Table of contents: 2023
Evaluation of commercially available chemical reagents and electroporation for insertion of nucleic acids into hard-to-transfect cells
Insertion of nucleic acids into cells unlocks the possibility of modulating gene expression; however, some cells such as primary cells and those that grow in suspension are hard-to-transfect. New therapies for cancer and possible autoimmune diseases, such as those that involve chimeric antigen receptor T cells, rely on the insertion of plasmids into lymphocytes, which fit into the hard-to-transfect category. In such cases virus-based transduction is usually applied, but the carrier vector tends to be incorporated into the cell’s own DNA in a stable manner, with unpredictable consequences. Thus, highly efficient non-viral gene/plasmid delivery is a sought-after technology. We evaluated several commercially available chemical transfection methods as well as electroporation in difficult-to-transfect cells, including a human lymphocyte cell-line (Jurkat) and fresh peripheral blood mononuclear cells (PBMCs), both grown in suspension. The cell-toxicity of the methods was also evaluated. Twenty-four hours after transfection of the plasmid pCMV-GFP, the proportion of GFP positive (GFP+) cells was evaluated by cytometry. The cationic polymer TurboFect yielded ~7.8% of GFP+ Jurkat cells on average, while the other reagents (Lipofectamine 3000, FuGENE HD and X-tremeGENE HP) presented <3% of GFP+ cells. In PBMCs, none of the chemical reagents yielded >3% transfected cells. Electroporation was more efficient, with ~45% of GFP+ in Jurkat and ~15.7% GFP+ in PBMCs. However, it proved to be highly toxic, with ~80% of the cells considered non-viable 24h after the procedure, while TurboFect showed little-to-no toxicity. In conclusion, it was found that despite its high toxicity electroporation was the only method with applicable transfection efficiency in PBMCs, while in Jurkat the reagent TurboFect can be applied with acceptable results. The strategy for insertion of nucleic acids needs to be fine-tuned for each target cell type and experimental condition.
The development of common bean plants with an improved root system can be a strategy for water and nutrient absorption in limiting environmental conditions. The objective of this study was to understand the influence of root phenotyping methods and phenological stages of evaluation on the selection of common bean genotypes for a highly branched root system. In the 2021/22 growing season, this study was initiated with 36 field treatments, consisting of the combination of three genotypes, two parents (Mesoamerican and Andean gene pools) and one progeny, two methods of root phenotyping (Shovelomics and WinRHIZO) and six growth stages (R1-6: four and eight trifoliate leaves, flower bud, full flowering, pod formation and grain filling). The field treatments were randomized in a simple lattice design. Five plants from each experimental unit were evaluated, considering the genotype and phenotyping methods in each developmental stage. The genotype x method x stage interaction was significant. The partitioning of the simple effects of the factors indicated that the root system variables of the parents could be distinguished from those of the progeny at R6. At this stage, the recommended phenotyping method differs according to the genetic origin of the genotypes. Plotting of the standardized canonical scores for the triple interaction showed that the Shovelomics and WinRHIZO phenotyping methods are adequate for the Andean and Mesoamerican genotypes, respectively, in view of their high scores with high discriminative power, allowing treatment discrimination. Specific phenotyping methods were indicated for Mesoamerican versus Andean genotypes in view of the root development trait intrinsic to each gene pool. We conclude that improving root phenotyping for the development of cultivars with a finely branched root system is a useful strategy to maintain common bean yields in environments under stressful conditions.
Mutation analysis of the GJB2 gene of patients with non-syndromic hearing impairment in the Kurdish population in Sulaimani province, Iraq
Approximately 60% of all cases of congenital bilateral sensorineural hearing impairment are due to genetic factors, and about 50% of hearing impairment cases at a later stage are caused by a mutation in a single gene. Because of the high frequency of gap junction beta-2 protein gene (GJB2) mutations, mutation analysis of this gene is widely used in hearing impairment research and diagnosis. This study aimed to determine the prevalence of common GJB2 mutations in patients with profound non-syndromic sensorineural hearing impairment. Sixty-one patients (32 male and 29 female) included in this study had above 90 decibels of bilateral sensorineural hearing impairment. Patient DNA was isolated from buccal cells. The 1st and 2nd exons of the GJB2 gene were amplified with specific primers after gel purification of both regions. Sanger DNA sequencing analysis was used for investigation of changes in these gene regions. The pathological variant was found in nine patients (15%). This variation involved a frameshift mutation in GJB2 (homozygous 35delG) of the 2nd exon; no mutation was detected in the 1st exon. This study is the first report of a genetic investigation of hearing impairment in the Kurdish population in Sulaimani province, northeastern Iraq, near the Iraq-Iran border. The results show that 35delG mutation has a high prevalence in patients with non-syndromic sensorineural hearing impairment.
Genetic diversity and structure of largemouth bass (Micropterus spp.) populations in reservoirs of northeastern Mexico
The largemouth bass (belonging to the genus Micropterus) is one of the most important freshwater fish for sport fishing; the native range of Micropterus salmoides extends into the northeastern Mexican drainages, providing important economic benefits for communities with thriving bass populations. However, the genetic diversity of this species is progressively declining due to various factors, including direct human impacts and alteration of natural ecosystems. In this study, the genetic diversity and structure of largemouth bass from the main reservoirs of northeastern Mexico were assessed. A total of 350 Micropterus spp. dorsal fin samples collected from seven reservoirs were genotyped using a panel of 10 microsatellite markers. The individual samples were genotyped and the different genetic diversity parameters and population structure analysis were evaluated. The microsatellite markers used in this study were highly informative. All the populations exhibited Hardy Weinberg disequilibrium, with some degree of inbreeding within populations. The populations showed moderate genetic differentiation, allowing the establishment of three genetic clusters. Structure analysis indicated that four ancestral populations is most likely. The populations are characterized by low genetic diversity, reduced effective population sizes and a high probability of inbreeding. This study highlights the importance of genetic diversity studies to manage native largemouth bass in the main reservoirs of northeastern Mexico.
Detection of blaZ and mecA genes and antimicrobial susceptibility in Staphylococcus aureus colonizing multipurpose boxes of dentistry students
Cross-contamination between patient and dentist is a real threat that has not been adequately studied. Staphylococcus aureus, through its characteristic genetic plasticity, has managed to develop multiple virulence and antibiotic resistance proteins. The antibiotic susceptibility profile and the presence of the blaZ and mecA genes that encode resistance to penicillin and methicillin, respectively, were analyzed in strains isolated from multipurpose boxes used by dental students at the Catholic University of Cuenca. These boxes are used to transport instruments and material. From the universe of study (249 boxes) 139 samples were obtained from boxes of the students who accepted and signed a consent to participate. Eight strains of S. aureus were identified, of which, through antibiogram analysis, it was found that seven were resistant to penicillin and two strains resistant to cefoxitin (MRSA strains). In molecular analysis, the mecA gene was identified in two strains, while the blaZ gene was found in all of them. It was concluded that the rate of S. aureus found in this study was low due to various factors, possibly including increased vigilance and cleanliness due to the COVID-19 pandemic during the study.
Fimbristylis ovata (Cyperaceae) extract alleviates neuronal degeneration and death through AGEs/RAGE/JNK regulation in human neuroblastoma cell line, SH-SY5Y
Clinical evidence has implicated advanced glycation end products (AGEs) as one of the risk factors for neurological dysfunction and death. However, the neurodegeneration regulation initiated by external AGEs has not yet been fully identified. We investigated the neuroprotective effect of Fimbristylis ovata extract, a medicinal plant utilized in Thai traditional medicine, against neuronal cell death in response to AGEs-induced cellular stress stimuli. SH-SY5Y, a human neuroblastoma cell, was used to investigate the relationship between AGEs induction and neuronal cell damage. The herbal extract derived from F. ovata, a member of the Cyperaceae family, was tested for its properties against AGEs-induced neuronal stress through changes in RAGE, Mn-SOD, p38 MAPK, and JNK protein expression. APP-related genes, including APP, ADAM10, BACE1, PSEN1, and TACE, were also tested. We found that F. ovata extract exerted its antioxidant activity by normalized AGEs-mediated RAGE elevation and increased Mn-SOD levels. Total apoptotic cells were also decreased by F. ovata extract treatment; this perhaps was due to its ability to suppress JNK activation. Additionally, reduction of expression of APP-related genes including APP, PSEN1, and TACE (or ADAM17) was observed in the F. ovata extract-treated groups. These results provide evidence that F. ovata extract has potential as a neuroprotective agent through its antioxidant and anti-apoptotic properties.
Effects of the drugs gliclazide and metformin on solute carrier family 2 member 2 (SLC2A2) gene expression in type 2 diabetic patients in Vietnam
In recent years, the number of people with type 2 diabetes mellitus has increased rapidly and it has become an important public health problem in Vietnam. To provide valuable information on the efficacy and safety of diabetes medications (metformin and gliclazide), we evaluated how they affected the expression level and mutations of the SLC2A2 gene in the liver of T2DM patients. The SLC2A2 gene was analyzed in 165 patients with T2DM and 54 control subjects. Anthropometry, clinical parameters, molecular analysis, and gene expression were examined. We discovered high levels of mRNA SLC2A2 expression in the livers of people with T2DM who did not receive drug treatment. These levels were 1.5 times higher when compared to other groups that received drug treatment. Mutations of the SLC2A2 gene sequence were recorded in two T2DM patients belonging to the therapy group (c.1127T>G (p.Met376Arg) in exon 9 and c.609T>C (p.Ser203Ser) in exon 5). Finally, we found a strong and significant relationship between the glycemic control index and two enzymes, alanine aminotransferase and aspartate aminotransferase, with mRNA SLC2A2 expression under therapy. Our findings identified mutations of the SLC2A2 gene and a positive correlation between SLC2A2 gene expression and the effects of gliclazide and metformin in the liver of T2DM patients. This might contribute to a better understanding of the SLC2A2 gene, and the safety and tolerability of metformin and gliclazide therapy.
Comparison of genomic DNA extraction methods for Colossoma macropomum fish fin clippings for single nucleotide polymorphism genotyping
Ideal DNA extraction techniques must be efficient in terms of time, labor, and costs, optimizing yield and quality of the DNA for the desired applications. We tested six DNA extraction methods: DNeasy® Blood & Tissue Kit, Cetyltrimethylammonium bromide (CTAB), Modified salting-out protocol (SA), Boiling Tissue, Proteinase K (PK) and Mini Kit Applied Biosystems, on fin clippings from tambaqui (Colossoma macropomum). DNA yield, purity, quality, and cost of each method were evaluated. The effectiveness of extraction was evaluated by PCR amplification and genotyping efficiency, repeatability and accuracy. DNA yield and purity were quantified using NanoDrop absorbance ratios. Cost was estimated in terms of time and material expenses. The results showed differences between the tested methods, with the PK method having the best performance, followed by SA and CTAB. PK was identified as the most economical and efficient technique in terms of time, cost, and scalability/potential automation, while also generating DNA of good quality for performing PCR amplification and SNP genotyping with tambaqui samples.
Detection via PCR is a fast, sensitive, and highly specific method. However, the cost for testing through this technique is quite high, mainly because of the costs of the kits that are used. We looked for the best cost-effective alternative for Dengue virus (DENV) detection via PCR through the evaluation, optimization, and comparison of RT-PCR (Reverse Transcription - PCR) and RT-qPCR (Reverse Transcription–qPCR) detection kits. The biological material was samples of blood serum collected from 40 Brazilian patients suspected of DENV infection. Two reaction final volumes were tested for diagnosis via RT-PCR, 12.5 µL and 25 µL, and diagnosis via RT-qPCR was performed using the two-step approach with the Sybr Green detection system. An analysis of the associated cost for each approach was also made. Analysis via RT-PCR allowed viral RNA amplification from 27 samples, independent of the final reaction volume tested. Diagnosis via RT-qPCR enabled virus identification from 33 samples. The costs per reaction for the RT-PCR technique were US$ 2.91 and US$ 2.41 American dollars for the final reaction volumes of 25 µL and 12.5 µL, respectively. For the RT-qPCR technique, the reaction cost was found to be US$ 2.30. The comparison between the techniques showed that RT-qPCR was more sensitive, allowing virus detection in a larger number of samples. However, results indicated that RT-PCR (12.5 µL) can be used as a screening method, considering its lower reaction cost. The cost analysis showed that RT-qPCR had the best cost-benefit ratio, since it allowed virus detection from a larger number of samples with a cost similar to RT-PCR. We also found that optimization of the cDNA (complementary DNA) synthesis step can significantly affect the final diagnosis cost for both techniques.
Genetic polymorphisms of the folate pathway in amyotrophic lateral sclerosis and multiple sclerosis: a systematic review and meta-analysis
The folate cycle is a biochemical pathway that plays an important role in the development and maintenance of the nervous system. Biocompounds synthesized in this cycle must be carefully regulated, since the accumulation of some substances can be neurotoxic and increase susceptibility to neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) and multiple sclerosis (MS). The methylenetetrahydrofolate reductase (MTHFR), 5-methyltetrahydrofolate-homocysteine S-methyltransferase (MTR), and solute carrier family 19 member 1 (SLC19A1) genes encode important proteins for this regulation. In this systematic review and meta-analysis, we investigated the association of some polymorphisms in the MTHFR, MTR, and SLC19A1 genes and their associations with ALS and MS. The protocol of this systematic review is registered in the PROSPERO platform (CRD42021232352). We performed a search in EMBASE, Pubmed/NCBI, Scopus, Virtual Health Library (BVS), and Web of Science databases for studies that described polymorphisms in these genes, regardless of statistical association. Thirteen studies were included, and four polymorphisms were identified: C677T (rs1801133) and A1298C (rs1801131) in the MTHFR gene, A2756G (rs1805087) in the MTR gene, and A80G in the SLC19A1 gene. In the meta-analysis, the allelic and genotypic comparison for the C677T polymorphism showed a 1.5-fold increased risk for MS. Despite this significant result, we found a lack of association of most polymorphisms in the MTR, SLC19A1 and MTHFR genes and susceptibility for developing ALS and MS. Further studies are needed to clarify the role of polymorphisms in folate pathway genes in the susceptibility for developing these neurodegenerative diseases.
Lack of association of VEGF-A polymorphisms and susceptibility to amyotrophic lateral sclerosis and multiple sclerosis: A systematic review and meta-analysis
Amyotrophic lateral sclerosis (ALS) and multiple sclerosis (MS) are degenerative scleroses with unclear etiology. Vascular endothelial growth factor A (VEGF-A) is a growth factor that plays multiple roles in the central nervous system. Previous studies indicated a potential association between polymorphisms in this gene and the susceptibility of ALS and MS; however, the results have been inconclusive. Here, we conducted a systematic review and meta-analysis to elucidate the relationship between polymorphisms in the VEGF-A gene and these degenerative scleroses. We searched for observational studies in PubMed, Web of Science, EMBASE, Virtual Health Library (BVS) and SCOPUS, without temporal and language restrictions and 12 studies were included in the systematic review. Six polymorphisms were identified: C-1558T, A-1190G, G-1154A (rs1570360), C-2578A (rs699947), C-634G (rs2010963), and C936T (rs3025039). After a systematic literature search, a pooled odds ratio (OR) and 95% confidence interval (CI) were used to evaluate the association of C-2578A, G-1154A, and G-634C polymorphisms and ALS. Due to the small number of articles found in this review for MS, it was not possible to perform a meta-analysis for this disease. The meta-analysis for ALS included 1441 patients and 1978 controls for C-2578A, and 1134 patients and 1629 controls for G-1154A and G-634C polymorphisms. No SNP was significantly associated with risk for ALS. We conclude that polymorphisms in the VEGF-A gene may not be a major risk factor for the development of ALS and MS; However, associated with specific factors, such as sex or haplotype combinations, they may become a strong susceptibility factor. Although we found a lack of association in most VEGF-A polymorphisms and the susceptibility for developing ALS and MS, this review provides a comprehensive understanding for the potential role of this gene in degenerative sclerosis.
Aptitude of Brazilian oat cultivars for reduced fungicide use while maintaining satisfactory productivity
The aggressiveness of fungal diseases in oats compromises grain yield. Although fungicides are effective for control, there is a need for productivity with food and environmental safety. Thus, we seek cultivars responsive to more sustainable management. The objective of this study was to identify oat cultivars that are more responsive to reduced fungicide use and a longer application-to-harvest interval with satisfactory yields, and to identify relevant variables in the simulation of grain yield by multiple linear regression. The study was carried out in 2019 and 2020 in Augusto Pestana, RS, a prominent region for oat cultivation in Brazil. The experimental design was randomized blocks, with three replications in a 22 x 4 factorial scheme, for 22 oat cultivars, recommended and no longer suitable for cultivation and four conditions of sequential use of fungicide (no application; one application 60 days after emergence; two applications, 60 and 75 days after emergence; and three applications, 60, 75 and 90 days after emergence). The fungicide used was FOLICUR® CE, at a dosage of 0.75 liters ha-1. The variables analyzed were necrotic leaf area and grain yield. The Stepwise technique was used to identify potential variables for the multiple linear regression model. The cultivars FAEM 4 Carlasul, URS Altiva, URS Charrua and URS Guará show superiority in grain yield in the absence of fungicide. In a single application, 60 days after emergence, FAEM 4 Carlasul and URS Charrua showed productivity above 3000 kg ha-1 with a long interval (around 60 days) from application to harvest. The variables minimum average temperature, necrotic leaf area and number of fungicide applications were found to be suitable for the composition of a multiple linear regression model to simulate grain yield.
Standardization of ELISA with Senecavirus A recombinant VP2 protein and its use in swine herds in Brazil
Senecavirus A (SVA) is a nonenveloped, single-stranded RNA virusThe icosahedral viral particle is composed of four structural proteins: VP1, VP2, VP3 and VP4, among which VP2 is strongly involved in the antibody immune response. The virus causes vesicles on the snout and feet in pigs, which are clinically indistinguishable from other vesicular diseases such as foot-and-mouth disease. Outbreaks of SVA have been reported worldwide since 2014; however, its prevalence in Brazil remains unknown. In this study, the VP2 structural protein was produced and purified from E. coli, and recombinant VP2 (rVP2), based on the most recent Brazilian strain, was used to develop an indirect ELISA to identify antibodies against SVA in Brazilian swine herds. Sensitivity and specificity values of the rVP2 ELISA were determined using receiver operating characteristic analysis performed on 43 SVA positive and 219 negative serum samples. In addition, serum samples from pigs immunized with eight distinct Brazilian SVA inactivated strains were tested with the rVP2 ELISA. For the specificity of the assay, 17 serum samples from vesicular stomatitis virus (VSV) from naturally infected pigs were tested. The rVP2 ELISA was found to have 100% specificity and 74.4% sensitivity. The performance of the assay using samples collected during the SVA outbreak, had a sensitivity of 100%, and with those collected nine months after the outbreak it had a sensitivity of 73.4%. The rVP2 ELISA developed here was able to detect specific SVA antibodies in acute disease and recovered pigs, and no cross-reactivity with VSV was observed. This assay has potential as a useful tool for monitoring SVA infection and could help to improve disease diagnosis.
Lack of significant association between MTHFR gene C677T polymorphism and colorectal cancer in the Azerbaijani population
We evaluated methylenetetrahydrofolate reductase gene C677T polymorphisms in patients with colorectal cancer in a population-based case-control study in the Azerbaijan population. Genomic DNA was isolated from blood samples taken from 155 patients with colorectal cancer and 155 healthy individuals. The MTHFR gene C677T polymorphism was detected on agarose gel by PCR-RFLP. The frequencies of the CC, CT, and TT genotypes of MTHFR (C677T) were 54, 37, and 9% in the patients with colorectal cancer and 65, 29, and 6% in the healthy control, respectively. Heterozygote CT (OR = 1.422, 95%CI = 0.883–2.289, P = 0.147) and homozygous mutant TT (OR = 1.440, 95% CI = 0.619–3.348, P = 0.395) genotypes were more frequent in colorectal cancer patients compared to controls. No significant associations were observed between genotype and allele frequency of the MTHFR gene and the risk of colorectal cancer. Our findings suggested that MTHFR C677T polymorphism might not be associated with the overall risk of colorectal cancer in an Azerbaijani population. However, these conclusions need to be validated in a larger cohort study.
Anti-inflammatory and protective effects of royal jelly against hepatic and renal damage induced by valproic acid in rats
Valproic acid (VPA) is a drug that is often used to treat epilepsy, seizures, and similar diseases. However, it is known to have serious toxic effects on the liver and the kidney. Oxidative stress and other metabolites of VPA have been suggested to be responsible for VPA induced hepatotoxicity and nephrotoxicity. We evaluated the possible protective role of royal jelly (RJ) against the effects of VPA through toxicity tests on livers and kidneys of rats. Twenty-four male albino rats were separated into three groups; group (1): healthy control received no drug, group (2): administrated VPA (500 mg/kg/day by oral gavage), group (3): received VPA (500 mg/kg/day by oral gavage) one hour prior to RJ (500 mg/kg/day by oral gavage). After two weeks, the rats' livers and kidneys were removed for histopathologic investigation with hematoxylin and eosin staining while biochemical assessment was performed on blood samples. The VPA group had a significant increase in serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), urea, creatinine and pro-inflammatory cytokines (IL-1a, IL-1b and IL-6). Histopathological observations in liver and kidney tissues also were related with the biochemical parameters. VPA causes hepato-renal damage by promoting inflammation, oxidative stress, and fibrosis. RJ enhanced the functions of the liver and kidneys by reducing ALT, AST, urea and creatinine compared with the VPA treatment group and reduced serum pro-inflammatory cytokines. In addition, the histopathological impairment of liver and kidney tissues were reversed by RJ treatment. In conclusion, RJ can protect hepato-renal functions against VPA acid-induced organ damage.