two patients (4%), and the t(8;21) translocation was not present in any patient. Trisomy 8 was the most common numerical chromosomal abnormality [four patients (13%)].There were variations and similarities with similar ethninc Arab populations. The most common chromosomal abnormalities were t(15;17), +8 and inv(16). Further and larger crossborder studies are needed.

Background Both environmental and genetic factors contribute to individual susceptibility to initiation of substance use and vulnerability to addiction. Determining genetic risk factors can make an important contribution to understanding the processes leading to addiction. In order to identify gene(s) and mechanisms associated with substance addiction, a custom platform array search for a genetic association in a case/control of homogenous Jordanian Arab population was undertaken. Patients meeting the DSM-VI criteria for substance dependence (n = 220) and entering eight week treatment program at two Jordanian Drug Rehabilitation Centres were genotyped. In addition, 240 healthy controls were also genotyped. The sequenom MassARRAY system (iPLEX GOLD) was used to genotype 49 single nucleotide polymorphisms (SNPs) within 8 genes (DRD1, DRD2, DRD3, DRD4, DRD5, BDNF, SLC6A3 and COMT). Results This study revealed six new associations involving SNPs within DRD2 gene on chromosome 11. These six SNPs within the DRD2 were found to be most strongly associated with substance addiction in the Jordanian Arabic sample. The strongest statistical evidence for these new association signals were from rs1799732 in the C/−C promoter and rs1125394 in A/G intron 1 regions of DRD2, with the overall estimate of effects returning an odds ratio of 3.37 (χ2 (2, N = 460) = 21, p-value = 0.000026) and 1.78 (χ2 (2, N = 460) = 8, p-value = 0.001), respectively. It has been suggested that DRD2, dopamine receptor D2, plays an important role in dopamine secretion and the signal pathways of dopaminergic reward and drug addiction. Conclusion This study is the first to show a genetic link to substance addiction in a Jordanian population of Arab descent. These findings may contribute to our understanding of drug addiction mechanisms in Middle Eastern populations and how to manage or dictate therapy for individuals. Comparative analysis with different ethnic groups could assist further improving our understanding of these mechanisms.

Laith N AL-Eitan,1 Saied A Jaradat,2 Steve YS Su,3 Guan K Tay,1 Gary K Hulse4,51Centre for Forensic Science, 2Princess Haya Biotechnology Center, Jordan University of Science and Technology, Irbid, Jordan; 3School of Mathematics and Statistics, 4School of Psychiatry and Clinical Neurosciences, 5Unit for Research and Education in Alcohol and Drugs, Queen Elizabeth II Medical Centre, The University of Western Australia, Crawley, WA, AustraliaBackground: A number of research studies on the genetics of opiate dependence have focused on the µ-opioid receptor (OPRM1), which is a primary target for opiates. This study aims to identify genetic polymorphisms within the OPRM1 gene involved in response to the biopsychosocial treatment in opiate-dependent individuals of Arab descent.Methods: Unrelated Jordanian Nationals of Arab descent (N = 183) with opiate dependence were selected for this study. These individuals, all males, met the DSM-IV criteria for opiate dependence and were undergoing a voluntary 8-week treatment program at a Jordanian Drug Rehabilitation Centre. All individuals were genotyped for 22 single nucleotide polymorphisms (SNPs) within the OPRM1 gene using the Sequenom MassARRAY® system (iPLEX GOLD). Statistical analyses were carried out using the R package.Results: Patients receiving biopsychosocial treatment showed that there was a significant difference in their OPRM1 SNPs’ genotyping distribution between good, moderate, and poor responders to the treatment at two sites (rs6912029 [G-172T], and rs12205732 [G-1510A], P < 0.05, Fisher’s exact test).Conclusion: This study is the first report of an association between the OPRM1 G-172T and G-1510A polymorphisms and treatment response for opiate dependence. Specifically, this study demonstrated that the OPRM1 GG-172 and GG-1510 genotypes were more frequent among patients who were nonresponders to the biopsychosocial treatment. However, further pharmacogenetic studies in a larger cohort of opiate-dependent patients of Arab descent are needed to confirm these findings and identify individuals with increased chance of relapse.Keywords: OPRM1, association, opiates, dependence, treatment response, Arab

Background Cronobacter spp. is a newly emerging pathogen that causes meningitis in infants and other diseases in elderly and immunocompromised individuals. This study was undertaken to investigate surface antigenic determinants in Cronobacter spp. using monoclonal antibodies (MAbs) and MALDI-TOF Mass spectrometry. Results Spleenocytes from mice that were immunized with heat-killed (20 min, 80°C) Cronobacter cells were fused with SP2 myeloma cells. Five desirable MAbs (A1, B5, 2C2, C5 and A4) were selected. MAbs A1, B5, 2C2 and C5 were of IgG2a isotype while A4 was an IgM. Specificity of the MAbs was determined by using immunoblotting with outer membrane protein preparations (OMPs) extracted from 12 Cronobacter and 6 non-Cronobacter bacteria. All MAbs recognized proteins with molecular weight ranging between 36 and 49 kDa except for one isolate (44) in which no OMPs were detected. In addition, MAbs recognized two bands (38-41 kDa) in four of the non-Cronobacter bacteria. Most of the proteins recognized by the MAbs were identified by MALDI-TOF peptide sequencing and appeared to be heterogeneous with the identities of some of them are still unknown. All MAbs recognized the same epitope as determined by an additive Index ELISA with their epitopes appeared to be conformational rather than sequential. Further, none of the MAbs recognized purified LPS from Cronobacter spp. Specificity of the MAbs toward OMPs was further confirmed by transmission electron microscopy. Conclusions Results obtained in this study highlight the immunological cross-reactivity among Cronobacter OMPs and their Enterobacteriaceae counterparts. Nevertheless, the identity of the identified proteins appeared to be different as inferred from the MALDI-TOF sequencing and identification.

In this paper, we experimentally investigated the navigation system of the nonpathogenic strain of E. coli (AW405) and developed a simulator for the locomotion performance of these swimming nanorobots. The swimming behavior of these cells is sensitive to the chemical gradients in their medium. Tissue and disease cells might produce chemical signals in their surroundings. These chemicals have the potential to affect the locomotion behavior of the bacterial cells. Therefore, bacterial cells can be considered as self-navigator nanorobots that are able to discriminate between disease cells such as cancer. Our current experimental and theoretical work is considered as a platform to this novel idea of early detection of problematic diseases.

Bladder cancer accounts for nearly 5% of all newly diagnosed cancers in Jordan, with a much higher frequency in males. Recent studies have shown that activating mutations in FGFR3 are the most common findings in non-invasive low grade bladder tumors. In this study, we, retrospectively, investigated a cohort of 121 bladder cancer patients with various grades and stages of the tumor for molecular changes in FGFR3. Overexpression of FGFR3 was observed in 49%, 34%, 15%, and 2% of pTa, pT1, pT2, and pT3 cases, respectively. Further, FGFR3 expression was positive in 45%, 26%, and 30% of G1, G2 and G3 cases, respectively. Mutational analysis of exons 7, 10 and 15 of FGFR3 identified four previously reported mutations, namely R248C (n = 4; 10%), S249C (n = 23; 59%), Y375C (n = 7; 18%), G382R (n = 4; 10%), and one novel mutation, G382E (n = 1; 3%). Our results indicate that both mutations and overexpression of FGFR3 are correlated together, and are more prevalent in early stage (pTa and pT1) and low grade (G1 and G2) bladder tumors. Survival analysis showed no contribution of changes in FGFR3 on the patient's survival. Multivariate Cox proportional hazards model analysis of overall survival for the following variables: age, gender, stage and grade of tumor, and FGFR3 (expression and mutation) revealed that age, stage and grade of tumor are independent predictors of overall survival in patients with bladder cancer. Our work is the first to address the molecular status of FGFR3 in Jordanian patients with bladder cancer, and provides further support for FGFR3 as a key player in the initiation of bladder tumors.

This chapter discusses some technical aspects of genomic approaches such as complementary DNA (cDNA) analyses and expression profiling. It presents two examples of such applications: expression profiling and comparison among various types of stem cells; and expression profiling between cloned mice and normal mice. The fundamental information required for genomics approaches are the sequences and structures of the genes under study. This information is provided by Expressed Sequence Tag projects, in which cDNA libraries are first made from a variety of tissues and randomly selected individual cDNA clones are sequenced from their 5'- or 3'-ends. Two forms of microarrays are currently available: cDNA microarrays and oligonucleotide microarrays. Various bioinformatics and statistical analysis tools have been developed and utilized for the analysis of expression profiles of a large number of genes. In particular, hierarchical clustering, self-organizing map and k-means clustering analyses have been widely used.

We have isolated and characterized the human gene ( COX7AH ) for the contractile muscle isoform of cytochrome c oxidase (COX) subunit VIIa. This subunit is one of the 10 nuclear encoded subunits of the 13-subunit holoenzyme that carries out the terminal step in the electron transport chain. Using transient transfection assays, we have located a 5′-flanking region sufficient to direct high level, skeletal myotube-specific reporter gene expression. This 792 bp basal promoter, which contains the single transcription start but no canonical TATA or CCAAT boxes, contains one MEF2 site, three E boxes, and an Sp1 site that show binding to their cognate factors, and are all required for full expression. Mutation and transactivation analysis suggest that there is functional interaction between these binding sites.

cDNA microarray technology has been increasingly used to monitor global gene expression patterns in various tissues and cell types. However, applications to mammalian development have been hampered by the lack of appropriate cDNA collections, particularly for early developmental stages. To overcome this problem, a PCR-based cDNA library construction method was used to derive 52,374 expressed sequence tags from pre- and peri-implantation embryos, embryonic day (E) 12.5 female gonad/mesonephros, and newborn ovary. From these cDNA collections, a microarray representing 15,264 unique genes (78% novel and 22% known) was assembled. In initial applications, the divergence of placental and embryonic gene expression profiles was assessed. At stage E12.5 of development, based on triplicate experiments, 720 genes (6.5%) displayed statistically significant differences in expression between placenta and embryo. Among 289 more highly expressed in placenta, 61 placenta-specific genes encoded, for example, a novel prolactin-like protein. The number of genes highly expressed (and frequently specific) for placenta has thereby been increased 5-fold over the total previously reported, illustrating the potential of the microarrays for tissue-specific gene discovery and analysis of mammalian developmental programs.

Cytochrome c oxidase (COX) consists of 13 subunits, 3 encoded in the mitochondrial genome and 10 in the nucleus. Little is known of the role of the nuclear-encoded subunits, some of which exhibit tissue-specific isoforms. Subunit VIa is unique in having tissue-specific isoforms in all mammalian species examined. We examined relative evolutionary rates for the COX6A heart (H) and liver (L) isoform genes along the length of the molecule, specifically in relation to the tissue-specific function(s) of the two isoforms. Nonsynonymous (amino acid replacement) substitutions in the COX6AH gene occurred more frequently than in the ubiquitously expressed COX6AL gene. Maximum-parsimony analysis and sequence divergences from reconstructed ancestral sequences revealed that after the ancestral COX6A gene duplicated to yield the genes for the H and L isoforms, the sequences encoding the mitochondrial matrix region of the COX VIa protein experienced an elevated rate of nonsynonymous substitutions relative to synonymous substitutions. This is expected for relaxed selective constraints after gene duplication followed by purifying selection to preserve the replacements with tissue-specific functions.

In this paper, we experimentally investigated the navigation system of the nonpathogenic strain of E. coli (AW405), and we developed a simulator for the locomotion performance of these swimming nanorobots. The swimming behavior of these robotic cells is sensitive to the chemical gradients in their medium. Tissue and disease cells might produce chemical signals in their surroundings. These chemicals have the potential to affect the locomotion behavior of the bacterial cells. Therefore, bacterial cells can be considered as self-navigator nanorobots that are able to discriminate between disease cells such as cancer. We exploit Bayesian decision theory as a framework in predicting the locomotion behavior of the E. coli robotic cells. Obvious agreement has been achieved between the experimental performance of our moving robotic cells and its corresponding simulation. Our current experimental and theoretical work is considered as a platform to this novel idea of early detection of problematic diseases.

We have isolated and examined the gene for the heart isoform of cytochrome c oxidase subunit VIIa (COX VIIa-H) in mouse, an isoform gene previously thought to be lacking in rodents. Interspecies amino acid comparisons indicate that mouse COX VIIa-H protein displays 82.5 and 70.9% identity with the bovine and human heart isoforms of COX VIIa, but only 53.7% identity with the paralogous mouse liver isoform (COX VIIa-L). Expression in adult mouse tissues is limited to heart and skeletal muscle, as found in other species. In the early mouse embryo, Cox7al was the exclusive isoform expressed and Cox7ah mRNA was not detectable until day 17 postcoitum. That the mouse Cox7ah gene characterized in this study is orthologous to the human COX7AH gene was also suggested by its mapping to mouse chromosome 7, to a conserved region syntenic with the human chromosome location of COX7AH, 19q13.1. As a result, all three COX heart isoform genes in mouse group to chromosome 7. Interestingly, mapping of the mouse Cox7al to chromosome 9 suggests a new syntenic region between the mouse and the human genomes.

Objective: The aim of this study was to determine the incidence of HbS and glucose-6-phosphate dehydrogenase (G6PD) deficiency in Jordanian newborn. Study Design: A total of 181 male and female babies born at Princess Basma Teaching Hospital, randomly selected, and cord blood samples were collected, and the erythrocyte G6PD activity was measured, and the hemoglobin electrophoresis for blood lysate was conducted and scanned for HbS scanning. Results: The frequencies of two major red cell genetic defects, sickle hemoglobin (HbS) and deficiency G6PD was determined, of the studied subjects 10 (11%) females and 11 (12%) males were found to be deficient in the G6PD gene. The frequency of HbS carriers among the females was 4% while it was 6% among males. The coincidence of both G6PD deficiency and sickle cell hemoglobin in the samples was 1%. No coincidence was found between G6PD deficiency and hyperbilirubinemia. Conclusion: A better understanding of the distributions of these genetic disorders has the potential to aid in the more efficient utilization of health care resources and improved planning.