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.
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.
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.
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 DIAGNOSIS AND GENOTYPING OF CANINE VISCERAL LEISHMANIASIS IN THE NORTHERN PART OF THE WEST BANK, PALESTINE
(AL-Quds University, 2005-05-10) سهير ابراهيم محمدعريقات; Suhair Ibrahim Mohammed Oraiqat; زياد عابدين; Robin Abu-Ghazaleh; Moaen Kanan
Molecular Genetics Analysis Of MEFV Gene Mutations In Familial Mediterranean Fever (FMF) Among Palestinans In The West Bank
(AL-Quds University, 2001-05-01) رانية يوسف ابراهيم أبو سير; Raniah Yousef Ibrahim Abu Seir; هشام درويش; محمود ابو حديد
Molecular Identification and Characterization of Amoeba Species from Different Geographical Regions in The West- Bank, Palestine
(جامعة القدس, 2022-11-27) مالك محمد عوض شريف; Malek Mohammad Awad Shareef
ABSTRACT Amoebiasis is one of the most ten common intestinal parasitic diseases worldwide, with Entamoeba histolytica infecting around 500 million people causing 100,000 deaths each year. In Palestine, amoebiasis is reported to be a major health problem, it is routinely diagnosed using microscopy by identification of cysts and trophozoites in fresh stool samples. However, this diagnostic method may result in overestimating the patient numbers infected with E. histolytica, and leads to mistreatment of the nonpathogenic species of Amoeba (E. dispar and E. moshkovskii) that are morphologically indistinguishable from the pathogenic species. In this study we investigated the molecular epidemiology of Amoeba species among Palestinian population in different regions of the West Bank. In addition, we determined the sociodemographic and socioeconomic factors associated with Amoeba infection among patients. A total of 100 stool samples were collected from patients who have been presented to Palestinian Ministry of Health (PMOH) clinics and private labs, patients came with symptoms of intestinal infections (abdominal pain, diarrhea and / or dysentery). Sociodemographic data was collected using questionnaire for patients who were diagnosed with Amoeba infection. The samples were initially analyzed by direct wet mount microscopy and then by PCR with specific primers for detection of E. histolytica, E. dispar, and E. moshkovskii. The PCR results confirmed the diagnosis of E. histolytica in 74 samples, and E. dispar in 29 samples. Mixed infection of both E. histolytica and E. dispar was identified in 7 samples. In a comparison between microscopy and PCR methods for the identification of E. histolytica and E. dispar, 96 positive fecal samples were yielded by PCR while 100 positive samples diagnosed microscopically. Furthermore, PCR confirmed of 74% positive samples diagnosed microscopically are also positive for E. histolytica. The demographic data showed a significant correlation between E. histolytica infection and patient’s age and educational level; where the highest infection rates were found among school and preschool children. Our study highlights the need for additional representative large population-based molecular studies on the distribution and epidemiology of the diseases in Palestine. Further studies on the environmental and behavioral factors of patients should be performed on larger scale to determine the risk factors associated with amoebiasis infection in Palestine. يعدٌّ داء الأميبا أحد أكثر ألامراض الطفيلية والمعوية شيوعًا وانتشاراً في العالم، حيث تصيب الأميبا من نوع المتحولة الحالة للنُسج "Entamoeba histolytica" حوالي 500 مليون شخص حول العالم مسببةً ما يقارب 100,000 حالة وفاة كل عام. وفقاً لإحصائياتٍ أجرتها وزارة الصحة الفلسطينية فإن داء الأميبا يمثل مشكلة صحية كبيرة واسعة الانتشار بين السكان الفلسطينيين في الضفة الغربية وقطاع غزة. يتم تشخيص هذا المرض بشكلٍ روتيني باستخدام الفحص المجهري لفحص وجود الشكل الهاجع "الكيسات" والشكل النشيط "الأتاريف" في عينات البراز الحديثة الإخراج من المريض باستخدام المجهر الضوئي. في معظم الأحيان تؤدي طريقة التشخيص الروتيني باستخدام المجهر إلى زيادةٍ غير دقيقة في تشخيص المصابين بالأميبا المتحولة المسببة للمرض من نوع المتحولة الحالّة للنُسج “E. histolytica”. في هذه الدراسة، قمنا بفحص عينات براز لمرضى يعتقد أنهم مصابون بداء الأميبا من السكان الفلسطينيين المقيمين في تسع مناطق مختلفة من الضفة الغربية وهي الخليل، بيت لحم، نابلس، رام الله، جنين، طولكرم، سلفيت، اريحا والعيزرية باستخدام تقنيات البيولوجيا الجزيئية "PCR". إضافةً إلى ذلك، قمنا بتحديد العوامل الاجتماعية والديموغرافية والاقتصادية المرتبطة بالإصابة بداء الأميبا. بالمجمل، تم جمع 100 عينة براز من المرضى الذين قاموا بمراجعة عيادات وزارة الصحة والمختبرات الخاصة، والذين عانوا أعراض التهابات معوية تشمل الإسهال، ألم تشنجي أو مغص في البطن، الهزال، والحمى. علاوةً على ذلك، قمنا بجمع معلومات ديموغرافية خاصة بالمرضى لأهداف الدراسة باستخدام استبيان تم تعبئته من المعلومات التي قدمها المرضى المشمولين في الدراسة بشكل مباشر. بدايةً، تم فحص العينات باستخدام الفحص المجهري المباشر ثم بواسطة تقنية تفاعلات البلمرة المتسلسلة “PCR” لتحري المادة الوراثية للأميبا في البراز باستخدام مشرعات "Primers" محددة وموثقة للكشف عن الحمض النووي الخاص بالأميبا. أكدت نتائج تفاعلات البلمرة المتسلسلة تشخيص الأميبا من المتحولة الحالّة للنُسج. E. histolytica في 74 عينة والاميبا من نوع المتحولة المتغيرة E. dispar في 29 عينة. كذلك تم الكشف عن عينات تحتوي على نوعيّ الأميبا المذكورين سابقاً معاً في 7 عينات. بالمقارنة بين تقنية الفحص المجهري وتقنية تفاعلات البلمرة المتسلسلة لتشخيص الاميبا المسببة للمرض، أكدت تقنية PCRعلى أنّ 74٪ من العينات الإيجابية التي تم تشخيصها مجهريًا كانت إيجابية أيضًا باستخدام PCR. كما أظهرت البيانات الديموغرافية وجود ارتباط كبير بين العدوى بالأميبا من نوع E. histolytica والفئة العمرية للمريض والمستوى التعليمي له، حيث وجدت أعلى معدلات الإصابة بين أطفال المدارس ومرحلة ما قبل المدرسة. قامت دراستنا بتسليط الضوء على الحاجة الملحة لمزيدٍ من الدراسات باستخدام تقنيات البيولوجيا الجزيئية على عددٍ أكبر من العينات للحصول على صورة أكثر وضوحاُ حول انتشار وتوزيع داء الأميبا في فلسطين. كذلك توصي دراستنا بضرورة إجراء المزيد من الأبحاث حول العوامل البيئية والسلوكية للمصابين على نطاق أوسع لتحديد عوامل من المحتمل ان تكون مرتبطة بعوامل الخطر للإصابة بداء الأميبا في فلسطين
The Role of Angiotensin-Converting Enzyme 1 and Angiotensin-Converting Enzyme 2 Genetic Polymorphism in Severe Acute Respiratory Syndrome Coronavirus-2 in Palestinian Population
(Al-Quds University, 2023-08-19) Lama Khaled Hammad Abu Saleh; لما خالد حماد ابو صالح
As a key enzyme of the renin-angiotensin system (RAS), angiotensin-converting enzyme 2 (ACE2), has been validated as a SARS-CoV-2 receptor, linking RAS to COVID-19. It is likely that functional ACE1/ACE2 gene polymorphism cause the imbalance of ACE1/ACE2 ratio, causing a RAS imbalance that may contribute to the COVID-19 infection complications by causing higher lung damage and disease with severe symptoms. Herein, we developed a new genotyping method using next generation sequencing (NGS) to study four single nucleotide polymorphisms (SNPs), three for ACE1, rs4343, rs4342, rs4341 and one for ACE 2, rs2285666 in one multiplex PCR tube. Bioinformatics analysis was done using free online galaxy program (https://usegalaxy.org.au/). The association of ACE 1 (rs4343, rs4342, rs4341) and ACE 2 (rs2285666) polymorphisms with COVID-19 infection in Palestine were investigated. A total of 130 samples were collected, including 50 negative controls without COVID-19 infection, 50 positive controls with COVID-19 infection but not hospitalized, and 30 patients with severe COVID-19 infection in the intensive care unit. Results showed that the genotype distribution of ACE2 (rs2285666) polymorphism was significantly different between COVID 19 patients and the control group (P-value = 0.049, X2), while no statistical differences were observed between the ACE1 mutations (rs4341, rs4342, rs4343) and the control group (P-value > 0.05, X2). Individuals with ACE2 rs2285666 GG genotype were more prevalent in COVID-19 patients compared to control group (P-value = 0.049, X2). Age and comorbidities such as hypertension and coronary artery disease were independent risk factors for COVID-19 disease (P-value < 0.05, X2). Symptoms of COVID-19 patients such as fatigue, headaches, runny noses, and loss of smell were significantly higher in the positive cases COVID-19 (P-value < 0.05, X2), while dyspnea was more frequent in the ICU patients (P-value < 0.05, X2), In this study, we support the hypothesis that wild genotypes of ACE2 rs2285666 GG are associated with COVID-19 infection
The Role of Human Cytomegalovirus UL16 Glycoprotein and pp65 Phosphoprotein on Tegument Site, Membrane and Raft Association in Human Fibroblast Infected Cells
(AL-Quds University, 2009-06-30) خضر محمد ابراهيم زواهرة; Khader Mohammed Ibrahim Zawahreh; ميساء العزة; Musa Hindiyeh; Zaidoun Salah
The Influence of the Interaction and Allelic Location of the C957T and SNP19 Polymorphisms of the Dopamine Receptor-2 Gene on Feedback-Based Learning
(Al-Quds University, 2020-06-10) Anfal Ahmad Saleh Abu-Hilal; أنفال أحمد صالح أبو هلال
People vary in their cognitive performance. Multiple factors can perturb learning and forming associations. It has been shown that the neurotransmitter dopamine is an essential factor in modulating feedback-based learning. Dopamine exerts its effects via two families of postsynaptic receptors, type 1 and type 2. The release of dopamine is regulated by presynaptic dopamine type 2 receptors (DRD2). In this project, we pursued a multidisciplinary approach to examine the molecular and cognitive aspects of naturally-occurring variations in the dopamine system. We investigated feedback-based learning under the influence of two naturally occurring polymorphisms in the DRD2 gene, the C957T, which regulates binding affinity in postsynaptic DRD2 receptors; and SNP19, which modulates the number of presynaptic DRD2 autoreceptors. After using polymerase chain reaction (PCR), we utilized restriction fragment length polymorphism (RFLP) to identify the polymorphism variants. Further, we developed a PCR based technique to test whether the two polymorphisms are on the same allele or not. Our aim was two-fold: (1) to study the interaction between the C957T and SNP19 in modulating cognitive function, and (2) to investigate the cognitive effects of allelic location of C957T and SNP19 on cognitive performance. All heterozygotes for the two polymorphisms underwent amplification refractory mutation system (ARMS) PCR followed by ARMS to examine the allelic location of the two polymorphisms. We recruited a sample of 476 healthy undergraduate students at Al-Quds University, Palestine. All subjects completed a probabilistic categorical feedback-based learning task that differentiates learning from positive and negative feedback. Our results showed a gene dose effect, in which the T allele of C957T that is related to low expression of D2 postsynaptically is linked to the G allele of SNP19 that is related to high expression presynaptically. Also, we found that subjects with the lowest DRD2 postsynaptic affinity (CC-homozygotes for C957T) alongside the highest concentration of presynaptic DRD2 autoreceptors (GG- homozygotes for SNP-19) learned significantly better after receiving positive feedback or being in a potentially-positive state. These results are in line with previous findings that highlight the role of DRD2 in modulating tonic dopamine signaling. To our knowledge, this is the first project to examine the cognitive effects of the interaction of the C957T and SNP19 polymorphisms and their allelic locations. These results will further our understanding of the dopaminergic system and its regulation of cognition and involvement in a myriad of neurological and psychiatric disorders.
The Role and Expression Pattern of TET1 in Breast Cancer
(Al-quds university, 2019-12-18) Mahmoud Ali Mousa Al-Zahayqa; محمود علي موسى الزحايقة
Cancer is a genetic disease. Mutations and epigenetic alterations such as aberrant DNA methylation which results in altered gene expression is evident in all cancers studied, and is likely responsible for its major hallmarks. Methylation is maintained by DNA methyltransferases, however, methylation can be reversed by mechanisms that are poorly understood. Recently, functional demethylation was linked to hydroxylation of 5hmC by the TET family. TET1 is the most reduced member in a variety of human malignancies, suggesting a tumor suppressor function for this protein. In addition, published research showed controversial conclusions about TET1 function in breast cancer. Moreover, recent evidence showed that TET1 has more than one isoform. Thus, our hypothesis proposes the different TET1 isoforms may play different roles in breast cancer through differential expression pattern in different transformation contexts. In the present study, we tested the expression level and localization of TET1 enzyme in breast cancer samples using IHC, and the expression level using relative qRT-PCR in different breast cancer cell lines under different contexts. In addition, we tested the expression pattern of different TET1 isoforms using in vitro and in vivo cell transformation models. We also tested the effect of TET1 overexpression in MDA MB231 cells using lentivirus vector containing TET1 coding sequence on various cancer hallmarks. Our results demonstrate that TET1 has differential expression pattern in breast cancer embedded tissue samples compared to normal tissue. In addition, TET1 expression correlated with the differentiation level. From our hormone experiments, and in vitro as well as in vivo transformation studies, we clearly showed that different TET1 isoforms are differentially expressed under different physiological and transformation contexts, and different TET1 isoforms having different distribution pattern. Finally, we proved that TET1 full length is a tumor suppressor gene. In conclusion, our study demonstrates the role of TET1 in breast cancer is not straight forward one and this necessitates future studies to better characterize the TET1 function in breast cancer initiation and progression

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