Biochemistry & Molecular Biology الكيمياء الحيوية والأحياء الجزيئية

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Analysis of the Secondary Structure of the Transmembrane Domain of SARS CoV E Protein using FTIR Spectroscopy
(AL-Quds University, 2007-05-10) قاسم موسى هاشم أبو رميله; Qassem Mussa Hashem Abu Rmeleh; معتز عكاوي; Mohammed Abo-Alhaj; Sameir Alnajde
One of the major obstacles facing the field of structural biology in the postgenomic era is the inherent difficulty of solving the structure of membrane proteins under native conditions. Membrane proteins share a common property; part of their structure is embedded in the lipid bilayer. This feature makes them attractive drug targets, which requires a detailed knowledge of the secondary structure of their transmembrane domain. Both crystallography and NMR still encounter difficulties in handling membrane proteins, so there is an urgent need for new biophysical methods and new insights in the biophysics of membrane proteins to solve the secondary structure of such proteins. The outbreak of the severe acute respiratory syndrome (SARS) virus, July 2003, has presented a formidable challenge for the scientific community. As part of that effort, we decided to study the high resolution backbone structure of E transmembrane proteins of the SARS coronavirus, by Attenuated Total Internal Reflection (ATR) FTIR of eighteen of isotopically labeled sites with ( 13C=18O) of the synthesized sequence for the SARS coronavirus E protein transmembrane domain. ATR-FTIR spectroscopy is a wellestablished method for generating precise structural information on isotopically labeled membrane proteins embedded in a lipid bilayer. We used the new biophysical method site specific infrared dichroism (SSID), to investigate the structure and orientation of transmembrane. α-helical bundles We postulate in this work that the E protein of SARS CoV is α-helix, and it has 26 residues embedded in the lipid bilayer, and the SARS CoV E protein is not a regular helix, but it adopts a unique transmembrane helical hairpin model, and the E protein has two possible kinks at residue No. 26 and 31 Phe and Leu respectively within the lipid bilayer, which isreported for the first time in this thesis. And it also has a possible kink in residue No 15 too. All the results were confirmed experimentally.
APOE Gene variants and Risk of Hyperlipidemia in Palestinian Type 2 Diabetes Mellitus Patients
(Al-Quds University, 2021-01-16) Manal Mohammed Saleh Ghattas; منال محمد صالح غطاس
Diabetes is considered as one of the most popular metabolic diseases worldwide. Type 2 is the most common by which it affects 90% of all diabetic patients in the world. The problem with the disease is that insulin is not doing its job or it is not secreted at all. This elevates the sugar level in the bloodstream, as it is not entering the human cells. There are several contributing agents that participate in the development of T2DM, they could be environmental or genetic or both. Many genes have been shown to be involved in increasing the risk of T2DM. One of these genes is APOE gene which is responsible for producing apolipoprotein E that is important in lipid metabolism. The study is a cross sectional that was done in the period from January to April 2019. It consists of T2DM Palestinian subjects divided into two groups; lipidemic (case) and non-lipidemic (control). A total of 204 diabetic subjects were included in this study; 96 participants were lipidemic and 108 were non-lipidemic. All patients aged over 50 years and admitted in Ramallah hospital/Palestine. The lipid profile was performed during the hospital admission examination. Two SNPs within the exon 4 of the APOE gene and three SNPs in the APOE promoter region were identified by amplicon based next generation sequencing (NGS) and it is the first to be held on Palestinian population. The frequencies of these polymorphisms were assessed in the studied population. Further, the associations of APOE genotype and promoter SNPs with risk of hyperlipidemia in T2DM patients were investigated. Our results showed that the frequency of E3 allele was the highest (79.6%) among other alleles; E3/E3 genotype was also found to be highest in all T2DM subjects with a frequency of 78.1%. Our study population was stratified according to lipid status, the obtained results showed no statistical differences (P>0.05) of APOE genotypes frequency (APOE polymorphisms and promoter SNPs) among lipidemic and non lipidemic groups. Further, Logistic regression analysis adjusted for age, gender and BMI showed no association between APOE genotypes (epsilon and promoter polymorphisms) and risk of dyslipidemia. No significant association was observed between APOE genotypes and serum cholesterol, TG, HDL-C and LDL levels. The possible synergistic effect between the promoter SNPs and the common APOE polymorphism was also investigated. We found no significant differences between lipidemic and non lipidemic groups after stratification of the promoter SNPs with the APOE genotypes. In conclusion, association between known SNPs and diseases are varied among different ethnic groups. Our study showed no association between APOE genotypes and hyperlipidemia in type 2 diabetic patients in the Palestinian population. Further studies should be done on larger sample to confirm the study results.
Association of Angiotensin-Converting Enzyme Gene Polymorphism with the Progression of End-Stage Renal Disease in Dialysis Palestinian Patiants
(Al-Quds University, 2025-01-05) Marwa Mahmoud Ahmad Al-Salahat; مروة محمود احمد صلاحات
End-Stage Renal Disease (ESRD) is a significant public health issue globally. The progression of chronic kidney disease (CKD) to ESRD necessitating dialysis presents a considerable healthcare challenge. ESRD is a complex phenotypic structure of renal diseases affected by different etiologies. Although ethnic, social and environmental factors play a part in the development of the disease, to a large extent the cause of the disease is determined by genetic factors. One of these genes is ACE gene which is responsible for converting angiotensin I to angiotensin II, a potent vasoconstrictor. ACE polymorphisms appear to have significant impact on the progression of ESRD. The study is a cross sectional that was done in the period from June to September 2024. A total of 215 participants, including 110 patients diagnosed with end-stage renal disease (ESRD) and 105 healthy controls, were recruited from Beit Jala hospital. Clinical data were collected through medical records and questionnaires, while blood samples were obtained for molecular analysis. DNA extraction, quantification, amplification, and genotyping were conducted to investigate the relationship between ACE polymorphisms and clinical outcomes. Our results showed that the frequency of D allele was the highest (63%) and DI genotype was present in all subjects with a frequency of 58.1%. whereas, the DD genotype was present in approximately 34% of all subjects with slightly more frequency in ESRD patients (34.5%) compare to controls (33.3%). Moreover, the DI genotype is significantly more common in ESRD patients (63.6%) compared to controls (52.4%), with a p-value of 0.003. Significant differences were observed in ESRD patients with high systolic blood pressure (SBP) (163.7 ± 17.8 mmHg) compared to controls (126.1 ± 21.1 mmHg) , especially among those with the DD genotype There was no significant differences in diastolic blood pressure (DBP) or creatinine levels between genotypes. The mean age at first dialysis for ESRD patients with the DD genotype was 46.3 years, DI was 46.9 years, and II was 54.2 years, with no statistically significant differences (p-value = 0.126). The mean duration of dialysis was longest for DD (4.3 years) and shortest for II genotype (3.1 years). In conclusion, association between ACE I/D gene polymorphisms and progression to ESRD is varied among different ethnic groups. Our study showed a significant association between the DD genotype and increased risk of ESRD. Further research should be conducted using larger sample size to confirm the study results.
Association of OCT1, OCT2 and OCT3 gene polymorphisms with Type 2 Diabetes Mellitus risk in Palestine.
(Al-Quds University, 2025-01-08) Dua'a Shareef "Muhamed Subhi" Fakhouri; دعاء شريف "محمد صبحي" فاخوري
Type 2 diabetes mellites is a chronic condition when the body fail to balance blood glucose levels due to insulin are not secreted or not doing effectively its job, the insulin is considered as a key anabolic hormone which regulate carbohydrate, protein and fat metabolisms, so any defect in insulin actions or secretion lead to metabolic disorders such as Type 2 Diabetes. In addition, chronic blood glucose elevation can lead to serious complication which cause death. Worldwide, type2 diabetes accounted 95% of all diabetic cases, in average 15.3% of Palestinian individuals are affected according to last published reports. To date, there are many contributing agents including, one or combination of both environmental and genetic factors. A lot of genes may be implicated in onset and progression of type2 diabetes. Solute Carrier transporters22A (1,2 and 3) are belong to these genes, which are responsible of nutrient uptake, regulate ion gradient across membrane, drug metabolism such as metformin. The study is case control study, the data collection was done in 2024, June- September. The present study cosiest of two individual groups includes type 2 diabetic patients as case and healthy individuals as control. A total of 240 diabetic participants were included in this study 97 healthy participants. All patients aged over 40 years who attending Jericho Health MOH Center and/or AL-Ahili health care center, Palestine. The HbA1c, FBS, lipid profile was performed during the hospital admission examination. Four SNPs, OCT1 (rs628031), OCT2 (rs145450955), and OCT3 (rs3088442 and rs2292334) gene polymorphisms in T2DM cases and healthy controls were identified by amplicon based next generation sequencing (NGS), it is the first investigation to be held on Palestinian population. The frequencies of these polymorphisms were assessed in the studied population (Table 3.5). Moreover, the associations of OCT1 (rs628031), OCT2 (rs145450955), and OCT3 (rs3088442 and rs2292334) gene polymorphisms in T2DM cases and healthy controls were investigated. Our results showed that there was no significant association between the genotype in all variants between Case and Control P- value > 0.05 (Table 3.5). There was a significant relationship between gender of T2DM patients compared to those in control group (P=0.000), also significant relationship in the mean age among case and control groups was observed (P = 0.0001) The higher proportion of females in the diabetic group (137 females vs. 23 in controls) suggests a notable gender difference in diabetic prevalence within this specific study populations, show a significant difference in systolic blood pressure P- value = 0.001, also the results show diastolic blood pressure significant differences between case and control with P- value is 0.002 as it presents in higher amounts in non- diabetes individuals. In addition, significant differences between 2 groups in HbA1c and FBS with P-vale = 0.0001 and the higher value were present in diabetic patents. However, BMI, Cholesterol, TG, LDL and HDL showed no significant differences between diabetic and healthy groups P- value> 0.05. In conclusion, association between of OCT1 (rs628031), OCT2 (rs145450955), and OCT3 (rs3088442 and rs2292334) gene polymorphisms and type 2 diabetes mellitus diseases are varied among different ethnic groups. Our study showed no association between these variants and type 2 diabetic patients in the Palestinian population. Further studies should be done on larger sample to confirm the study results.
Design, Synthesis and In Vitro Studies of Lipophilic Mixed Aliphatic/Aromatic Platinum(II/IV) Derivatives that are Encapsulated in Novel Drug Delivery Systems
(Al-Quds University, 2024-05-11) Hebah Ibrahim Ali Damdoum; هبه ابراهيم علي دمدوم
Research on synthesizing new anticancer compounds with minimal side effects is ongoing, with platinum-based drugs renowned worldwide for their anti-tumor properties. The main hypothesis of our study is that the choice of suitable chelating ligands for the platinum center in both oxidation states (Pt(II) or Pt(IV)) significantly influences the processing of these molecules and the nature of the lipid-based nano-delivery system. The physicochemical properties of the ligand substituents coordinated to the Pt center also strongly affect the dispersion of Pt-based complexes in drug delivery systems, such as niosomal layers. This study focuses on the synthesis and investigation of new platinum(II/IV) derivatives with aliphatic and aromatic ligands encapsulated in niosomal vesicles. Characterization was performed using Attenuated Total Reflectance-Fourier Transform Infrared (ATR-FTIR) spectroscopy and nuclear magnetic resonance iv (NMR). The influence of the encapsulated ligands of platinum-loaded niosomal formulations on size, polydispersity, and loading efficiency was highlighted by using various novel types of platinum ligands. In vitro, studies on liver cancer cells (HepG2 and Hep3B) and the human hepatic stellate cell line (LX2) showed promising cytotoxic effects compared to those of cisplatin, miriplatin, and oxaliplatin. The results indicated that smaller and more compact ligands, such as NBA, have better loading efficiency in the niosomal formulation compared to bulkier ligands like DACH. There were also clear connections between compounds with high lipophilicity, such as myristate, and increased potency against cancer cell lines, likely due to interactions with lipidic compounds and non-ionic surfactants in the niosomes. In conclusion, this study introduces novel platinum(II/IV) compounds that show significant cytotoxic effects in liver cancer cells, highlighting the need for further investigation. This underscores the critical role of achieving well-balanced lipophilicity in drug delivery systems to enhance anticancer activity, minimize drug resistance, and ensure efficient tumor penetration. Further research is essential to understand and optimize these promising platinum-based formulations fully.
Detection of Familial Hypercholesterolemia Variants in Selected Patients with Premature Coronary Artery Disease from Hebron Region
(Al-Quds University, 2024-05-15) Enas Yousef Mustafa Sarahna; ايناس يوسف مصطفى سراحنه
Background: The Familial hypercholesterolemia disorder is prevalent but varies across ethnicities. Familial hypercholesterolemia is an autosomal dominant disease with 87% penetrance caused by pathogenic variants in the genes involved in cholesterol metabolism: LDL Receptor, apolipoprotein B or Proprotein Convertase Subtilisin/ Kexin 9 genes, resulting in impaired clearance of circulating Low Density Lipoprotein cholesterol, given that up to 90% of genetically confirmed familial hypercholesterolemia have LDL Receptor variants. It is a highly atherogenic metabolic disorder characterized by lifelong vascular exposure to LDL-C, leading to premature coronary artery disease. Purpose: This work aims to identify the pathogenic variant that cause this disease and verify cascade screening among the studied family. Methods: We selected a patient who fulfilled our inclusion criteria: (1) fasting plasma LDL-C level >190 mg/dL and triglycerides < 220 mg/dL; (2) presence of tendon xanthomata/xanthelasma/corneal arcus or premature coronary artery disease or a first degree relative, or a family history of hypercholesterolemia. The proband’s whole blood sample was sent to Whole Exome Sequencing. Then, blood samples were drawn for PCR and Sanger sequencing from his first-degree relatives. Any newfound case was treated as a new index, and his/her first-degree relatives were screened for that variant. Results: Findings showed that our proband has a heterozygous likely pathogenic missense NM_000527.2: c.1210A>G p. (Thr404Ala) variant in exon 9 of LDL Receptor gene. This variant has not yet been submitted to the Clinvar database. Screening results of his first-degree relatives showed that this variant was transmitted from his father (homozygous familial hypercholesterolemia: GG) to all of his brothers (heterozygous familial hypercholesterolemia: AG). First-degree relatives of affected individuals were screened; a pedigree was drawn. Sequence result and LDL-C level was significantly correlated. Conclusion: Familial hypercholesterolemia is underdiagnosed and undertreated in our population, and this increased the number of premature Atherosclerotic cardiovascular disease cases. Cascade screening is a beneficial and cost-effective process for the diagnosis and treatment of familial hypercholesterolemia early in life using lipid-lowering agents in order to decrease the burden of Atherosclerotic cardiovascular disease and prevent premature cardiovascular death among our population. The variant NM_000527.2: c.1210A>G p. (Thr404Ala) is highly suspected to be FH-causing as A>G substitution has a significant correlation with LDL-C mean level.
Detection of Genetic Mutations in Inherited Glanzmann Thrombasthenia Patients in Hebron-Palestine.
(Al-Quds University, 2025-01-05) Dalal ‘Mohammad Belal’ ‘Mohammad Dawoud’ Al-Hashlamon; دلال "محمد بلال" "محمد داوود" الهشلمون
Glanzmann Thrombasthenia (GT) is a rare autosomal recessive bleeding disorder caused by mutations in genes critical for platelet aggregation, particularly the ITGA2B and/or ITGB3 genes, leading to defective platelet function. This disorder affects platelet function through impaired expression or dysfunction of the αIIbβ3 integrin, which plays a key role in blood aggregation. GT can be classified into three types based on αIIbβ3 integrin expression: Type I, where expression is absent or below 5% of normal levels; Type II, where expression is 5–20% of normal levels; and Type III, where integrin is present but functionally abnormal. Diagnosis involves platelet function tests and sequencing of the ITGA2B and ITGB3 genes. Early diagnosis, management, and genetic counselling are vital for improving patient outcomes and preventing complications. The global prevalence of GT is approximately 1 in 1,000,000 individuals. However, in regions with high rates of consanguinity, this prevalence can rise to as high as 1 in 200,000, or even greater. Purpose: This study aims to validate the diagnosis of a clinically suspected GT patient, explore the feasibility of cascade screening for the genetic disorder, and identify pathogenic variants associated with GT within the Palestinian population. Methods: Patients meeting the inclusion criteria were selected based on the following criteria: (1) A lifelong bleeding tendency characterized by a normal platelet count, prolonged bleeding time, normal PT, and normal APTT. (2) Primary symptoms included mucocutaneous bleeding, frequent nosebleeds (epistaxis) and easy bruising. (3) Family history of GT, including being an immediate family member of a diagnosed GT patient. The proband’s whole blood sample was analysed using WES. Based on the WES findings, specific primers were designed, and RT-PCR was performed. Subsequently, first-degree relatives were screened for the identified variant. Results: WES revealed that the proband has a gain variant of uncertain significance (VUS) in the ITGA2B gene: NM_000419.5. This variant involves a duplication spanning exons from 3 to 12, causing an alteration in the reading frame. Notably, this duplication has not been reported in the ClinVar database, suggesting it may be specific to the local population. RT-PCR analysis of the proband's first-degree relatives identified two additional affected family members and six carriers of the variant. However, the results for the proband’s parents remain inconclusive, warranting further investigation. A pedigree was constructed to map the inheritance pattern within the family. Conclusion: GT remains underdiagnosed and undertreated within our population. Our study highlights the potential of WES as a powerful tool for uncovering the molecular basis of GT, suggesting its integration into diagnostic practices could significantly improve both the understanding and management of this condition. Additionally, the duplication mutation identified in our research, spanning multiple exons, may be unique to our population, as it has not been previously reported in genetic databases. However, further research is needed to confirm its pathogenic significance and explore its broader implications for genetic screening.
Development of ELISA immunodiagnostic test for COVID-19 detection in infected and vaccinated human sera
(Al-Quds University, 2023-06-03) Rawand Naji Talab Ajlouni; روند ناجي طلب عجلوني
Background: SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) is a novel coronavirus that has recently emerged and is causing a human pandemic. Despite the quick development of molecular diagnostic methods, validated serologic assays are necessary for detection, vaccination response study and epidemiological research. Our study's primary goal was to clone surface and membrane SARS-CoV-2 protein and use them to create an indirect ELISA so that we could screen immunity both qualitatively and quantitatively using the ELISA assay. Methods: Membrane (M) protein and spike (S) protein of SARS-CoV-2 were cloned using Pet28-a vector in Bl21 E. coli bacterial cells, then ELISA assay done step by step and adjusted in the lab to test the collected samples from infected, vaccinated and non-vaccinated patients using the newly expressed protein antigens in ELISA. Result: our study resulted in cloning of surface and membrane SARS-CoV-2 protein gene in Bl21 E. coli bacteria after the discovery of a point mutation. The newly cloned gene achieved a similarity of (99.3%) with sequences of SARS-CoV-2 genomes found in GenBank. The protein expression was inducted to have a crude protein with concentration of 0.5 mg\ml. The use of the recombinant S and M proteins in future screening purposes were tested using ELISA serological test, serum samples that were previously infected and then vaccinated have a higher rate of positivity (100%) using any of the two antigens, serum samples that were collected 2-3 months after vaccination have higher positive results (n= 19/25) when S antigen is used compared to M antigen (n=14/25). The number of positive results in the serum samples that collected at least 1.5 years after vaccination was 7/18 for S antigen and 9/18 for M antigen. The total samples that give positive ELISA result was 39 in S antigen and positive samples in M antigen was 36, the antibody titers related to S antigen is higher than M antigen. The titer of antibodies was higher in the samples that were previously infected and then vaccinated, then samples that were collected 2-3 months after vaccination, then lastly, the samples collected 1.5 years after vaccination. Conclusion: The recombinant S and M antigens were cloned successfully, we recommend to use S antigen in ELISA test rather than M protein, or use them together. We recommend also to do further studies for the sensitivity and specificity, or epidemiological study. :
Development of loop mediated isothermal DNA amplification (LAMP) test for the detection of active brucellosis infections among diary animals
(Al-Quds University, 2021-05-29) Ahmad Msallam Ibrahim Makhamreh; أحمد مسلم إبراهيم مخامره
Brucellosis is zoonotic a disease caused by bacterial genus Brucella. It affects a wide range of mammalian species and is transmissible to humans, making it a major socioeconomic problem. Because of the unspecific symptoms and signs that are shared with other febrile diseases, diagnosing brucellosis can be challenging and due to the slow growth rate in blood culture. Rose Bengal test is an internationally recommended test for the screening of brucellosis. They are not always specific as the antibodies can cross-react with other gram-negative bacteria, these tests are not of high value in detecting infection at early stages, May give a false negative result, for a short period after infection, Low sensitivity particularly in long chronic cases, relatively low specificity in endemic areas, since generation and disappearance of antibodies requires some time. To diagnose brucellosis, we developed a LAMP assay. The reaction takes 60 minutes to complete at 63 ° C. Its highly sensitive method , simple, quick, specific, and cost-effective. The strand displacement reaction is carried out at a constant temperature and is characterized by the use of four different primers, each of which is designed to recognize six distinct regions on the target gene. There was no cross-reactivity with the other bacteria. Its More effective than traditional PCR (Less expensive, easier, faster, no hardware required, and with the same accuracy as PCR). The blood test was carried out for 5000 sheep. out of which 200 (4%) were positive. We used the positive results (200) samples and re-examination using the LAMP method that we have developed. Whereas, by developing the LAMP method, we were able to eliminate the interference with other bacteria, and get rid of the false positive result due to the presence of antibodies to Brucella in the blood as a result of a previous infection. Whereas, The LAMP method, unlike the serology approach, is based on the detection of DNA for bacteria rather than antibodies to the bacterium, In addition to removing all of the barriers to detecting brucellosis that existed using serological approaches.
Establishment of Vitamin D Reference Values in the Palestinian Society And Investigation of Cultural, Behavioral, and Socioeconomic Effects
(Al-Quds University, 2022-08-21) Mohammed Ibrahim Issa Almahariq; محمد ابراهيم عيسى المحاريق
تساهم هذه الدراسة في مناقشة و توثيق نسبة فيتامين د والعوامل المرتبطة به، ويعتبر التعرض لأشعة الشمس هو المصدر الرئيسي لإنتاجه، حيث يوفر التعرض لاشعة الشمس بشكل يومي وكافي ما يقارب ال 90% من التركيز الكلي في الجسم، بينما يعتبر تناول الأطعمة الغذائية ذو مساهمة قليلة و ثانوية في زيادة نسبة الفيتامين في الدم . في العقد الأخير، هناك زيادة واضحة في تناول المكملات الغذائية وعمل الفحوصات المخبرية الخاصة بقياس نسبة فيتامين د عالميا، ويعزى هذا الإزدياد لعدم وجود مستويات مرجعية واضحة وثابتة خاصة بتركيز فيتامن د في أمصال دم الأشخاص الأصحاء. حيث تبلغ نسبة التركيز الكافي الموصى بها عالميا ≥30 نانوغرام / مل (75 ≥نانومول/ لتر)، ولكن يعتبر المستوى الدال على نقص التركيز ≤ 20 نانوغرام/مل (≤ 50 نانومول/لتر)، بينما يعتبر التركيز ما بين 20 و 30 نانوغرام/مل كمية غير كافية من الفيتامين في الدم. تهدف هذه الدراسة المقطعية إلى تحديد المستوى الطبيعي لفيتامين د الخاص بسكان الضفة الغربية في دولة فلسطين، و تحديد الإعتبارات البيئية، والاجتماعية والاقتصادية والسلوك الشخصي ومدى تأثيرها على تركيز فيتامين د حيث تم دراسة 300 عينة من كلا الجنسين بالغين وذو صحة سليمة، و لايعانون من أي تاريخ مرضي خاص بالكلى و الكبد و سوء الإمتصاص في الأمعاء، و تتراوح أعمارالمشاركين بين 19 و 65 عام. حيث تم إختيار 123 ذكر و 177 أنثى بطريقة عشوائية من مدن وقرى ومخيمات الضفة الغربية. و كذلك تم إستخدام إستبانة خاصة مثبتة لجمع المعلومات الديمغرافية و العوامل الخطرة المتعلقة بفيتامين د حيث تم جمع عينة الدم و إستخلاص المصل لقياس نسبة التركيز بإستخدام جهاز chemiluminescence immunoassay analyzer (ARCHITECT i1000SR). حيث كانت النتائج تشير الى أن نسبة تركيز فيتامين د في الدم تساوي 13.4 نانوغرام / مل ، مع ملاحظة أن 92% من الأشخاص هم دون مستوى ال 30 نانوغرام / مل، و 79.3 % أقل من 20 نانوغرام / مل، في حين أن 61% من الأشخاص المشاركين كانت نسبهم أقل من 12 نانوغرام/مل . كما تبين أن الأشخاص ذوو الفئات العمرية الأصغر (18- 28عام) كانت لديهم نسبة تركيز فيتامين د أقل من الأشخاص في الفئات العمرية العليا مثل (29-40)،(41-50)،(51-65)عام. كما أوجدت الدراسة أنه لا يوجد إرتباط بين مستويات تركيز فيتامين د والجنس والسمنة وإستخدام كريم واقي الشمس و التعرض لأشعة الشمس و كذلك إرتداء الحجاب للنساء المشاركات في الدراسة. و بالإضافة لذلك فقد تم دراسة عادة تناول أنواع محددة من الوجبات الغذائية، و قد تبين أنه لا يوجد اختلاف في مستويات فيتامين د لدى الأشخاص الذين يتناولون وجبات غذائية تحتوي على كميات كافية من الجبنة و الزبادي و كذلك تناول كميات كافية من البيض والحليب والسمك والخبز من قبل الاشخاص المشاركين في الدراسة. في الخلاصة، ووفقًا لعدم وجود أدلة تربط بين أمراض جهاز الهيكل العظمي وغيره من الأجهزة الرئيسية في الجسم مع المستويات الموصى بها لتركيز فيتامين د (≥ 20 نانوغرام / مل ، أو 30≥ نانوغرام / مل) ، وفي ضوء دراستنا لتحديد المستوى المرجعي لفيتامين د لدى البالغين الأصحاء ، نوصي بالنظر إلى المستويات التي تقل عن 20 نانوغرام / مل وقريبة من 14 نانوغرام / مل كمستوى مرجعي لفيتامين د لعامة السكان في مجتمع الضفة الغربية. ومع ذلك ، فإن عينة دراستنا محدودة بعدد صغير من المشاركين ، وبالتالي هناك حاجة إلى مزيد من التحقيق والبحث للتوصل إلى استنتاجات أكثر شمولية فيما يتعلق بالمستويات المرجعية والعوامل ذات الصلة بهذا الفيتامين.
Evaluation of Reverse transcription loop mediated isothermal amplification (RT-LAMP) compared to polymerase chain reaction and Covid-19 antigen tests among COVID-19 Patients
(Al-Quds University, 2023-05-28) Ayat Ismail Mohammad Halabeya; ايات اسماعيل محمد حلبية
Background: As of August 31, 2020, the recently discovered coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is the cause of coronavirus disease 2019 (COVID-19), up-to-date reports showed 767 million confirmed cases and over 6.9 million deaths have been reported globally.. To handle the pandemic, COVID-19 diagnosis must be precise and fast. Reverse transcription loop-mediated isothermal amplification (RT-LAMP) is a nucleic acid amplification test that could be an alternative to reverse transcription polymerase chain reaction (RT-PCR), in being a simpler, less expensive, and can be performed in water bath or heating blocks. The primary goal of this study is to evaluate the diagnostic potential of RT-LAMP compared to reverse transcription polymerase chain reaction (RT-PCR) among suspected infected individuals and to see if there is a correlation of DNA based detection tests with the use of Covid-19 quick antigen tests based on horizontal immunochromatography dipsticks. Methods: About 80 nasopharyngeal samples from suspected individuals were collected and tested for COVID 19 SARS virus infectivity. The total nucleic acid material was extracted from nasopharyngeal samples of the assigned people, followed by cDNA synthesis. Primers for two PCR systems were designed that are both targeting the Covid-19 spike gene, and they were employed in amplification of the viral genetic materials from the collected nasopharyngeal samples (80 samples). Commercially available LAMP kit was used as well to amplify the viral genetic material from positive and negative samples that were determined by PCR systems 1 and system 2. Finally, the presence of Covid-19 antigen in nasopharyngeal samples were detected using commercial quick dipsticks. Results and conclusion: Among the 80 PCR-tested samples using PCR system 1 and system 2; PCR system 2 revealed more positivity (53/80) compared to PCR system 1 positivity (36/80). The obtained shared positive samples by both PCR systems were 33 samples, from which 20 samples were randomly chosen to be tested by LAMP commercial kit and at the same time another 20 negative samples identified by both PCR systems were tested as well by LAMP method. Using LAMP commercial kit it showed positive amplification results from 19 smaples out of 20 PCR confirmed positive samples and positive amplification of 2 samples that were previously determined as negative by both PCR systems. It was difficult to draw any correlation between DNA based amplification of COVID 19 PCR and LAMP tests compared to Covid-19 antigen test, iv since only four of the ten positive tested samples were found to be positive by antigen test, and none of the negative tested samples were found to be positive by Covid-19 antigen test. Based on the fact that PCR system 2 gave higher number of positivity compared to PCR system 1, we recommend to optimize system 2 to amplify only one bands which enables its DNA sequence analysis and determines the specificity of this PCR system. On the other hand LAMP COVID 19 test could be appropriate diagnostic tool especially in low settings laboratories and in poor areas, since it only needs a water bath and the products can be directly visualized by chemo fluorescence DNA binding dyes.
First molecular detection and genotyping of Neospora caninum in naturally infected cattle and sheep in Palestine
(Al-Quds University, 2024-05-05) Heba suleiman salem alfarajeen; هبة سليمان سالم الفراجين
Neosporosis has become one of the most common diseases causing abortion in dairy cattle globally. The clinical signs of Neospora caninum have been reported in sheep, goats, deer, and horses. Fetal abortion induced by N. caninum is a common reproductive problem that causes significant economic loss in cattle and sheep husbandry. This study aimed to detect and identify N. caninum in intermediate hosts such as cattle and sheep from Palestine using polymerase chain reaction (PCR) followed by DNA Sanger sequencing and to study the genetic diversity of the study samples, targeting the MS10 microsatellite, using phylogenetic analysis. A total of 124 brain tissue samples were collected from 106 (85%) cattle and 18 (15%) sheep from a slaughterhouse in Jericho-Palestine. The PCR technique was used to identify the Neospora DNA in the brain samples based on the Nc-5 gene. N. caninum was detected in the brain samples; out of 124 samples, 30 (24.19%) samples were positive for N. caninum. The frequency of N. caninum in cattle (25.47%) was higher than that of sheep (16.66%). The infection caused by N. caninum was confirmed by DNA Sanger sequencing of ( 13 ) random positive samples. BLAST analyses of the Nc-5 gene revealed more than 92% to 99% matching with N. caninum sequences available in GenBank. Microsatellite MS10 repeats were used to study the genetic diversity of the Palestinian isolates. The microsatellite genotyping was done by next-generation sequencing (NGS). It was applied to 30 positive samples, and successful results were achieved in 15 samples. Our study sequences displayed 100% similarity with N. caninum isolated from dogs in Liverpool and 99.34% similarity with N. caninum isolated from cattle in Japan. The phylogenetic analysis of 15 N. caninum samples from Palestine and 83 microsatellite (MS10) repeats obtained from different countries and different hosts showed that N. caninum isolates are genetically diverse and distributed into v two main clusters. Locally, the Palestinian isolates are distributed in two clusters and share the same repeats with samples from Asia and Europe. The present study provided the first estimate of the frequency of N. caninum in cattle and sheep from Palestine. Additionally, it was also the first time for the investigation of the phylogenetic analysis of N. caninum based on microsatellite markers.
Follow-up of Tobamovirus infection in cultivated tomato crop using NGS analysis
(Al-Quds University, 2024-05-18) Maram Hatem Adel Jaafreh; مرام حاتم عادل جعافره
The Tomato vegetable plant (Solanum lycopersicum), which bears edible fruit, is a common component of the diet, and it is economically importance across the world. Tobamovirus group (family: Virgaviridae) are destructive agricultural diseases that infect plants. The main goal of the current research is to identify the most tomato cultivars that show the best resistance to viral infections. This outcome could help farmers to avoid many financial losses by choosing specific disease tomato resistance varieties. This study is designed to detect the presence of Tobamovirus relied on next generation sequencing (NGS) technology. The study was performed after collection of leaves, fruit tomato plants and soil samples from 4 different greenhouses located in Jericho district over a period of four months starting from February to May 2023 till the end of the samples collection period, a total of 155 of soil and plant samples were collected. For each collected sample total nucleic acid extraction was done, and processed for genetic analysis. First single stranded cDNA was synthesized from the produced total nucleic acid, followed by amplification via polymerase chain reaction. For each sample two PCR systems were applied for the viral detection and later its DNA sequence analysis was performed by NGS method. All files were uploaded on Galaxy platform program (usegalaxy.org), quality filtered, and analysis were achieved. Among the 155 PCR-tested samples using PCR system 1 and system 2; there was consistency in the PCR results of the two systems. Samples that were seen to have faint or weak amplification were different among the two used PCR systems. It was clear more positive results were seen among the tested samples using PCR system 1 compared to PCR systems 2. From the total tested samples using PCR system 1, 86 samples revealed to have moderate strong PCR results reflected by showing strong PCR bands on agarose gel electrophoresis of Tobamovirus compared to about 46 samples of the same criteria were obtained by PCR system 2. On the other hand, the samples that showed a faint or weak amplification were calculated to be 45 samples applying PCR system 1 and 86 samples applying PCR system 2. All other samples were revealed to be negative by the two used PCR systems. IV Using NGS analysis, the sequences showed 99% similarity to different isolates of Tomato brown rugose fruit virus including the Jordanian tomato Tobamovirus isolate (TBRFV-Jo), (GenBank accession no. KT383474.1).
Genetic Analysis and Population Structure of Phlebotomus sergenti Sandflies; Vectors of Leishmania tropica in Palestine
(Al-Quds University, 2024-05-19) Bushra Abdel Qader Tahboub Al-Amawi; بشرى عبدالقادر طهبوب الأموي
Phlebotomus (Ph.) sergenti is a widespread sandfly, it is considered a vector of leishmania tropica in Palestine and other countries; it is the cause of cutaneous leishmaniasis. This study aimed to investigate and analyze the phylogenetic and phylogeographic structure of Ph. sergenti among separated populations in the West Bank; Palestine. Population analysis of these sandflies is necessary for developing models that may provide better understanding of their current geographic distribution. The genetic population structure analysis of fifty Ph. sergenti sandflies were collected from West Bank, Palestine. This study inspected the genetic differentiation between Ph. sergenti populations using a 442 bp fragment of Cyt. b mtDNA gene. Maximum parsimony tree and median joining network were constructed depending on the identified haplotypes. Additionally, one hundred Ph. sergenti sequences previously deposited in the GenBank from ten countries (Iran, Pakistan, Afghanistan, Turkey, Cyprus, Greece, Syria, Lebanon, Morocco and Spain), were included in the study. Nine haplotypes were obtained from the Palestinian populations; six of them were private haplotypes, suggesting the presence of genetic isolation. The global analysis exhibited forty-five global haplotypes and grouped according to their geographical location. Pairwise FST values ranged between 0.58533 and 0.89288, indicating a high genetic differentiation and low genetic flow. Population genetic analysis of Ph. sergenti is considered crucial and may be needed to design an appropriate insecticide spraying programs and for sandfly control models, to restrict the transmission of leishmania parasites in the endemic areas of cutaneous leishmaniasis.
Genetic Mutation in Metastatic Bresat Cancer in Lumnial A Tumors, which may cause a Resistant to Hormonal Therapy in Palestine
(Al-Quds University, 2023-08-06) Diala issa salem hammad; ديالا عيسى سالم حماد
Molecular classification for breast cancer has many important diagnostic therapeutic and prognostic implications for breast cancer patients, which depends on molecular types presented or missing in cancerous cells. The Luminal A subtype, contain Estrogen receptors (E.R.), Progesterone receptors (P.R.) while missing Human epidermal growth factor-2 (HER2). Those patients are treated by hormonal therapy that targets these receptors to reduce or stop cancer cell growth and survival. In general, these patients have the best prognosis and the disease is more treatable than other subgroups. Some patients may develop hormonal therapy resistance for many reasons. In this research, we want to study the most common genetic mutation that causes hormonal therapy resistance. Material and method: This research contains two parts. First, we review a result tested in a tissue sample by Next generation sequencing (NGS) in sixteen patients diagnosed with metastatic breast cancer, Luminal A, treated with hormonal therapy, and developed a disease progression through the treatment management to know the most common mutation in the population. Second, tested three patients that were positive in the first part and targeted these mutations by Polymerase chain reaction (PCR) and sanger sequencing in a blood sample. Results The most common mutation that causes hormonal therapy resistance are ESR1 and PIK3CA mutations, and these mutations cannot be detected by PCR method. Conclusion These mutations need a specific method that covers all mutations that cause hormonal therapy resistance and disease progression in breast cancer patients. We need a specific method such as ddPCR to detect the mutation in ctDNA liquid biopsy.
Genetic Screening of Xeroderma Pigmentosum Disorder in a Palestinian Family
(Al-Quds University, 2023-08-13) Farouq Ziyad Rashad Nather; فاروق زياد رشاد ناظر
Xeroderma pigmentosum (XP) is an uncommon autosomal recessive genetic disorder characterized by an extreme sensitivity of the skin to sunlight, particularly UV light, and an elevated risk of skin cancer. Some patients with XP also exhibit neurological symptoms. The majority of XP cases are attributed to mutations in eight specific genes (XPA through XPG and XPV). The XP-V subtype of the disease results from mutations in a gene called XPV, also known as POLH, responsible for encoding Pol eta, a member of the Y-DNA polymerase family. XP variant represents a milder form of XP caused by variants in the POLH gene. POLH encodes an error-prone DNA polymerase eta, which plays a crucial role in synthesizing past UV-induced photoproducts. In the current study, we aimed to look for the underlying molecular cause of XP in a Palestinian family with multiple affected individuals suffering from the disease. WES analysis in the Proband led us to the identification of a homozygous frame-shift mutation c.106_118del p. (Val36Asnfs*8) in POLH which is responsible for the disease. Subsequently, clinical investigations and familial segregation analysis using Sanger sequencing were performed to check which family members are homozygous/ heterozygous (genotype) for the frameshift mutation. Interestingly, we discovered that a 60-year-old family member without any medical history is a homozygous for the mutation. In conclusion, this study enabled us to establish the genetic diagnosis of XP in a Palestinian family by the identification of a new disease-causing mutation in the DNA polymerase eta (POLH gene). This Shows the significance of molecular diagnosis for accurate identification of the disease and provides valuable information for proper genetic counseling in families affected by XP.
Identification and Analysis of Cutaneous Leishmaniasis in Northeastern Libya from Giemsa-Stained Tissue Smears between 2014 and 2020
(Al-Quds University, 2023-08-05) Hemam Adel AbdulAziz Doudin; همام عادل عبد العزيز دودين
Cutaneous leishmaniasis (CL) is a prevalent skin infection caused by the transmission of a protozoan parasite through the bite of a phlebotomine sandfly. This study aimed to evaluate the epidemiological aspects of CL in patients attending the dermatology clinic of the main referral hospital in Zliten, Libya. Data from 355 patients diagnosed with CL between 2014 and 2020 were analyzed to determine the incidence of CL and its distribution based on age, sex, residence, season, and affected body sites. In addition, this study aimed to identify the species of Leishmania using the ITS1-RFLP PCR technique and to compare the sensitivity and specificity of different PCR-based assays targeting the Ribosomal Internal Transcribed Spacer 1(ITS-1), Hexokinase (HK), and Phosphoglucomutase (PGM) genes on Gimza stained tissue smears. Furthermore, the study assessed the current spatiotemporal distribution of CL cases and projected the future incidence of the disease. The findings revealed a higher risk of CL in the coastal regions of Libya. The projected trends until 2060 indicated an increasing incidence of CL in the north-western part of Libya, a spread along the coastal region, and pthe otential emergence of new endemic areas in the north-eastern districts. These findings highlight the need for health authorities to develop appropriate effective control programs. The majority of patients in this study came from Zliten and its suburban areas, with a minority from neighboring cities.
Investigating the Molecular Basis of Immotile Sperms and Azoospermia Associated with Male Infertility in Palestinian Individuals
(Al-Quds University, 2023-01-03) John Edward Tawil; جون إدوارد طويل.
Infertility in men seems to be very obscure and lacks appropriate explanations in terms of genetic causes, though as a definition infertility, in general, is the inability of couples to conceive within a year of trying to achieve pregnancy without using contraceptives. Considerations in the definition means that the male is incapable of producing healthy sperms and/or sometimes no sperms at all that can fertilize an egg. The quality and the quantity of the produced sperms is very important, and a sperm should owe to specific criteria in order to be fertile, it needs a good tail or flagellum that can provide capacity to move, a good head that provide capacity to penetrate the egg in a quick manner and enough mitochondria in the neck, all with an integrated morphological feature and avide quantity. In term of Quality for instance, Multiple morphological abnormalities of the sperm flagellum (MMAF) is a disorder that results in the inability of sperm to move effectively. It encompasses a range of flagellar disorders. The genetic basis of MMAF is complex and could be owed to multiple genes. And, in terms of quantity oligospermia and azoospermia refer to decreased sperm quantity, which plays a critical role in increasing the chances of conception. Advances in genetic and molecular research are helping to address this issue and improve reproductive outcomes. In Palestinian community investigation is worthy because we have many consanguineous marriages which is set at a rate of ~ 40% this is according to the central bureau of Statistics in Palestine, which may help in finding variations in genes associated to MMAF that is not related to environmental factors. In this study, we utilized whole exome sequencing (WES) to analyze two independent individuals with different forms of infertility: asthenospermia and non-obstructive azoospermia. Our WES analysis led to the identification of several candidate genetic variations that may contribute to these phenotypes. In the asthenospermia case, we identified variants in four candidate genes (MROH8, MUC4, FADS6, TAS2R43) that may explain the MMAF phenotype. In the azoospermia case, we identified a homozygous missense variation in the MDM1 gene (NM_017440.4: c.1981G>A: p.Ala661Thr), which may explain the observed spermatogenesis failure. Segregation analysis revealed that the affected brother of the proband is also homozygous for the same MDM1, further supporting its potential role in the azoospermia observed in this family. Additionally, we identified variations in other four candidate genes (MAGEC1, OSBP2, NAT10, CD248) in the azoospermia case that may also play a role in infertility. We believe that there are still many unknown genes that causes azoospermia and asthenospermia. Trying to find a signature that exist in our Palestinian community would make innovative solutions in the future in the world of genetics practical.
Molecular and phylogenetic analysis of Enterobius- vermicularis from appendectomy specimens in the West Bank, Palestine
(Al-Quds University, 2024-12-22) Issam Bassam Ahmed Jawabra; عصام بسام احمد جوابره
Background A parasitic nematode known as Enterobius vermicularis (human pinworm) frequently causes gastrointestinal symptoms and, in some cases, acute appendicitis. Despite its widespread prevalence, Palestinian populations have limited molecular data on E. vermicularis. This study aims to investigate the molecular and phylogenetic characteristics of E. vermicularis in appendectomy specimens across Palestine. The study will concentrate on the COX1 gene to comprehend the genetic diversity and transmission patterns. Methods A number of 55 formalin-fixed, paraffin-embedded (FFPE) appendectomy specimens previously collected from 2018-2023, were confirmed for E. vermicularis infection. Samples originated from three government hospitals: Queen Alia Hospital in Hebron, Rafidia Hospital in Nablus, and Beit Jala Hospital in Bethlehem. DNA extraction using FFPE sections was carried out, followed by polymerase chain reaction (PCR) targeting the COX1 gene. Phylogenetic, sequence, and haplotype analysis were performed... Results The male-to-female ratio was 1.2:1, with 54.5% of patients being male and 45.5% female. The average age of patients was 13.3 years. There are no sequence variations among the sequenced samples, suggesting the conservation of this population's genetic structure. The phylogenetic analysis revealed the existence of Type B E. vermicularis in the Palestinian samples, which is exclusively associated with human hosts. Similarly, the haplotype analysis using median-joining network group all Palestinian samples in Group B. Statistical analysis of demographic and clinical variables; including age, gender, WBC, and neutrophil counts, showed no significant correlations with the infection Conclusion The study revealed a strong genetic uniformity among E. vermicularis samples from appendectomy samples in Palestine, suggesting limited transmission dynamics. All samples were identified as Type B, exclusive to human hosts when compared with global patterns. No significant associations were found between demographic or clinical manifestations and infection, indicating stable infection patterns across the population. The findings of the current research provide baseline molecular information about E. vermicularis infection in Palestine and may direct public health efforts by the Palestinian Ministry of Health in future research on the transmission and epidemiology of Enterobius.
Molecular Characterization of Borrelia Species as Causative Agents for Tick-Borne Relapsing Fever from Ixodidae and Argasidae in Palestine
(Al-Quds University, 2025-01-09) Abbas Khader Hussin Masalma; عباس خضر حسين مسالمة
Introduction: Tick-Borne Relapsing Fever (TBRF) is primarily caused by several Borrelia species that are transmitted through the bites of ticks. It is mainly characterized by multiple recurrences of nonspecific signs and symptoms, including fever, headache, myalgia, arthralgia, and even some neurologic complications. These Borrelia species are mostly vectored by hard ticks like Rhipicephalus genus and soft ticks like Ornithodoros tholozani which is known as the main vector of B. persica in Palestine. However, Molecular based data on Borrelia species and their hosts in Palestine are very scarce as B. persica is the only Borrelia species that had been studied.Objectives: This study aimed to investigate the molecular epidemiology of Borrelia species in ectoparasite hard and soft ticks, collected from different animal hosts throughout Palestine.Methodology: Ticks samples were collected from different animal hosts residing in different districts through Palestine, all ticks were identified at the genus and species levels based on taxonomic keys. DNA extraction was carried out, then screening for the presence of Borrelial DNA was carried out by PCR targeting flagellin (fla B) gene. All positive samples were confirmed by 16sRNA – PCR. PCR products were loaded onto agarose gel for band detection at 250 bp for fla B and 523 bp for 16sRNA, positive samples were sequenced and analyzed for BLAST identification. Statistical analysis was carried out using IBM SPSS v27.0 software. Results: Among all tick samples: 86% (n= 734) were identified as Ixodidae (hard ticks) and 14% (n= 117) were Argasidae (soft ticks). Overall, 76% of the identified hard ticks belonged to the genus Rhipicephalus (92% R. sanguineous and 5.5% R. Turanicus), 12% of Haemaphysalis ticks (80% H. parva and 20% H. adleri), 11% were Hyalomma ticks (84% H. dromedarri, 6% H. impeltatum, and 5% H. eagyptium). All collected soft ticks (n= 117) were identified as Ornithodoros tholozani. Out of 734 hard ticks, 2% (n= 13) were detected to be positive for Borrelia DNA by PCR. Sequence analysis revealed the presence of B. persica in 77% (n=10) of positive samples, while 15% (n=2) were B. turcicae, and 8% (n=1) was B. Lonestari. On the other hand, 9% of soft ticks (n=17) were found to harbor B.persica.Conclusions: Our results highlight the presence of Borrelia persica among hard ticks despite its’ usual presence among soft ticks, this could indicate an incidental acquisition or ecological overlaps. B. persica genotype was also observed in soft ticks. Notably, in Palestine, R. sanguineus is the major hard ticks which vector Borrelia species among hard ticks, while O. tholozani is the major one among soft ticks.

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