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Sep 2023 DOI 10.14302/issn.2692-1537.ijcv-23-4660
SARS-CoV-2 real-time reverse-transcription PCR (rRT-PCR) is the most effective testing system available to combat COVID-19, given the absence of any specific treatment or vaccine. Moreover, numerous SARS-CoV-2 rRT-PCR kits have been approved under emergency-use-authorization (EUA) worldwide. In this article, we present a comparison of important performance features among five commercial RT-PCR assays. A total of consecutive nasopharyngeal (NPS) samples and oropharyngeal (OP) swabs were collected from 50 COVID-19 patients to analyze sensitivity and specificity. The results showed variations in sensitivity among all the RT-PCR kits examined. The Pishtaz teb assays demonstrated the highest positive percent agreement (PPA) of 95.2% (40/42), followed by Covitech (90.5% - 38/42), DaAn Gene (83.3% - 35/42), Sansure (66.66% - 28/42), and Power check of SARS- CoV-2 panel (64.3% - 27/42). Conversely, all five molecular assays demonstrated a negative percent agreement (NPA) of 100% (8/8). These findings provide a technical baseline for assessing the performance of five distinct commercial PCR assays for detecting SARS-CoV-2. They could prove practical and useful for laboratories seeking to purchase any of these assays for further clinical validation. Highlights ·Compared five COVID-19 RT-PCR kits approved and available by Iran Ministry of Health. ·Pishtaz teb's kit identified the highest number of positive clinical samples.
Feb 2019 DOI 10.14302/issn.2471-7061.jcrc-18-2526
There is currently no validated micro(mi)RNA diagnostic stool test to screen for colon cancer (CC) on the market because of the complexity of fecal density, vulnerability of stool to daily changes, and the presence of three sources of miRNAs in stool (cell-free from fecal homogenates, exsosomal miRNAs from fecal exosomes, and fecal colonocytes). To address these complexities, we have first carried out a microarray miRNA experiment, using Affymetrix GeneChip miRNA 2.0 Arrays, on immunocaptured and enriched stool colonocytes of 15 subjects (three healthy controls and twelve colon cancer patients [three TNM stage 0-1 (e.g., polyps◻ ³ 1 cm, villous or tubvillous, or with high grade dysplasia), three stage 2, three stage 3, and three stage 4 in triplicates to select a smaller panel of 14 preferentially expressed mature miRNAs associated with colon cancer (12 Up-Regulated, miR-19a, miR-20a, miR-21, miR-31, miR-34a, miR-96, miR-106a, miR-133a, miR-135b, miR-206, miR-224 and miR-302; and 2 Down-Regulated, miR-143 and miR-145). In a subsequent validation study carried out on total small RNA extracted by immunocapture, followed by RT that employed TaqMan® miRNA Reverse Transcription (RT) Kit and a Custom TaqMan RT Primer Pool, absolute quantification of miRNAs, in copies/µl, was measured using a chip-based Absolute QuantStudio 3D Digital PCR analysis. To ensure that we have chosen human and not bacterial small total RNA, we have carried out coextraction protocols with E. coli K1 strain RS18, compare Agilent electrophoretic patterns, and also sequenced random samples throughout this research using mRNA/miRNA sequencing. Our initial quantitative dPCR miRNA data presented herein showe that the quantitative changes in the expression of a few mature miRNA genes in stool, which are associated with right and left colon cancer, would provide for a more convenient, sensitive and specific diagnostic screening markers thatare more useful than those test markers currently available on the market, such as the low-sensitivity (<15%) fecal occult blood test (FOBT); result in better compliance; and is more economical than the invasive and expensive colonoscopy exam in colon cancer, which can be cured if that cancer is detected at the early TNM stages, and that becomes incurable and deadly if not diagnosed before metastasis. Initial test performance characteristics of the miRNA approach showed that the test has a high numerical predictive value in colon cancer. Moreover, underpinning of the miRNA markers as a function of total RNA showed that the test can numerically differentiate between control subjects and colon cancer patients, particularly at the early stages of that curable cancer. We propose to extend our initial research results to a larger prospective and randomized five-years nested case-control study, to validate the expression of the above 14 miRNAs, in stool of 180 individuals in an epidemiologically designed study, using (30 controls and 150 colon cancer patients (thirty precancerous polyps (stage 0-1), forty five stage 2, and seventy-five colon cancer stages 3 or 4). chosen randomly by an epidemiological method from 900 control and CC subjects to allow for an adequate time to collect the required 900 stool samples, as well as allowing for statistically valid analysis, standardized test conditions, and to provide a mean for determining the true sensitivity and specificity of a miRNA-screening approach in noninvasive human stool. Power-analysis has indicated that a total of 180 individuals, which will take us 5 years to enroll in testing, is an appropriate number of subjects to standardize and validate our proposed miRNA screening test. We may find out at the end of the proposed validation study in stool that fewer miRNAs, or even one miRNA, may suffice to serve as an efficient and a quantitative marker for the non-invasive diagnostic screening of colon cancer in human stool. The above approach when combined with bioinformatics analysis, to correlate miRNA seed data with our previously published messenger (m)RNA target data in stool, allows for a thorough mechanistic understanding of how miRNA genes regulate mRNA expression, and would offer a better comprehensive diagnostic screening test for the non-invasive early detection stage (0-1) of colon cancer. In order to show the clinical sensitivity and specificity of the proposed miRNA test, the absolute miRNA PCR values, in copies/µl, will be correlated with FOBT, colonoscopy, and pathology data. Standardization will establish test’s performance characteristics (sample selection, optimal sample running conditions, preservation and storage) to ensure that the assay will perform the same way in any laboratory, by any trained personnel, anywhere in the World. Ultimately, a smaller number of selected validated miRNAs (<10) showing increased and reduced expression could suffice to give quantitative miRNAs colon cancer expression values, useful for the early diagnostic screening of that curable cancer.
Aug 2018 DOI 10.14302/issn.2690-4721.ijcm-18-2217
Background Overuse of beta-lactam antibiotics has lead to selection for extended-spectrum β-lactamase (ESBL) producing Enterobacteriaceae, a major cause of antibiotic resistant urinary tract infections (UTIs). Standard detection methods are time-consuming, with disputed accuracy. This study describes a novel real-time PCR method to detect CTX-M, SHV, OXA and TEM. Methods 179 Enterobacteriaceae isolates from UTIs were collected from the Leicester Royal Infirmary, UK. A multiplex Plexor®-based real-time PCR assay detected ESBLs using their specific amplicon melting temperature, during each cycle, removing the need for a melt-curve analysis. Validation was achieved by end-point PCR and disk diffusion. Results The method was able to produce rapid and accurate results, achieving a sensitivity and specificity of 94.9% and 72% respectively, and the assay can differentiate between the different ESBL genes, with ease. Conclusions With further investigation, a Plexor®-based assay could form the basis of a high-throughput kit that health services could use to detect ESBLs or other antibiotic resistance genes.
Nov 2025 DOI 10.14302/issn.2326-0793.jpgr-25-5573
Advancements in proteomic and genomic technologies have transformed molecular biology by enabling comprehensive analysis of biological systems at the molecular level. This literature review explores the evolution, methodologies, and practical applications of key proteomic and genomic techniques. In proteomics, tools such as two-dimensional electrophoresis, mass spectrometry, Western blotting, Edman degradation, and functional protein microarrays have facilitated high-throughput protein identification, post-translational modification analysis, and biomarker discovery. Similarly, genomic methodologies like PCR, recombinant DNA technology, gel electrophoresis, and Southern blotting have revolutionized gene detection, manipulation, and expression profiling. The review also highlights the interdisciplinary impact of these technologies across clinical diagnostics, oncology, autoimmune disorders, infectious disease surveillance, cardiovascular research, and personalized nutrition. Integrative approaches combining proteomics and genomics are enabling the discovery of novel therapeutic targets, improving disease classification, and advancing precision medicine. Despite current limitations, such as the absence of amplification techniques for proteins and challenges in data interpretation, ongoing innovations promise to bridge these gaps. This synthesis underscores the pivotal role of molecular techniques in deepening our understanding of human biology and accelerating biomedical advancements for improved healthcare outcomes.
Mar 2024 DOI 10.14302/issn.2997-2108.jcc-23-4838
Among the reproductive cancers cervical cancer has special place, because the second most frequent cause of cancer-related death among women worldwide. The studies suggested that the PI3K/mTOR/AKT signaling pathway is associated with certain reproductive tumors. A lot of research is ongoing for understanding this pathway evidence of its role in promoting tumorigenesis and recent progress in the development of therapeutic agents that targeted PI3K/AKT. In this a single-arm study included 34 Azerbaijan population woman with HPV-negative cervical tumors. The core genes of PAM signaling pathway were analyzed using RT-PCR method. Our preliminary results suggested that tumorgenesis of HPV-negative cervical cancer patients approximately 25% associated with dysregulation of PAM signaling pathway reason which are core genes alteration. The overall survival times in the PAM-active and PAM-stable patients were not significantly varies. However, the main factor for overall survival times were treatment strategy: both PAM-active and PAM-stable patients who received radiation therapy alone had a shorter overall survival than patients who received radiation plus chemotherapy. The patients with alteration of ATK1 and mTOR genes in PAM signaling pathway had poor prognosis then patients with PIK3CA and PTEN mutation
Dec 2023 DOI 10.14302/issn.2324-7339.jcrhap-23-4634
Introduction Human Immunodeficiency Virus (HIV) remains a persistent global public health challenge. In 2020, approximately 37.9 million individuals were living with HIV globally, including 1.7 million children <15 years old, with a global HIV prevalence of 0.8% among adults. A larger portion of people living with HIV are found in low-and middle-income countries, and Sub-Saharan Africa (SSA) is home to about 68% of people living with HIV in the world. Strikingly, with increased uptakes in PMTCT, challenges in ART programs, and high viremia among children and adolescents in SSA, the success rate of ART might be quickly compromised, with possible HIVDR emergence, particularly after years of paediatric ART exposure. Therefore, monitoring ART response in children and adolescents in terms of HIVDR patterns and other socio-economic determinants of disease progression might help achieve better treatment outcomes at individual levels. At a programmatic level, this can guide further optimization of treatment options for SSA especially Zimbabwean rural where there is paucity of information on HIVDR prevalence in children and adolescents. Methods We enrolled 89 children and adolescents experiencing virologic failure from Chidamoyo Christian Hospital in Hurungwe. We managed to amplify all the 89 using nested PCR and 32.5% (29) had resistance to at least one ART drug and analysis was done using the 29 samples. Results Among the 89 participants with virologic failure,29 were resistant to at least one of their ART drugs. 39.2% of males and 23.07% of females had HIV-1 with resistance to at least one medication. Among 29 participants with HIVDR mutations, the prevalence of at least one HIVDR mutation to protease inhibitors (PIs), Nucleotide Reverse Transcriptase Inhibitors (NRTI), and Non-Nucleotide Reverse Transcriptase Inhibitors (NNRTI) were 6.47% ,46.76% and 46.76% respectively. Of the 29 participants who had HIVDR 19 (65.5%) had resistance to a drug they were currently taking and they needed to be switched to a better effective ART regimen Conclusion Use of HIVDR testing in guiding and monitoring development of HIVDR at the start of ART or at 1st failure can be very important in treatment options and patient management.
Oct 2023 DOI 10.14302/issn.2575-1212.jvhc-23-4532
Camels are a significant source of income for nomadic populations in many developing countries, including Ethiopia. Camels are well adapted to dry and semi-dry regions, providing income, food security, and transportation. However, camel production and productivity are constrained by infectious diseases, such as brucellosis, which is a highly infectious bacterial disease that affects camels and humans worldwide. Brucellosis causes significant economic losses due to abortion, low herd fertility, and decreased milk production. In Ethiopia, the prevalence of camel brucellosis varies depending on factors related to the host, agent, climate, and management system, with a reported prevalence ranging from 0.5% to 11.9%. Accurate diagnosis of camel Brucellosis is essential for herd-based screening of animals. Although culturing the pathogen is the preferred method for diagnosis, serological tests such as Rose-Bengal plate test (RBPT), Enzyme-linked immunosorbent assay (ELISA), and Complement fixation test (CFT) and polymerase chain reaction (PCR) assays have been developed. Implementing effective diagnosis and surveillance systems to control the spread of brucellosis in animals and humans is very important, on top of awareness campaigns, vaccination programs, and suitable laboratory establishment recommended. Continued research is essential to maintain the health and productivity of camel populations, particularly in pastoral areas where camels play a significant role in the livelihood of communities. Therefore, the present paper views the seropositive prevalence and potential risk factors associated with camel brucellosis in Ethiopia.
Jul 2023 DOI 10.14302/issn.2575-1212.jvhc-23-4510
Dermatophytosis affect companion animal’s skin and keratin appendages as cats and dogs, resulting in red, scaly, itchy, bald, and raised patches like ring. The three main groups are Microsporum, Trichophyton and Epidermophyton. This study collected samples of skin scrapping and hairs from 130 cats and 70 dogs, using common mycological approach samples were examined. Antifungal agar disc diffusion and broth microdilution assays were utilized on some of the isolates. Three groups of Guinea pigs (6 in each) were then infected with one isolate of M. canis or T. mentagrophytes fungi, another skin scrapping samples of virulent fungi was isolated on the 7th and 14th days, blood samples were collected at 14th day. Reverse transcription-PCR to detect 98 bp protease gene. Resulting in 45% of cats and dogs tested positive for Microsporum and Trichophyton species. Agar disc diffusion revealed that the antifungal medication griseofulvin was the most effective against tested isolates. The best results for MIC test were griseofulvin (0.98 µg/ml) followed by acetic acid (0.28 µg/ml). Differential leukocytic count of Guinea pigs showed that monocyte levels remained unchanged, while neutrophil and lymphocyte levels had increased. The active (isolates from Guinea pigs skin scrapping) and dormant cells (isolates from keratin free media) were distinguished by Reverse Transcriptase-PCR. Collectively, qPCR is a successive and feasible method for the diagnosis for Microsporum and Trichophyton species.
Jun 2023 DOI 10.14302/issn.2377-2549.jndc-23-4615
A literature review was undertaken with a focus on 1) identifying the research gaps regarding CECs, 2) identifying the most common ones, and 3) identifying the typical analytical methods/technologies employed, for their analysis. A total of 214 papers were noted, with a total of 21 review articles (9.8%). Of this total, a surprisingly high number were from South Africa alone: 117 (54.7%), of which 44 (20.6%) reports were associated with South Africa’s Water Research Commission (WRC). The top three CECs research gaps were (decreasing rank: Number of “gaps”, %): 1) Toxicity/Risk/Impact (260, 21.5%), 2) Analysis/Tests/Methods (118, 9.8%) and 2) Future research/studies (118, 9.8%), and 3) Monitoring (89, 7.4%). The common classes of CECs that were reported on, were : (i) Chemical: pharmaceuticals, personal care products, steroids, chlorinated and brominated contaminants, PAHs, PCBs, phthalates, alkyl phenols, herbicides, organochlorine pesticides, engineered nanomaterials and (ii) “Microbiological”: antibiotic resistance genes, human enteric bacteria and viruses, microbial pathogens (e.g., E Coli, rotavirus, Crypto, etc.), infectious biological water contaminants (e.g., E Coli isolates), cyanobacterial blooms (Microcystis). Common test methods used for analysis of the chemical contaminants were found to be chromatography (gas, liquid)-mass spectrometry; for the microbial contaminants, they were culture-based methods, ELISA, fluorescence microscopy, qPCR, RT-qPCR, gel electrophoresis, Raman spectroscopy, and also chromatography (largely liquid)-mass spectrometry, were also used. Some proposals were additionally made to address the very common, significant research gaps noted in CECs research, especially the standardization of analytical chemical test methods, based on chromatography-mass spectrometry, for quantification.
May 2023 DOI 10.14302/issn.2575-1212.jvhc-23-4521
Background The emergence and spread of carbapenem-resistant gram-negative bacteria pose a serious threat to human health. Currently, little is known about the molecular mechanisms underlying carbapenem -resistance and their prevalence among APEC in Egypt. The aim of this study was to detect APEC in clinically diseased broiler chickens collected from broilers farms located at Dakahalia governorates, asses their virulence –associated genes, detect the antimicrobial susceptibility of recovered isolates and to detect genes encoding carbapenemase resistant. Methods A total of 100 organ tissue samples subjected to conventional culture technique for isolation of E. coli. The confirmed E. coli were subjected to disc diffusion method for detection their susceptibility to antimicrobials. Polymerase chain reaction (PCR) was used for detection of APEC virulence genes (hlyA, iutA, ompT, iss, iroN) and six carbapenem- resistant genes namely, blaIMP, blaVIM, blaKPC, blaOXA-48 blaGES and blaNDM,. Results Forty isolates were confirmed to be E. coli among them, three or more APEC virulence- genes were detected from all isolates. The hlyA gene was detected in 90% (36/40), iroN in 95% (38/40), ompT in 97.5% (39/40), iutA in 92.5% (35/40) and iss was detected in 95% (38/40) of APEC isolates The tested isolates exhibited a remarkable resistance to ampicillin (97.5%), cefuroxime (92.5%), clindamycin (90%), chloramphenicol (62.5%), doxycycline (45%), amikacin (25%) and ciprofloxacin (12.5%). While, the retrieved isolates displayed 100 % sensitivity against imipenem, meropenem, ertapenem, ceftazidime and colistin. Concerning carbapenemase-encoding genes, blaIMP, blaVIM, blaKPC, blaOXA-48, blaGES couldn’t be detected among the E. coli isolates, while, blaNDM was confirmed in three isolates . Conclusion The detection of NDM as one of the carbapenem resistant genes reveals that the resistant strains are not only capable of infecting humans, but that carbapenams- resistant E. coli (CREC) has also started to pose a threat to poultry farm and other livestock animals. This may give rise to worries that these food-carrying creatures could infect humans or colonize them.
Apr 2023 DOI 10.14302/issn.2576-6694.jbbs-22-4390
Background The genetic material of the genetically modified crop has been altered to develop the necessary insect resistance features by introducing genes from the Bt (Bacillus thuringiensis) bacterium. The objective of this study was to find smuggled GM Bt crops in the Metema farming area and examine its environmental effects. Method An experimental; Completely Randomized Design (CRD) was used to collect crop samples in the study area. The CTAB (Cetyltrimethyl ammonium bromide) technique was used to isolate DNA from all transported samples, and the purity was determined using a Nano Drop spectrophotometer. Conventional PCR with particular primers for different Bt gene events was used to detect the presence of genes. Furthermore, utilizing Bt cotton specific primer sets, the prevalence of GM cotton was measured, and amplified fragments were confirmed using agarose gel electrophoresis. Result The PCR results revealed that 15 (33.3 percent) of the samples were Bt cotton smuggled from Sudan. The PCR assay also revealed the presence of GM maize. Moreover, the effects of GM genes on the environment were studied in diseased samples, and no transgenes were found. Furthermore, domestic and indigenous crops were used to determine horizontal gene transfers of GM genes to other crops, and the transgene was not found in any of the samples analyzed. Conclusion: In the current study, 28 (13.4%) of the 209 (100%) total analyzed samples were GM crops which indicated the presence of unauthorized GM seeds in the study area. Environmental impact studies and horizontal gene transfer data similarly revealed that the Bt gene was not transferred to other crops and had no harmful environmental effects. For a better understanding of the Impact of imported unauthorized GM seeds, more additional detection of GM events should be done by expanding the sampling site and sample types.
Sep 2022 DOI 10.14302/issn.2692-1537.ijcv-22-4328
Objectives To evaluate the diagnostic accuracy of chest CT for the diagnosis of COVID-19 associated with the clinical presentation and in relation to the PCR-RT. Sensitivity, specificity, positive predictive value and negative predictive value, gender, age group and degree of lung involvement will be evaluated. Methods We evaluated 1545 patients with chest CT, delineating the age range and degree of lung involvement, and 306 patients with chest CT and PCR-RT. Results Of the 1545 examinations, 53% were men and 47% were women, there was greater involvement in the 50-59 age group. In the pulmonary study, 55.05% were COVID-19. In the degree of lung involvement 37.70% were mild, 35.76% were moderate, and 26.54% were severe. In the distribution by age, there was a greater involvement between 50-59 years with 56% between moderate (27.6%) and severe (28.0%). Between tomography and PCR-RT, the sensitivity was 68.8%, specificity 59.5%, accuracy 91.3%, with prevalence 31.9%, positive predictive value 44.3% and negative predictive value 80.3%, in females, sensitivity 55.3%, positive predictive value 37.1%, negative predictive value 75.3%, in males, sensitivity 81.6%, positive predictive value 50, 6 and negative predictive value 86.6%.The sensitivities are different between the genders with p of 0.005 and specificity of 0.938, with age effect, starting at 45 years we have a p of 0.057 that decreases to 0.006 at 80 years for sensitivity and specificity. Conclusions The sensitivity and accuracy of CT scan in relation to PCR-RT was significant. Sensitivity increases with prevalence and in the older age group and in men.
Aug 2022 DOI 10.14302/issn.2835-2165.jfsh-22-4263
In Pakistan, production of poultry has been evolved as a good alternate of beef and mutton. In this study multiplex PCR approaches were adopted to differentiate and detect avian mycoplasma in a single PCR reaction as a step forward in the economization of PCR detection. From the total of 25 samples, 25 (100%) had a positive Mycoplasma isolation result. The biochemical test yielded the highest number of isolations, with MG accounting for 10/25. Commercial DNA-based PCR kits have been developed as a result of recent advancements in diagnostic techniques for Mycoplasma infections. Tetracyclines, fluoroquinolones, tilmicosin, tylosin, spiramycin Infections should be reduced as much as possible.
Mar 2022 DOI 10.14302/issn.2572-3030.jcgb-22-4121
Introduction Genomic mutations in TP53 gene in association with etiological risk factors have been associated with oral carcinogenesis. Herein, we screened for genomic variants of TP53 predisposing to oral cancers in Senegalese patients. Methodology 88 patients with confirmed diagnostic were recruited after informed consent. Blood samples were collected from each patient to perform DNA extraction, PCR amplification of all coding exons of TP53 followed by Sanger Sequencing of PCR products. Nucleotide sequences were analysed with Genalys software. 94 blood donors with no cancer diagnosis were also recruited as controls for association study between the most common variants identified in patients and predisposition to oral cancers. Results Sequence analysis showed that 52.27% of patients carry at least one mutation in TP53. Eleven genomic variants were identified, 7 variants already reported in databases and 4 new variants. The most recurrent variants in this study already reported as cancer-related variants were Pro72Arg (rs1042522; Arginine frequency estimated at 31.26%) and a 16 bp insertion in intron 3 (rs59758982; allelic frequency estimated at 26.25%). Haplotype analysis between these variants showed a strong linkage disequilibrium (D’ = 0.999, r2 = 0.153 and p-value < 0.05). However, association study did not find any significant association with susceptibility to oral cancer (p-value > 0.05). Conclusion Our study highlighted that despite the absence of association between the two most common cancer-related variants in Senegalese patients diagnosed with oral cancer, their strong LD suggested that they could be transmitted together in a common haplotype which may be implicated in oral carcinogenesis.
Dec 2021 DOI 10.14302/issn.2692-1537.ijcv-21-4045
Introduction In December 2019, cases of serious illness causing pneumonia and death were first reported in Wuhan, China.2 The clinical features of Corona Virus Disease-19 (COVID-19) are ranging from asymptomatic to multi organ dysfunction. The disease can progress to pneumonia, respiratory failure and death.4 Thus, a tool is needed that can predict the severity and in-hospital mortality risk of a patient with COVID-19 Pneumonia. The PIRO (predisposition, insult, response, and organ dysfunction) scoring was developed for use in the emergency department to risk stratify sepsis cases.15 Eventually it was adapted in pneumonia cases to predict its severity. Objective To validate PIRO score as an assessment tool for COVID-19 mortality risk among patients with confirmed COVID-19 RT-PCR test among patients aged 19 and above admitted in World Citi Medical Center from March 2020 to August 2020 Methods This study included 93 patients aged 19 and above admitted in World Citi Medical Center with a primary diagnosis of COVID-19 Confirmed with pneumonia between March 2020 to August 2020. The patients’ charts were retrieved from the hospital medical records and case notes were reviewed. A severity assessment score was developed based on PIRO score (Predisposition comorbidities and age; Insult multilobar opacities and viremia; Response shock and hypoxemia; Organ Dysfunciton) were extracted. The patients were stratified in four levels of risk: a)Low,0-2 points; b)Mild,3 points; c)High,4 points; d)Very High,5-8 points. The PIRO score and the clinical outcome were compared. The discriminative ability of PIRO score to predict mortality risk was evaluated under receiver operating characteristic curve (AUC). Results The PIRO score had an excellent predictive ability for in-hospital mortality (AUC0.9197). Analysis of variance showed that higher levels of PIRO scores were significantly associated with higher mortality (p<0.001). Patients with Mild PIRO risk category were 98.65% less likely to expire (p<0.001, 95%CI 0.0015) and High PIRO risk category were 94.47% less likely to expire (p<0.001, 95%CI 0.0124), both compared to patients with Very high PIRO risk category. Finally, Very High PIRO risk category were more than 44 times likely to expire compared to patients with Low, Mild and High PIRO risk category (p<0.001, 95%CI 11.738). Conclusions The PIRO score is a valid risk model that can be used to predict in-hospital mortality, that can help clinicians provide timely and accurate assessment, and hence appropriate management to patients with COVID-19 Pneumonia.
Dec 2021 DOI 10.14302/issn.2575-1212.jvhc-21-4034
In bovine tuberculosis (bTB), cellular, humoral, or both types of immune responses have been observed. The purpose of this study was to examine the immune status of tuberculous cows based on the differential cytokine gene expression associated with Th1 (IFN-γ, IL-2), or Th2 (IL-4, IL-10) responses. Twenty-three (23) cows belonging to a dairy herd located in a rural region of the State of Hidalgo, México, were selected for the study. Single Intradermal Comparative Cervical Tuberculin (SICCT) Test, Interferon-Gamma (IFN-γ) Release Assay (BOVIGAM), and Enzyme-Linked Immunosorbent Assay (ELISA) were used for detection of cattle infected by M. bovis. Thirteen cows were positive to all the tests (Group 1); ten cows were positive only to ELISA (Group 2), and the remaining Group (Group 3, control) included cows negative to all the tests. Peripheral blood mononuclear cells (PBMC) from animals were in vitro stimulated by bovin purified protein derivative (PPD), avian PPD, and Concanavalin A (Con A) mitogen for 72h. Changes in the levels of expression of mRNA of the respective cytokines was measured by Reverse Transcription-Polymerase Chain Reaction (RT-PCR) using β-actin gene as internal control. In group 1, PPD bovis and Con A-stimulated cells exhibited high production of IFN-γ, IL-2 and IL-4, but not IL-10. In contrast, PPD avium-stimulated cells displayed a low production of cytokine transcripts. In group 2, cells showed a significant production of IL-10 in response to bovine PPD (P< 0.001). In the control group, a high production of IFN-γ and IL-2 was observed only in Con A-stimulated cells. Post-mortem examinations in animals of group 1 showed slight and medium lesions in lymph nodes, whereas in group 2, the lesions were more extensive. Results indicate differences on gene expression levels of cytokines considered to determine balance in Th1/Th2 response among the evaluated groups. In addition, high levels of antibodies against M. bovis and high IL-10 expression in PBMC together are indicators of progressive bTB when both tuberculin test and IFN-γ assay are negative in tuberculous anergic cattle. Inclusion of serology and IL-10 cytokine expression in in the diagnosis checklist improves detection of infected cattle to help control bovine tuberculosis.
Aug 2021 DOI 10.14302/issn.2692-1537.ijcv-21-3924
This study measures the impact of chloroquine (CQ) therapy in reducing SARS-CoV-2 viral load in infected individuals and hence its transmissibility by describing changes in nasopharyngeal SARS-CoV-2 RNA kinetics in patients receiving standard of care (SOC) or CQ +/- ritonavir-boosted lopinavir (LPV/r). The nasopharyngeal (NP) samples were collected from mild COVID-19 patients admitted at Bamrasnaradura Infectious Diseases Institute between March and April of 2020. These patients either received SOC, or a high dose of CQ with loading dose, or high dose of CQ plus LPV/r. The samples were tested at AFRIMS using a quantitative RT-PCR assay. Levels of CQ in the plasma were measured 6 days post initiation of their treatment. In some instances, viral isolation was attempted to determine SARS-CoV-2 viability. Analyses of the clinical outcomes showed that CQ +/- lopinavir did not contribute significantly to decreasing the number of days with detected SARS-CoV-2 RNA. Viral NP GEs declined faster in the CQ group, but benefits diminished rapidly with delays in treatment initiation. Funding Global Emerging Infections Surveillance, Armed Forces Health Surveillance Branch (GEIS-AFHSB) for all research-related activities at the AFRIMS
Aug 2021 DOI 10.14302/issn.2326-0793.jpgr-21-3917
50 years ago the Enzyme Immunoassay Enzyme-Linked Immunosorbent Assay, mostly known as ELISA was developed. This is a powerful but simple method that is very widely used in the diagnostic practice, as well as in biomedical research. During this time a number of ELISA modification were developed that significantly increased its properties, especially the senstivity, such as avidin-biotin assay, immuno-PCR, nano-ELISA and finally, the digital ELISA. This short review describes the principles of ELISA and the evolution from a conventional assay to the modern ultra-sensitive method. Most of the immunological methods have two components: antigen and antibody. The high specificity of their interaction gives a possibility to detect one of them if other one is included in the reaction as a specific partner. The simplest method for antigen detection in the presence of the antibody is immune diffusion (radial immune diffusion in that case), which practically the formation of precipitate of the “antigen-antibody” complex, when the target antigen diffuses from well into agarose containing the specific antibody. Unfortunately, this assay, as well as other traditional methods, like hemagglutination or complement fixation, have a low sensitivity and are unwieldy.
May 2021 DOI 10.14302/issn.2690-4721.ijcm-21-3835
Objective Real-time surveillance of SARS-CoV-2 variants of concern (VOC) is of essential public health importance. Rapid Antigen Detection Tests (RAgDT) have become first-line COVID-19 diagnostic methods in many regions, but this strategy can hamper the surveillance of the virus variants due to their decentralized performance. The aim of this study was to assess the usefulness of the remaining sample of a widely used RAgDT (Panbio) for the surveillance of the B.1.1.7 VOC using molecular methods. Methods Symptomatic individuals and asymptomatic close contacts of confirmed cases were routinely screened for SARS-CoV-2 infection using the RAgDT in Primary Health Care Centers. After performing the test, the extraction tubes containing the remaining biological material of RAgDT-positive cases were sent to the clinical microbiology laboratory where RT-PCRs detecting key mutations of the VOC were conducted. Results A valid result was obtained in 1770/1812 (97.7%) RAgDT-positive cases. Variant B.1.1.7 was detected in 34.7% of the patients, increasing from 0% to 87.7% between the weeks beginning January 4 and March 15, 2021. Conclusion The sample remaining after performing the Panbio RAgDT allowed to monitor the emergence and circulation of the B.1.1.7, greatly improving the population screened for the molecular study of SARS-CoV-2 variants.
Apr 2021 DOI 10.14302/issn.2372-6601.jhor-21-3801
Lennert lymphoma (lymphoepitheloid lymphoma) is an extremely rare variant of peripheral T-cell lymphoma, not otherwise specified. Here we report a case of Lennert lymphoma diagnosed in a 57-year-old woman. She had a three-year history of waxing and waning lymphadenopathy with a rapid increase in size in the past four months before presentation. A needle biopsy and a fine needle aspiration were non-diagnostic due to extensive necrosis. The patient underwent a right neck lymph node excisional biopsy which showed the lymph node architecture was effaced by numerous and sometimes confluent clusters of epithelioid histiocytes and infiltration of small lymphocytes. Extensive necrosis was present. Immunohistochemical stains revealed a mixed population of B- and T-cells with the T-cells showing diminished T-cell markers CD3, CD5, and CD7. Flow cytometric analysis detected a small population (7% of total lymphocytes) of CD4-positive T-lymphocytes with loss of CD3, CD5, and CD7 expressions. PCR-based T-cell receptor gene rearrangement studies showed positive results (clonal peaks) in both gamma and beta genes. Stains for microorganisms were negative. The overall findings indicate Lennert lymphoma. To our knowledge, this is the first reported case of Lennert lymphoma with extensive necrosis. The patient is undergoing chemotherapy. The diagnosis of Lennert lymphoma can be challenging, particularly in cases with extensive necrosis. Our case highlights that adequate sampling is important in the investigation of patients with suspected Lennert lymphoma. A careful pathologic examination with ancillary studies including flow cytometry, immunohistochmistry, and cytogenetic and molecular studies leads to the accurate diagnosis.
Mar 2021 DOI 10.14302/issn.2575-1212.jvhc-21-3767
Antibodies and antibody fragments, especially single-domain antibodies known as nanobodies, are important tools in diagnostics, research, and therapeutics. In a conventional antibody, light and heavy chains contribute to the formation of the antigen binding site. In addition to conventional antibodies, old and new world camels also have heavy-chain antibodies (hcAbs), which lack the light-chain antibodies that usually bind to the antigen, as well as single domain antibodies, the VHH domain, which are the smallest antigen-binding fragments and have high solubility, stability, and specificity. A VHH library against E. coli lipopolysaccharide (LPS) was produced using the camel immune system. E. coli strains from dead camel calves were isolated to extract the LPS and used to immunize a 2-year-old female camel. After isolating mononuclear lymphocytes for RNA extraction and amplification of the VHH gene, the PCR product was cloned into the pF1AT7 Flexi vector and transformed into JM109 E. coli competent cells by heat shock, resulting in a comprehensive VHHs library with 6.9 × 104 cfu/µg. The VHHs were expressed and screened with ELISA and PCR. Eleven colonies were positive by PCR, six of which were sequenced and submitted to Genbank compared with GenBank data to confirm the production of nanobodies with a similarity >90%.
Sep 2020 DOI 10.14302/issn.2379-7835.ijn-20-3369
Background Since swine flu has been declared pandemic in 2009 it has become a major challenging public-health problem associated with high morbidity and mortality. 25(OH)D deficiency is also pandemic and has been reported to be clinically correlated with decreased immunity and respiratory infections. The possible role of vitamin D in infections is implied from its impact on the innate and adaptive immune responses. This study is planned to evaluate clinical significance of 25(OH)D status on course and outcome in hospitalized cases of swine flu and to compare it with normal healthy subjects living in the same vicinity to evaluate if vitamin D is having any protective effect. Material & Methods Present prospective cross-sectional study was conducted on 79 RT-PCR confirmed cases of swine flu admitted during recent epidemic. All patients were evaluated thoroughly by clinical history physical examination and laboratory investigations as per Performa and followed-up during hospital stay. 25-hydroxyvitamin D (25(OH)D) estimation was done by electro-chemiluminescent Assay in all the cases and it was also done in 36 normal healthy family members of study patients living in the same vicinity (control group). Results High prevalence (70.9%) of low (≤30ng/ml) status of 25(OH)D was observed in cases of swine flu as compared to control group. 25(OH)D status was associated with severity of illness. Mean value of 25(OH)D in mechanically ventilated patients was 9.81±6.43 while it was 22.76±11.35 ng/ml in patients who do not required ventilation (p<0.05). Mean 25(OH)D level in patients who stayed in hospital for <5 days was 28.60±8.79 ng/ml, 24.18±11.67 for 6-10 days and 8.23±2.12 for >10 days (p<0.01). Mean value of 25(OH)D in patients who died was 9.59±5.90 ng/ml as compared to 23.13±11.62 ng/ml who survived (p<0.01). Conclusion Our study suggests that 25(OH)D may have preventive role for swine flu infection. Low level of 25(OH)D is associated with high morbidity in terms of increase requirement for mechanical ventilation, multiorgan dysfunction and long duration of hospital stay. 25(OH)D deficiency is associated with high mortality in swine flu. 25(OH)D status should be given due consideration in high risk patients especially during winter season.
Sep 2020 DOI 10.14302/issn.2575-1212.jvhc-20-3477
A total number of 100 samples from ten random broiler chicken carcasses (breast and thigh) were collected from an automatic poultry slaughtering plant in Ismailia city, Egypt. The mean values of Enterobacteriacae count were 5.9x104±9.7x103 cfu/g and 7.1x 104 ± 1.1x104 cfu/g for chicken breast and thigh samples respectively. The prevalence of E.coli were 12% and 9% breast and thigh samples examined, respectively. They are serologically identified as 33.35 and 22.2% O157:H7 (EHEC) , 16.6% and 11.1% O114:H21(EPEC), 16.6% and 33.3 %O127:H6 (ETEC) , 0% and 0% O126 (ETEC) and 33.3% and 0% O26 (EHEC) for breast and thigh samples, respectively. The incidence of E.coli O157:H7 was 100% in both serological and PCR methods from biochemical positive E.coli samples. Culture is specific and cheap whereas PCR is sensitive and expensive, hence, we recommend both culture and molecular methods, which improve sensitivity and specificity, to enhance detection of foodborne pathogens including E.coli.
Sep 2020 DOI 10.14302/issn.2372-6601.jhor-20-3552
Plasma cell neoplasms of the thyroid gland are uncommon. They may occur either as a primary extraosseous (extramedullary) plasmacytoma or as secondary involvement by multiple myeloma (MM). Here, we report the case of a 62-year-old female, presenting with goiter and Hashimoto’s thyroiditis, in whom the histologic diagnosis of extraosseous plasmacytoma was unexpected. Histology of the total thyroidectomy specimen showed a diffuse infiltration of well-differentiated plasma cells against a background of Hashimoto’s thyroiditis. By immunohistochemistry, the majority of the plasma cells are positive for IgG heavy chain and kappa light chain (kappa:lambda ratio was about 6-7:1). PCR analysis of the immunoglobulin heavy and kappa chain (IGH, IGK) gene rearrangements showed clonal IGH and IGK gene rearrangements. MM was ruled out by lack of MM-related end organ damage and negative serum protein electrophoresis, immunofixation, and bone marrow biopsy. Although rare, plasmacytoma should be considered in patients presenting with enlarging thyroid gland and autoimmune thyroiditis. Histologic diagnosis and differential diagnoses are comprehensively discussed.
Jul 2020 DOI 10.14302/issn.2576-6694.jbbs-20-3450
Background Hyperphenylalaninemia (HPA) combined with neurological signs due to impaired catecholamine, dopamine and serotonin synthesis. Symptoms may appears in first week of life but most seen in age of 4 months. Atypical PKU disease caused mainly by deficiency in 6-pyruvoyltetrahydropterin synthase (PTPS) involved in synthesis of BH4. Clinical symptoms may include poor sucking, impaired tone, ataxia, and seizures. The purpose of this study was to analyze the genotype-phenotype relation among BH4 deficient patients because of PTPS mutations in different state of Egypt. Methods Suspected PKU patients loaded with phenylalanine/Kuvan, and the level of phe and phe/tyrosine ratio determined using tandem mass spectrometry by dried blood spots. Blood samples of 13 unrelated Egyptian patients were collected for total RNA extraction, amplification of PTPS gene by PCR followed with sequencing by Sanger method and finally mutations were recorded for genetic analysis. Results The mean value of phe in 13 patients decreased after loaded of phenylalanine from 482.5μmol/L to 270.63 μmol/L as well as phe/tyrosine ratio was decreased from 13.4 to 6.36 after 24hour of treatment with Kuvan. Sanger sequencing of PTPS gene of those patient showed 21 SNPs and Indels mutations. The most repeated mutation is a novel 23 base pair homozygous deletion in 12/13; c.200C>T in four patients, a novel c.86A>T in two patients and three different mutations located once in three different patients (novel c.22C>T; novel c.273G>A and 405T>C) among patients. On amino acid predicted sequences 4 different types of mutations on protein level were presented, 1 deletion mutation in seven amino acid and 3 different missense mutations in addition to 2 silent mutations among 13 patients. Conclusion Patients were the first case of clinical diagnosis as hyperphenylalaninemia (HPA) undergoing genetic diagnosis for PTPS deficiency in Egypt. The sever HPA patients with severe nervous system damage mainly accompanied with deletion mutations and should pay more attention to the BH4 deficiency. While mild HPA is associated with base substitution mutations with mainly transition mutations (7/9; 78%). Next-generation sequencing technique can increase the mutation detection rate when the hereditary diseases are highly suspected in clinic.
May 2020 DOI 10.14302/issn.2575-7881.jdrr-20-3343
Background Anemia of chronic disease is anemia found in certain chronic disease states, is typically marked by the disturbance of iron homeostasis or hypoferremia. Chronic renal failure is currently known as Chronic Kidney Disease (CKD) or Chronic Renal Insufficiency (CRI) implies long-standing, progressive and irreversible renal parenchyma disease resulting in diminished renal function up to 40 to 60%. Often, chronic kidney disease is diagnosed as a result of screening of people known to be at risk of kidney problems, such as those with high blood pressure or diabetes and those with a blood relative with chronic kidney disease. This disease may also be identified when it leads to one of its recognized complications such as cardiovascular disease, anemia, or pericarditis. Methods Sysmex kx21 used to CBC and the Cobase411 used to iron profile. Enzyme-Linked immunoassay (ELISA) was used to determine the level of serum hepcidin. Sample preparation and PCR detection of HAMP DNA Polymorphisms: Restriction digestion of PCR products was done using Fast Digest. (Figure 1). Results Serum hepcidin levels higher in patients with anemia of chronic kidney disease compared with healthy controls mean. The polymorphisms of the hepcidin gene promoter in Sudanese patients with ACKD showed that the hepcidin HAMP AA genotype 70, AG 23, and GG 7 in 100 patients dialysis-dependent and AA 83, AG 17 and GG 0, and the allele A are more frequent in patients affected by ACKD. Significant statistical association observed between the hepcidin level and end-stage kidney disease. Conclusion This study evaluates for the first time the association between anemia of chronic kidney disease and hepcidin genes promoter polymorphisms and show that the hepcidin HAMP AA genotype and the allele A are more frequent in patients affected by ACKD, further investigation is needed, our data support the hypothesis and hepcidin HAMP are important in the pathophysiology of ACKD.
Jul 2019
Co-infection of HIV with Mycobacterium tuberculosis is a common event, particularly in developing countries. The emergence and spread of multidrug resistant tuberculosis (MDR-TB) is an increasing public problem in India. The drug-resistant M. tuberculosis strains are posing a significant challenge to TB control. This study used PCR to characterize mutations inside the rifampicin resistance-determining region (RRDR) of the rpoB gene in the rifampicin-resistant M. tuberculosis co-infected with HIV. All the rifampicin-resistant strains had missense mutations. Sequence analysis detected a single or multiple hotspot mutations in the RRDR region of the rpoB gene at codons 516, 512 and 531, in most strains. Furthermore, mutations also occur at codons 512, 514, 517 and 526. The results suggest that hotspot mutations in the rpoB gene are not the sole contributors to MDR-TB co-infected with HIV.
Nov 2018
Pre-adipocytes are the precursors with the potential to make new fat cells during adipose tissue expansion. Nevertheless, the pre-adipocytes behaviors, and their possible roles in energy homeostasis have long been overlooked. Our previous study implicates that interleukin-4 (IL-4) plays a positive metabolic role by promoting insulin sensitivity and inhibiting lipid accumulation. Besides, abundant evidence shows the involvement of matrix metalloproteinase-2 (MMP-2) in the process of adipose tissue expansion. The present study aimed at examining the cross talk among glucose, insulin and IL-4 on regulating MMP-2 expression and activity in 3T3-L1 pre-adipocytes. Effects of insulin and/or IL-4 on MMP-2 expression and activity were examined in pre-adipocytes under euglycemic or hyperglycemic environment by RT-PCR and gelatin zymography, respectively. Our results revealed that glucose level is a pre-requisite for pre-adipocytes responding to insulin and/or IL-4 treatment. In high glucose-containing environment, short-term acute insulin treatment (AI) and long-term chronic insulin exposure (CI) showed opposite regulation to MMP-2 expression and activity. Interestingly, the dominant MMPs regulatory role of CI under euglycemic condition was attenuated in cells exposed to high glucose concentration. Our results suggest pre-adipocytes may participate in the process of increasing adiposity, diabetic onset and diabetic complications through ECM alterations resulted from the insulin- and/or glucose- mediated changes of MMP-2 activity. The present study uncovers novel observations regarding pre-adipocytes behaviors.
Aug 2018 DOI 10.14302/issn.2641-9181.ijnr-18-2195
Objectives: An association between the measles virus and Hodgkin lymphoma has been disclosed by our laboratory in Beer-Sheva, starting in 2003. We question the refutation of our study and the absence of interest among experts. Methodology: It was based on immunohistochemistry with commercial, as well as experimental anti-measles antibodies. It relied also on RT-PCR and in situ hybridization evidence of measles virus RNA. Key Results: At this stage (2004), the link between the virus and the lymphoma was essentially descriptive. The first and last response to our challenge appeared in 2007, in the form of doublet articles, in the same issue of a major cancer journal. The two European research groups responding, rejected categorically our findings by proposing different arguments. Major Conclusion: As reservations to these reactions became soon apparent, a series of papers from our laboratory were published. These articles concerned the evidence of a relationship between the measles virus and additional categories of cancers. Different malignancies in which this virus was not expressed at all, were also described. A further study suggested a mechanism by which the measles virus may activate lymphomagenesis in classic Hodgkin lymphoma. To our dismay, and in spite of repeated calls to verify the various results, no further response was obtained from international experts.
May 2018 DOI 10.14302/issn.2637-6075.jpae-18-2062
Estimates of the genetic diversity of Large Japanese field mouse Apodemusspeciosus populations and identification of their plant food resources were conducted in an industrial green space, where were constructed on reclaimed land and belonged to the Aichi Refinery of Idemitsu Kosan Co., Ltd., in Aichi Prefecture, Japan. A total of six mitochondrial D-loop haplotypes were identified in 50 mice. Habitat condition with the highest number of captured individuals had abundant broad-leaved trees and understory vegetation. A minimum spanning network, which did not form a ring-shaped network, revealed that the hereditary population structure was weak. The low genetic diversity observed in the study area was thus attributed to isolation from other populations once the population in the study area by sea and road, which is more than 30 m wide. In order to identify which plant food resources were utilized by mice captured inside the industrial green space, partial chloroplast rbcL sequences were amplified by PCR from DNA extracted from 43 feces samples. Calculations of sample completeness curve revealed that 25 of the taxa identified in this study comprised approximately 90% of the food plant resources in the study area. Of the 21 plant families identified from the obtained rbcL sequences, members of the Rosaceae (28.0%), Fagaceae (17.2%), Lauraceae (14.2%) and Oleaceae (7.7%) were dominant. To ensure the continued survival of A. speciosuspopulation in this industrial green space would be to preferentially conserve plant species that are used as food resources by this species.
May 2018 DOI 10.14302/issn.2690-4829.jen-18-2048
Rooting of cuttings is very important for production of economically important plants. We produced thousands of plantlets in Taxus chinensisvar. mairei using the technology of rooting of cuttings and identified two types of rooted cuttings, one with low rate of root formation and another with high rate of root formation. To determine the physiological role of antioxidative enzymes and microRNAs during the process of rooting, we measured the levels of these antioxidative enzymes and microRNAs in the stem portion, needles, roots, and basal portion of cuttings. Compared to the cuttings with low rate of root formation, cuttings with high rate of root formation had higher expression of polyphenoloxidase (PPO), catalase (CAT), peroxidase (POD), ascorbate peroxidase (APOX), glutathione reductase (GR), and superoxide dismutase (SOD) in the adventitious roots and basal portion of the rooted cuttings 77 days after planting. In the basal portion of cuttings, the content of thiobarbituric acid reactive substances (TBARS) and total phenols were decreased and the content of antioxidants was increased, but they did not changed in the needles of cuttings during planting. Analysis of microRNAs by quantitative realtime PCR demonstrated that expression of miR162, miR408, and miR857 increases in the basal portion of cuttings, but not in the stem portion of cuttings, 77 days after planting. Expression of miR408 and miR857 were also increased in the needles of cuttings 77 days after planting. Changes of these antioxidative enzymes and microRNAs associated with the rooting features of T. chinensisvar. maireicuttings and their functions have been discussed.
Mar 2018 DOI 10.14302/issn.2578-2371.jslr-18-1994
Tuberculosis involving the liver in the absence of active pulmonary tuberculosis is very rare. The inflammatory pseudotumoral form is an entity difficult to diagnose. We report a case of an inflammatory pseudotumor of the liver due to tuberculosis, who didn’t underwent hepatectomy because of the size of the tumor. The diagnosis of tuberculosis was made on biopsy and Polymerase Chain Reaction (PCR).
Nov 2017 DOI 10.14302/issn.2638-4469.japb-17-1838
Strawberry powdery mildew, caused by Podosphaeraaphanis is a major fungal disease that affects strawberry yield and quality. In the model plant species Arabidopsis and the crop plants barley, tomato and pea, the Mildew resistance locus O (MLO) proteins have been found to be required for powdery mildew susceptibility. The present study, based on the sequence of a wild plum (Prunus americana) MLO protein, identified 16 MLO genes within the genome of woodland strawberry, Fragaria vesca and examined their expression pattern in response to powdery mildew infection in three diploid strawberry cultivars. Phylogenetic analysis showed that the FvMLO genes can be classified into six clades. Four FvMLO genes were grouped into clade III, which comprises MLO genes from Arabidopsis, tomato and grapevine that mediate powdery mildew susceptibility. A RNA-seq analysis of two diploid strawberry cultivars, F. vescassp. vesca accession Hawaii 4 (HW) and F. vesca f. semperflorens line “Yellow Wonder 5AF7” (YW) at 1 d (1 DAI) and 8 d (8 DAI) after infection showed the expression of 12 out of the 16 FvMLO genes. The comparison of Fragments Per Kilobase of transcript per Million mapped reads (FPKM values) detected by RNA-seq and expression values of qRT-PCR for FvMLO genes showed substantial agreement. The FvMLO3 gene, which was grouped in clade III and orthologous to the Arabidopsis,tomato and grapevine genes, was highly expressed in YW compared to other FvMLO genes across varieties. The results showed that FvMLO genes can be used as potential candidates to engineer powdery mildew resistance in strawberry based on MLO suppression or genome editing.
Nov 2017 DOI 10.14302/issn.2576-6694.jbbs-17-1744
Aim: Colorectal cancer is one of the most commonly diagnosed cancers in the world. Cell adhesion molecules play an important role in the progression of various cancers. It has been shown that the high level expression of some Cell adhesion molecule could be a new diagnostic factor for several cancers. Vascular cell adhesion molecule 1(VCAM1) is a cell surface glycoprotein that is expressed in the endothelium activated by cytokine. Generally, VCAM-1 expression level is very poor in normal adult tissue endothelial cells. According to the above explanation, this study was conducted to investigate the expression of VCAM-1 in tumoral tissues and adjacent normal tissues in Iranian colorectal cancer patients to its relation with clinicopathological Features in patients with cancer. Methods: In this study, 60 tumoral tissues and 39 adjacent normal tumor tissues were evaluated using reverse transcription-polymerase chain reaction (RT-PCR) technique. Conclusion: A significant correlation was found between VCAM-1 expression level and the stage, lymph nodes involvement, tumor progression factor of cancer and sex. Interestingly, VCAM-1 expression not observed in tumors with stage0. No association was seen between VCAM-1 expression and other clinical features such as age, size of the tumor, metastasis and the number of lymph nodes. These findings suggest that VCAM-1 expression level may reflected disease progression and elevation in VCAM-1 has prognostic significance in patients with colorectal carcinoma.
Aug 2017 DOI 10.14302/issn.2576-2818.jfb-14-553
All independent experimental data on epithelial and glandular cells lines of human endometrium support the evidence for a rapid production of eicosanoids from the LH/hCG receptors when exposed to the hCG hormone. Prostaglandins rapidly act on the surrounding endometrial stromal cells throughout the adenylyl cyclase enzyme leading to very large amounts of cAMP and angiogenic factors (VEGF) production. The cAMP is the most important intracellular second messenger and along with progesterone accomplishes the full process of decidualization and acquisition of receptivity after estrogenic priming of the endometrium. The status of uterine receptivity lasts few days only and timing for successful embryo-signal transduction system activation by the endometrium is probably short. In absence of in vivo embryonic signals it is impossible to predict, on individual bases, how the intensity of all the complex interlinked molecular changes of decidualization might ever be in case of exposure to native hCG. In other terms, amount of prostaglandins and cAMP produced in response to variably glycosylated hCG are all, in vivo, not measurable variables and should be viewed as a “wave” of biochemical chain reactions. Embryonic hCG is secreted in form of multiple isomers having an unpredictable variable level of glycosylation and control of this variable remains elusive. During cycles of ovarian stimulation many drugs (FSH, LH, HCG) interact with different G-protein coupled receptors (GPCRs) making it possible to alter the prostaglandins-mediated decidualization process ready to be elicited only by hCG of pregnancy. Since the molecules (cAMP and progesterone) controlling endometrial stromal cells differentiation into decidual cells are critical for successful implantation and placenta formation, the evidence of fast eicosanoids production associated with endometrial LH/hCG receptors exposure to hCG and the potential by human endometrium to produce, in response, very large amounts of cAMP has biological and clinical relevance.
Oct 2016 DOI 10.14302/issn.2572-3030.jcgb-16-1190
Background: Gastric cancer (GC) ranks as the fourth most common cancer and the second leading cause of cancer deaths worldwide. Epstein-Barr virus is a well-known oncogenic virus, it is responsible for 10% of gastric carcinomas across the world. The aim of study was to determine the prevalence of EBV associated with GC in Sudanese patients. Method: Fifty Paraffin embedded blocks of gastric biopsy specimens diagnosed as gastric carcinoma were collected from Soba university hospital and Ribat teaching hospital, Khartoum, Sudan. DNA was extracted from the paraffin-embedded tissue, and then Epstein-Barr virus gene was detected by polymerase chain reaction (PCR). Result: Among the gastric biopsy specimens 27 (54.0%) were of male and 23(46.0%) were of female. Eleven EBV positive samples were found in gastric carcinomas (22.0%), 8 (72.7%) were of male and 3(27.2%) were of female. The mean age of the patients was 56 years, the most positive cases were between 50-59 years old, and (10%) of them are alive in Khartoum. Conclusions: There exists an association between EBV and gastric carcinoma in some Sudanese patients.
Jun 2016
Earlier studies reported significant association of obesity, hypertension and Type2 Diabetes Mellitus (T2DM). Genetic and many disease-associated alleles have been identified through GWAS and applied to T2DM and indicated roles of renin-angiotensin system (RAS) in insulin signaling pathway and insulin resistance has been well documented. Angiotensin converting enzyme (ACE) gene catalyzes the conversion of angiotensin I to angiotensin II and also inactive the vasodilatation and hence renin-angiotensin system (RAS) in insulin signaling pathway and insulin resistance has been reported. To best of the knowledge we are reporting for the first time regarding association of ACE gene polymorphism with body composition, physiological and metabolic variables among any endogamous ethnic group (Kurmis) from of West Bengal, Eastern India. To achieve the purpose, total 197 (male 99 and female 98) randomly selected apparently healthy unrelated adult individuals of Kurmi population of Purulia District, West Bengal, India were incorporated in the present study. Anthropometric variables, physiological variables (blood pressure) and metabolic variables (PP blood sugar) have been collected using standard techniques. Extracted genomic DNA was PCR amplified and genotyped to understand ACE gene I/D polymorphism. The result demonstrated significant (p<0.05) sexual dimorphism in PBF. MAP and PP blood sugar found to be in normal range among the Kurmis. ACE gene polymorphism showed no deletion of the Kurmis and hence, only the prevalence of ACE II (insertion-Insertion) genotype has been noticed. The present study vindicated on the basis of body composition in terms of fat patterning, physiological and metabolic variables and ACE gene polymorphism that there is very low or no risk of T2DM among the Kurmis of West Bengal, India.
Mar 2016 DOI 10.14302/issn.2470-0436.jos-15-739
Ciliary neurotrophic factor (CNTF) is a well-tested, neuroprotective agent that has been shown to retard photoreceptor degeneration in several animal models of retinitis pigmentosa. The molecular mechanisms underlying CNTF-mediated neuroprotection are currently not understood. CNTF could act directly on photoreceptors or it could act indirectly by stimulating Müller glial cells to produce photoreceptor neuroprotective agents. To better characterize CNTF action on Müller cells, we have studied signaling pathways activated by CNTF using an established retinal Müller cell line, rMC-1. RNA was isolated from CNTF-treated cultures, and suppressor of signal transducer and activator of transcription (SOCS3) and Glial fibrillary acidic protein (GFAP) transcript levels were assessed by quantitative real-time PCR. Immunoblotting was used to examine activation ofmitogen activated protein kinase (ERK1/2/MAPK) and phosphoinositide 3-kinase (PI3-K)/Aktpathways in response to CNTF. Additionally, the level of5' AMP-activated protein kinase (AMPK), an enzyme that plays a key role in cellular energy homeostasis levels, was determined by immunoblotting. CNTF treatment resulted strong upregulation of SOCS3 and GFAP transcripts that were blocked by expression of a dominant-negative STAT3 mutant. CNTF treatment also resulted in transient activation of ERK1/2/MAPK but not PI3K/Akt signaling pathway. There was no change in activation of AMPK. We conclude that CNTF treatment leads to stimulation of JAK-STAT and MAPK signaling pathways but not the PI3K/AKT pathway, associated with cell death, in Müller cells.
Feb 2016 DOI 10.14302/issn.2572-5424.jgm-15-815
Background: Polymorphonuclear leucocytes are the first line of defence against foreign invaders and constitute the major cell type involved in certain types of acute and chronic inflammatory diseases. Aim of the Work: The aim of the present study was to investigate the changes in expression of BCL-2 and BAK genes by real time PCR and to study whether they were involved in the accelerated neutrophil apoptosis which might be responsible for the recurrent bacterial infections seen in chronic renal disease and hemodialysis patients. Subjects and Methods: This study was conducted on sixty two subjects. Patients were selected from those admitted to Theodor Bilharz Research Institute (TBRI). Patients under study were classified into three groups; CKD patients (group I) kept on conservative treatment (22 cases), ESRD patients (group II) maintained on dialysis therapy, HD (20 cases). In addition, twenty healthy individuals served as a control group (group III) were involved. Results: There was significant increase in level of BAK gene in both patients' groups compared to control group with more increase in CKD group than ESRD group. Significant difference between the 3 groups was encountered with a higher expression level in CKD and ESRD groups than controls. There was decrease in level of BCL2 gene in both groups less than control group with more declines in ESRD group than CKD group. Conclusion: Bcl2 and Bak genes could have a role in survival and apoptosis of the studied groups and suggested their impact in controlling the inflammatory mechanisms and eventually their therapeutic potential.
Feb 2016 DOI 10.14302/issn.2575-7881.jdrr-15-849
Reverse Transcription Polymerase Chain Reaction (RT-PCR) using new designed primers pair for Heat Shock Protein70 homologue (HSP70h) of Olive leaf yellowing-associated virus revealed 667 amplified product of 10 olive accessions collected from various olive-growing regions in Tunisia. Amplicons were cloned and sequenced. The sequences were deposited in the international databases. Pairwise sequence comparisons among 10 Tunisian isolates along with a reference sequence (AJ440010) extracted from GenBank revealed a nucleotide identity of 86.06-99.40 and an amino acid similarity of 91.89-99.55. Sequence multiple alignments were searched for evidence of recombination using three methods, ie. Differences of Sums of Squares (DSS) implemented in TOPALi v2.5 software and Single Breakpoint (SBP) along with GARD, a genetic algorithm, both incorporated in HyPhy package. All used methods pointed out the presence of putative breaking points in partially sequenced HSP70h-coding gene. Since failing to account for recombination can mislead the phylogeny inference and can elevate the false positive error rate in positive selection assessment, the use of GARD resulted in the reconstruction of different phylogenies on the left as well as on the right sides of putative recombination breaking points, and the 11 accessions were distributed into at least three clusters compared to MEGA6 software which delineated only two clades. Nonetheless, by dividing the aligned sequences at breakpoints into separate sequence sets, MEGA6 delineated a clustering pattern different from the former two. As a result, recombination reshuffled the affiliation of the different accessions to the clusters. Analysis of selection pressures exerted on HSP70h encoded protein using different models (SLAC, IFEL, FEL, REL, PARRIS, FUBAR, MEME, GA Branch, and PRIME) taking into account recombination, and implemented in HyPhy package, revealed that it underwent predominantly purifying selection as confirmed by Tajima’s D, Fu and Li’s D and F tests, and SNAP algorithm. However, a few sites were also under positive selection as assessed by various models such as FEL, IFEL, REL, MEME, and PRIME.
Jan 2016 DOI 10.14302/issn.2379-8572.joa-15-814
Background and Objective: The etiological factors for nasal polyps include infection, inflammation or an imbalance of a metabolic pathway. This study was designed to compare serum Helicobacter pyloriantibodies and H. pylori–DNAs between cases of nasal polyp and controls (nasal fracture). Patients and Methods: This case control study was carried out in ENT Department of Rasul Hospital in Tehran (2007-2008), upon nasal polyp tissues in 62 cases and inferior nasal turbinate mucosa in 25 controls. H. pylori–DNAs were searched by qualitative polymerase chain reaction (PCR) and serum specific H. pylori antibodies (ELISA IgG and IgA). Comparative tests were performed for the 2 groups, and P value < 0.05 was considered as statistically significant. Results: The mean age of cases and controls were 37.5 ± 13.7 and 31 ± 11.5 years, respectively. H. pylori–DNA was found in 32.3% (20/62) of the cases and 4% (1/25) of the controls (P value = 0.005). Serum H. pylori antibody (IgA) was found in 14.5% (9/62) of the cases and 4% (1/25) of the controls (P value = 0.27). However, previous immunity (IgG) was higher in 71% of the cases and 32% of the controls (P = 0.001). Conclusion: H. pylori infection may play a key role in the formation of nasal polyps. We recommend the PCR as the best method of searching for H. pylori infection. However, from the data obtained in this investigation it could not be determined whether or not H. pylori play a pathogenic role. Long-term antibiotics treatment in cases with nasal polyp, especially in cases with severe chronic rhinosinusitis where patients do not respond to surgery or steroids, may be useful. More randomized controlled trial (RCT) studies are necessary to validate the role of H. pylori infection in nasal polyp and the effect of antibiotics for eradication of H. pylori infection.
May 2015 DOI 10.14302/issn.2471-7061.jcrc-14-574
Colon cancer has a five-year survival of 64.7%, and about 50,000 people are expected to die from colon cancer this year. Patients with metastatic colorectal cancer have a significantly worse prognosis, a 12.9% five-year survival. This emphasizes the need for strategies to inhibit the growth and metastases of colorectal cancer. Prostate apoptosis response protein 4 (Par-4) is a pro-apoptotic protein that has been shown to mediate apoptosis in response to stimuli, such as chemotherapeutics and radiation. Recombinant Par-4 protein has been shown to reduce the occurrence of Lewis lung carcinoma metastases in-vivo; however, the mechanism by which Par-4 can inhibit metastasis has not been elucidated. In this study, human colon cancer cell lines - SW480 and SW620 - were transfected with Par-4 plasmid or anti-Par-4 shRNA, and the effect on metastasis was examined. Par-4 overexpression inhibited cell migration and invasion, while Par-4 knockdown promoted it. Moreover, the morphology of SW620 cells was altered when Par-4 levels were increased. The change was characteristic of a mesenchymal-to-epithelial transition (MET) in these cells. MET can be induced by upregulation of E-cadherin expression, and RT-PCR and Western blot analyses showed that E-cadherin mRNA and protein levels, respectively, were increased in the Par-4 overexpressing cells concomitant with a decrease in vimentin. The results of this study demonstrate the potential of Par-4 in colon cancer therapy, not only in primary tumors but also in metastatic cells.
Feb 2014 DOI 10.14302/issn.2372-6601.jhor-13-358
Acute lymphoblastic leukemia (ALL) is a rapid form of leukemia characterized by clonal proliferation and accumulation of immature hematopoietic stem cells of the lymphoid lineage in the bone marrow as well as peripheral blood. Chromosomal aberrations identified in childhood ALL have an important role in disease diagnosis, prognosis and management. We present the results of hematologic, immunophenotypic, cytogenetic, FISH and Multiplex RT-PCR analysis of a 6-year-old boy diagnosed with B-cell precursor Acute Lymphoblastic Leukemia (BCP- ALL). In this study, we identified a novel chromosomal translocation t(10;15)(q22;q22) by cytogenetic and FISH analysis. To the best of our knowledge, this is the first report of this novel chromosomal translocation in this subset of ALL and has not yet been reported elsewhere. This rearrangement may include certain cancer associated tumor suppressor gene(s) or genes involved in apoptosis and transcription regulation, which on loss of normal function may lead to leukaemogenesis.
Jan 2014 DOI 10.14302/issn.2326-0793.jpgr-13-355
FIP-2 is a multifunctional protein which is involved in various cellular processes. Using different approaches we investigated its regulatory activity. The microarray analysis has shown that FIP-2 substantially altered the expression of 75 genes (35+/40-) from different functional groups with maximal presentation in “Signal transduction” and “Transcription regulation”. Real time RT-PCR indicated significant elevation in the transcription of chemokines, particularly IL-8 (CXCL8). Production of IL-8 in HEK293 cells dramatically increased with FIP-2 overexpression. We also demonstrated that FIP-2 induced activation of IL-8 promoter activity through NF-kB binding site. Additionally, we showed that FIP-2 could interact with PAK3 and increase its kinase activity. Overall, we demonstrated the role of FIP-2 in the regulation of chemokines (IL-8, MPIF-1, MCP-1) and kinases (PAK3, ALK).