Journal, genetic engineering & biotechnology最新文献

Background: The identification of miRNAs as well as characterization of miRNA-mRNA interactions in SARS-CoV-2 infection is important to understand their role in disease pathogenesis. Therefore the aim of the present study was to measure the expression levels of hsa-mir-18a-5p in the sera of severe COVID-19 Egyptian patients admitted to ICU to investigate its roles in the pathogenesis and severity of COVID-19 disease.

Methods: A total of 180 unvaccinated severe COVID-19 patients were enrolled in our study. Besides the routine laboratory work, the expression level of hsa-mir-18a-5p was done using reverse transcription quantitative real-time PCR (RTqPCR) technique. Also, target genes of hsa-mir-18a-5p were explored by using online bioinformatics databases.

Results: The expression level of hsa-mir-18a-5p decreased in nonsurvival severe COVID-19 patients (0.38 ± 0.26) when compared to the survival ones (0.84 ± 0.23). While as a prognostic tool for the prediction of bad prognosis and mortality among severe COVID-19 patients, our results showed that the serum hsa-mir-18a-5p expression level is a good sensitive and specific marker. By using bioinformatics tools, our results revealed that the decreased hsa-mir-18a-5p expression level may have a crucial role in COVID-19 pathogenesis and severity through decreased immunological responses (interpreted as lymphopenia) or increased inflammation (interpreted as increased serum levels of IL-6, CRP, LDH).

Conclusion: Taken together, the decreased expression level of hsa-mir-18a-5p could be a bad prognostic marker and therapeutic overexpression of hsa-mir-18a-5p could be a novel approach in the treatment of COVID-19 disease.

Background: The deletions of azoospermic factor regions (AZF) are considered risk factor of spermatogenic failure. AZF duplications or complex copy number variants (CNVs) were rarely studied because STS-PCR could not always detect these changes. The application of multiplex ligation-dependent probe amplification (MLPA) as a valuable test for detection of the deletion and or duplication was introduced to investigate the AZF sub-region CNVs. The MLPA technique is still not applied on a large scale, and the publications in this area of research are limited. The aim of this work was to evaluate the efficacy of MLPA assay to detect AZF-linked CNVs in idiopathic spermatogenic failure patients and to evaluate its importance as a prognostic marker in the reproduction outcome.

Results: Forty infertile men (37 with azoospermia and 3 with severe oligozoospermia) and 20 normal fertile men were subjected to thorough clinical, pathological, and laboratory assessment, chromosomal study, MLPA, STS-PCR assays, histopathology study, and testicular sperm retrieval (TESE). Out of the 40 patients, 7 patients have shown CNV in the AZFc region, 6 patients have partial deletion, and one patient has partial duplication. Only one of the normal control has AZFc duplication. STS-PCR was able to detect the deletion in only 4 out of the 7 positive patients and none of the control.

Conclusion: We concluded that MLPA should be applied on a larger scale for the detection of Y chromosome microdeletion as a rapid, efficient, and cheap test.

Background: Asthma is a chronic and complex pulmonary condition that affects the airways. A total of 250,000 asthma-related deaths are recorded annually and several proteins including chymase, spleen tyrosine kinase, and prostaglandin D2 receptor have been implicated in the pathophysiology of asthma. Different anti-inflammatory drugs have been developed for the treatment of asthma, particularly corticosteroids, but the associated adverse reactions cannot be overlooked. It is therefore of interest to identify and develop small molecule inhibitors of the integral proteins associated with asthma that have very little or no side effects. Herein, a molecular modeling approach was employed to screen the bioactive compounds in Chromolaena odorata and identify compounds with high binding affinity to the protein targets.

Results: Five compounds were identified after rigorous and precise molecular screening namely (-)-epicatechin, chlorogenic acid, ombuine, quercetagetin, and quercetin 3-O-rutinoside. These compounds generally showed impressive binding to all the targets understudy. However, chlorogenic acid, quercetagetin, and quercetin 3-O-rutinoside showed better prospects in terms of triple-action inhibition. Further pulmonary and oral pharmacokinetics showed positive results for all the reported compounds. The generated pharmacophore model showed hydrogen bond donor, hydrogen bond acceptor, and aromatic rings as basic structural features required for triple action inhibition.

Conclusion: These findings suggest that these compounds could be explored as triple-action inhibitors of the protein targets. They are, therefore, recommended for further analysis.

Background: Nitrogen is one of the most important mineral nutrients for plants and is absorbed by the root system mainly in the inorganic form (NH⁺₄ and NO^-₃). Plants absorb nitrogen as a food source for growth, biomass production, and development. Nitrogen is mainly absorbed as nitrate, which is the most common source of nitrogen available to higher plants. One of the unique features of nitrate transport is that NO^-₃ is both a substrate for transport and an inducer of NO^-₃ transport systems in genes and at physiological levels.

Methods: In the present study, morphological and physiological traits (chlorophyll a/b, total chlorophyll, and carotenoid, antioxidant enzymes, and protein content), correlation between traits and gene expression, and principle component analysis of traits among five barley cultivars were measured in response to nitrogen deficiency (ND). The starved plants were transferred to a nutrient solution containing 0.2 mM and 2 mM NO^-₃ up to 7 and 14 days after ND application and non-stressed conditions, respectively.

Results: Gene expression analysis revealed that the 10 HvNRT2 genes were induced in the leaf and root tissues at 7 and 14 days after ND treatments in five barley cultivars. Expression of NRT2 genes by relative quantitative qRT-PCR analysis for 10 HvNRT2 genes were determined. Based on the gene expression, HvNRT2.1, HvNRT2.2, and HvNRT2.4 were strongly induced by NO^-3, peaking at 7 and 14 days after ND treatment. In contrast, the HvNRT2.4 showed only moderate induction in both leaves and roots. From our results, the Reyhan cultivar showed a significant increase in root fresh weight (RFW), protein content, and antioxidant enzyme activity in roots at 7 and 14 days after ND treatment as compared to the non-stressed condition. A highly positive correlation was observed between root catalase (CATr) and HvNRT2.2/2.5/2.6 leaves.

Conclusion: The expression of HvNRT2.4 is increased during long-term nitrogen starvation, while the expression of HvNRT2.1 and HvNRT2.2 are transiently increased by ND. Based on physiological and morphological traits and molecular mechanisms, the Reyhan is considered a tolerant cultivar under ND condition.

Background: One of the 11 recognized mucopolysaccharidosis (MPS) diseases is Sanfilippo. It is autosomal recessive in its mode of transmission. There are four subtypes of Sanfilippo (A, B, C, and D). The most worldwide prevalent subtypes of mucopolysaccharidosis type III (MPS III) are A and B followed by C and D subtypes. To estimate the frequency of MPS IIIA among MPS III patients, we diagnose and compare their clinical features with those of MPS IIIB and also compare the prevalence of MPS IIIB versus MPS IIIA among diagnosed cases at the Biochemical Genetic Department at NRC. For every case that was referred, the quantitative determination of urine Glycosaminoglycans (GAGs) was assessed. Two-dimensional electrophoresis (2DE) of GAGs extracted from urine was performed on all cases with high urinary GAG levels. Both N-sulphoglucosamine sulphohydrolase (MPS IIIA) and N-alpha-acetylglucosaminidase (MPS IIIB) enzyme activity were determined fluorometrically.

Results: From November 2019 to May 2022, 535 cases were referred to the National Research Centre's Biochemical Genetics Department. 233 (43%) MPS cases were diagnosed with high urinary GAG levels for their ages. 73 (31.3%) MPS III cases were diagnosed by 2DE out of the 233 MPS cases. Plasma N-alpha-acetylglucosaminidase enzyme assay was insufficient in 36 (49.3%) patients (Sanfilippo type B), while N-sulphoglucosamine sulphohydrolase enzyme activity was deficient in 15 (20.6%) patients. The other 22 (30.1%) patients are either Sanfilippo type C or D.

Conclusion: N-sulphoglucosamine sulphohydrolase enzyme activity was measured for the first time in Egypt. Thirty-one percent of all diagnosed MPS cases during the last 3 years were MPS type III, making Sanfilippo the most common MPS type among the referred cases to our Biochemical Genetics Department. MPS IIIA accounts for 20.6% of MPSIII cases in this study. Still, MPS type IIIB is the commonest type among diagnosed patients.

Background: Obesity is one of the most serious problems over the world. MicroRNAs have developed as main mediators of metabolic processes, playing significant roles in physiological processes. Thus, the present study aimed to evaluate the expressions of (miR-15a, miR-Let7, miR-344, and miR-365) and its relationship with the different classes in obese patients.

Methods: A total of 125 individuals were enrolled in the study and classified into four groups: healthy non-obese controls (n = 50), obese class I (n = 24), obese class II (n = 17), and obese class III (n = 34) concerning body mass index (BMI < 30 kg/m², BMI 30-34.9 kg/m², BMI 35-39.9 kg/m² and BMI ≥ 40 kg/m², respectively). BMI and the biochemical measurements (fasting glucose, total cholesterol, triglycerides, HDL and LDL, urea, creatinine, AST, and ALT) were determined. The expressions of (miR-15a, miR-Let7, miR-344, and miR-365) were detected through quantitative real-time PCR (RT-qPCR).

Results: There was a significant difference between different obese classes and controls (P < 0.05) concerning (BMI, TC, TG, HDL, and LDL). In contrast, fasting glucose, kidney, and liver functions had no significant difference. Our data revealed that the expression of miR-15a and miR-365 were significantly associated with different obese classes. But the circulating miR-Let7 and miR-344 were not significantly related to obesity in different classes.

Conclusion: Our study indicated that miR-15a and miR-365 might consider as biomarkers for the obesity development into different obese classes. Thus, the relationship between regulatory microRNAs and disease has been the object of intense investigation.

Background: Cryptococcus neoformans is a fungal pathogen that can cause serious meningoencephalitis in individuals with compromised immune systems due to HIV/AIDS (human immunodeficiency virus/acquired immunodeficiency syndrome), liver cirrhosis, and transplantation. Mannoproteins (MPs), glycoproteins in the C. neoformans capsule, crucially impact virulence by mediating adhesion to lung cells and modulating immune response via cytokine induction and phagocytosis influence. Therefore, creating a vaccine that can generate targeted antibodies to fight infection and prevent fungal illnesses is essential.

Results: This research aims to create a unique, stable, and safe vaccine through bioinformatics methodologies, aiming at epitopes of T and B cells found in the MP of C. neoformans. Based on toxicity, immunogenicity, and antigenicity, this research predicted novel T cells (GNPVGGNVT, NPVGGNVTT, QTSYARLLS, TSVGNGIAS, WVMPGDYTN, AAATGSSSSGSTGSG, GSTGSGSGSAAAGST, SGSTGSGSGSAAAGS, SSGSTGSGSGSAAAG, and SSSGSTGSGSGSAAA) and B cell (ANGSTSTFQQRYTGTYTNGDGSLGTWTQGETVTPQTAYSTPATSNCKTYTSVGNGIASLALSNAGSNSTAAATNSSSGGASAAATGSSSSGSTGSGSGSAAAGSTAAASSSGDSSSSTSAAMSNGI, HGATGLGNPVGGNVTT, TMGPTNPSEPTLGTAI, GNPVGGNVTTNATGSD, and NSTAAATNSSSGGASA) epitopes for a multiple-epitope vaccine and constructed a vaccine subunit with potential immunogenic properties. The present study used four linkers (AAY, GPGPG, KK, and EAAAK linkers) to connect the epitopes and adjuvant. After constructing the vaccine, it was confronted with receptor docking and simulation analysis. Subsequently, the vaccine was cloned into the vector of Escherichia coli pET-28a ( +) by ligation process for the expression using the SnapGene tool, which confirmed a significant immune response. To assess the constructed vaccine's properties, multiple computational tools were employed. Based on the MP sequence, the tools evaluated the antigenicity, immunogenicity, cytokine-inducing capacity, allergenicity, toxicity, population coverage, and solubility.

Conclusion: Eventually, the results revealed a promising multi-epitope vaccine as a potential candidate for addressing global C. neoformans infection, particularly in immunocompromised patients. Yet, additional in vitro and in vivo investigations are necessary to validate its safety and effectiveness.

Background: The bleach-boosting capability of xylanases is well-known. The use of xylanase pre-treatment before the application of chemical bleach has multiple advantages including (i) lesser use of polluting chemicals of the traditional bleaching process; (ii) less damage to the cellulosic fibers, therefore better recyclability; and (iii) better brightness of chemical bleach. The major impediment in the application is the availability of commercial enzymes that are active at the elevated temperature and pH that exist during the industrial pulping process. In the present paper, xylanase having suitability for application in deinking is reported.

Results: The xylanase used showed high deinking potential. Optimal deinking was obtained at the xylanase dosing of 20U/g of the dried pulp at 60℃ for a treatment time of 1h. It could bring about a 50% reduction in the usage of chemical bleach that was applied after xylanase pre-treatment. The comparison of FTIR spectra showed changes in intensity without significant changes in the functional group signatures implying that there is negligible damage to the fiber strength in the xylanase pre-treatment process as compared to the chemical bleach process.

Conclusion: The xylanase used in this study was effective in deinking paper pulp and can be used for bio-bleaching of recycled paper.

Background: The quick and accurate identification of viruses is essential for plant disease management. Next-generation sequencing (NGS) technology may allow the discovery, detection, and identification of plant pathogens. This study adopted RNA-sequencing (RNA-Seq) technology to explore the viruses in three potato plants (S3, S4, and S6) growing under field conditions.

Results: Potato-known infecting viruses, such as alfalfa mosaic virus (AMV), potato leafroll virus (PLRV), and potato virus Y (PVY), were identified using bioinformatics programs and validated using RT-PCR. The presence of these potato viruses was also confirmed by visual inspection of host symptoms. In addition, the nearly complete genome of PLRV and the complete or partial genome sequence of multipartite virus segments have been identified. Besides the three major potato viruses that BLASTn analysis revealed were present in our samples, BLASTx analysis revealed some reads are derived from other potato viruses, such as potato virus V (PVV), Andean potato latent virus (APLV), and tomato chlorosis virus (ToCV), which are not frequently reported in potato field screenings in Egypt. Other microbial agents, such as bacteria and fungi, were also identified in the examined sample sequences. Some mycovirus sequences derived from ourmia-like viruses and Alternaria alternata chrysovirus were also identified in sample S4, confirming the complexity of the potato microbiome under field conditions.

Conclusion: NGS quickly and accurately identifies potato plant viruses under field conditions. Implementing this technology on a larger scale is recommended to explore potato fields and imported plants, where symptoms may be absent, unspecific, or only triggered under certain conditions.

Background: Microsatellites are important markers for livestock including ducks. The development of microsatellites is expensive and labor-intensive. Meanwhile, the in silico approach for mining for microsatellites became a practicable alternative. Therefore, the current study aimed at comparing whole-genome and chromosome-wise microsatellite mining approaches in Muscovy and Mallard ducks and testing the transferability of markers between them. The GMATA software was used for the in silico study, and validation was performed using 26 primers.

Results: The total number of the detected microsatellites using chromosome-wise was 250,053 and 226,417 loci compared to 260,059 and 238,462 loci using whole genome in Mallards and Muscovies. The frequencies of different motifs had similar patterns using the two approaches. Dinucleotide motifs were predominant (> 50%) in both Mallards and Muscovies. The amplification of the genomes revealed an average number of alleles of 5.08 and 4.96 in Mallards and Muscovies. One locus was monographic in Mallards, and two were monomorphic in Muscovies. The average expected heterozygosity was higher in Muscovy than in Mallards (0.45 vs. 0.43) with no significant difference between the two primer sets, which indicated the usefulness of cross-species amplification of different primers.

Conclusion: The current study developed a whole-genome SSR panel for ducks for the first time, and the results could prove that using chromosome-wise mining did not generate different results compared to the whole-genome approach.