Biochemistry & Molecular Biology

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    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.
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    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.
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    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.
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    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. :
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    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.