Agarose Gel Research Articles

Patterns of KIR-genotypes in women with preimplantation losses after assisted reproductive technologies

Aim. To analyze the frequency and distribution of KIR genotypes in women with repeated preimplantation losses during ARTs compared to women with control group. Methods. DNA isolation from leukocytes by the salting method, PCR-SSP, agarose gel electrophoresis; methods of statistical analysis. Results. The molecular genetic determination of KIR genes in the group of women with recurrent implantation failures and in the control group of women was performed. The frequency and distribution of KIR genotypes in the study groups were determined. In women with RIF, a slightly shifted distribution of the frequency of KIR genotypes was found, towards the homozygous genotype by haplotype "A", the frequency of which was almost half of all identified genotypes (42.86 %). The distribution and frequency of KIR genotypes in the control group was normal and described by the Hardy-Weinberg principle. A comparative analysis of the study groups using statistical methods showed a significantly higher frequency of the KIR-AA genotype (χ2=8.875; p<0.005) in the group of women with RIF compared to the control group of women. The calculation of the odds ratio indicates a 2-fold increase of risk for recurrent implantation failures after ARTs (OR=1.94; CI 95 %: 1.25–3.00) in women with the KIR-AA genotype. Conclusions. We regard the KIR-AA genotype as a significant risk factor for preimplantation loss. KIR genotyping is considered an important molecular genetic test for determining the risk of fetal rejection by the maternal immune system, which will help to correctly direct treatment interventions to keep the pregnancy alive.

CD14 gene polymorphism in COVID-19 convalescents: analysis of (C-159)T

Cluster of differentiation 14 (CD14) is a pattern recognition receptor for lipopolysaccharide of gram-negative flora and has a close relationship with toll-like receptor 4 (TLR4). The interaction between CD14 and TLR 4 leads to the synthesis of cytokines such as interleukin-1, interleukin-6 and tumor necrosis factor-á. The TLR4 receptor is associated with the penetration of the new coronavirus infection virus into the cell, additionally stimulating the expression of the angiotensin converting enzyme 2 molecule and suppressing apoptosis in infected cells. Work on the study of the (159C)T polymorphism of the CD14 gene in COVID-19 is represented by only a few publications describing observations only in the Western European region. The purpose of our study was to study the association between single nucleotide polymorphism (SNP) of the CD14 gene – (159C)T (rs2569190) and susceptibility to a new coronavirus infection in the population of the Republic of Crimea. The study included 158 patients (87 women (55%) and 71 (45%) men, average age 45.6±6.14 years) who had COVID-19. Verification of previous COVID-19 was based on anamnestic data and data from studies performed at the time of illness (PCR). The control group consisted of 85 respondents who had not suffered a new coronavirus infection. To analyze the (C-159)T polymorphism of the CD14 gene, allele-specific PCR with electrophoretic detection in a 3% agarose gel (NPF "Litekh", Russia) was used. To compare the frequencies of allele combinations, the ÷2 test and odds ratio (95% CI) were used. The distribution of CD14 mutations followed Hardy-Weinberg equilibrium (p 0.05). The results of the study showed that the distribution of CD14 gene genotypes did not differ significantly in all study groups. The dominant genotype was the heterozygous genotype CT of the (C-159)T polymorphism of the CD14 gene. The analysis showed that the presence of CT and TT SNPs was not associated with the risk of developing a new coronavirus infection (p 0.05). Conclusions. The CD14 SNP (C-159)T is not associated with a higher risk of COVID-19 infection. The distribution of occurrence of SNP variants in the group of recovered individuals did not differ significantly from that in the control group (p 0.05).

Study of the role of polymorphic variants G919A and A2039G of the follicle stimulating hormone receptor gene FSHR in the genesis of male infertility

Aim. To determine the distribution of genotypes of polymorphic variants G919A and A2039G of the gene FSHR (follicle-stimulating hormone receptor) among men with azoospermia. Methods. DNA from peripheral blood leukocytes was isolated and purification using a modified salting out method. Extracted DNA was amplified by Polymerase chain reaction (PCR). The PCR products were subsequently digested with the restriction enzyme for identify polymorphic variants of the follicle-stimulating hormone receptor gene FSHR. Electrophoresis of PCR products was performed in a 2 % agarose gel. Results. Given that idiopathic infertility is overwhelmingly caused by genetic factors, it seemed necessary to conduct a set of cytological and molecular genetic studies in a group of men with azoospermia. The genetic component was verified in 28 men with azoospermia, which is 40 % of all subjects. Molecular genetic studies were performed and the distribution of genotypes of polymorphic variants A919G and A2039G of the FSHR gene among men with azoospermia was determined. Conclusions. A slightly higher frequency of homozygous genotypes GG of polymorphic variants A919G and A2039G of the follicle stimulating hormone receptor gene FSHR was found among men with azoospermia compared to the group of fertile men.

Analysis Pork Contamination in Beef Meatballs through Polymerase Chain Reaction in Palu City

The need for halal products is essential, particularly among various processed beef products like meatballs, which are popular among nearly the entire community. These products are susceptible to issues of meat counterfeiting for greater economic gain. This study aims to determine the smallest DNA concentration that can still be detected and to assess whether there is any pork contamination in meatballs circulating in Palu City using Polymerase Chain Reaction (PCR). This test uses four DNA samples obtained from fresh meat to test the specificity of the primer: wild boar, rat, and goat DNA samples as comparisons, and pig DNA as a positive control. The presence of DNA bands in the agarose gel electrophoresis visualization results indicates that DNA amplification has occurred, meaning that the sample can be detected. The analysis results showed that the primer used (Cyt-b) had good specificity for detecting pork DNA, while the absolute pork DNA concentration of 0.01 ng/µL and the reference meatball DNA with a concentration of 0.01% are the smallest limits that can still be detected using PCR. Of the four meatball samples from Palu City, one sample produced a DNA band, indicating that the sample contains or is contaminated with pig DNA. This method is valid as it meets the specificity test and demonstrates a good detection limit.

Development of a Bio-Selecting Agent Based on Immobilized Bacterial Cells with Amidase Activity for Bio-Detection of Acrylamide

Actinobacteria cells Rhodococcus erythropolis 4-1 and Rhodococcus erythropolis 11-2 and Proteobacteria Alcaligenes faecalis 2, which have amidase activity, were immobilized by entrapping barium alginate and agarose into the gel structure, as well as by obtaining biofilms on thermally expanded graphite (TEG). The operational stability of such immobilized biocatalysts after storage in frozen and dehydrated form was determined, and a prototype of a conductometric acrylamide biosensor based on such a bioselective agent was developed. The most preferred method for storing immobilized cells was freezing at temperatures from –20 to –80°C; long-term storage is also possible wet at 4–25°C. It was shown that these cells were most preferable for the biodetection of acrylamide A. faecalis 2, immobilized in an agarose gel structure. An agarose gel with bacterial cells immobilized in its structure had greater mechanical strength and stability during successive cycles of conversion of acrylamide into acrylic acid compared to barium alginate gel. The mechanical strength of barium alginate gel can be enhanced by the addition of carbon nanomaterials during cell immobilization. Growing biofilms on carbon materials used for manufacturing electrodes is also promising. Biofilms of R. erythropolis 11-2 on TEG are capable of converting acrylamide into acrylic acid in more than 20 reaction cycles while maintaining at least 50% amidase activity.

An Improved Method and Device for Nucleic Acid Isolation Using a High-Salt Gel Electroelution Trap.

The success of DNA analytical methods, including long-read sequencing, depends on the availability of high-quality, purified DNA. Previously, we developed a method and device for isolating high-molecular-weight (HMW) DNA for long-read sequencing using a high-salt gel electroelution trap. Here, we present an improved version of this method for purifying nucleic acids with high yield and purity from even the most challenging biological samples. The proposed method is a significant improvement over the previously published procedure, offering a simple, fast, and efficient solution for isolating HMW DNA and smaller DNA and RNA molecules. The method utilizes vertical gel electrophoresis in two nested, partially overlapping electrophoretic columns. The upper, smaller-diameter column has a thin layer of agarose gel at the bottom, which separates nucleic acids from impurities, and an electrophoresis buffer on top. After the target nucleic acid has been gel-purified on the upper column, a larger-diameter column with a layer of high-salt gel overlaid with electrophoresis buffer is inserted from below. The purified nucleic acid is then electroeluted into the buffer-filled gap between the separating gel and the high-salt gel, where excess counterions from the high-salt gel slow its migration and cause it to accumulate. The proposed vertical purification system outperforms the previously described horizontal system in terms of ease of use, speed, scalability, and compatibility with high-throughput workflows. Furthermore, the vertical system allows for the sequential purification of several nucleic acid species from the same sample using interchangeable salt-gel columns.

Association of VDR Polymorphisms with Muscle Mass Development in Elite Young Soccer Players: A Pilot Study.

The vitamin D receptor (VDR) is an important candidate gene in musculoskeletal phenotypes. Polymorphisms in the VDR have been previously associated with several pathologies and muscular strength in athletes and elderly people; however, the literature reported contradictory results. The object of this research was to verify the association between the most studied VDR variants (rs2228570, rs7975232, and rs1544410) and the increase in muscle mass in elite young soccer players. A sample of 55 soccer players (15-18 years old) from a professional team were selected for this study. DNA was extracted by the salting-out method, and polymorphisms were genotyped by PCR-RFLP, followed by 2% agarose gel electrophoresis. To test the effect of the three SNPs (single nucleotide polymorphisms), a logistic regression analysis was applied. The body composition determination was carried out through the skinfold thickness method, and the muscular area of the arm and lower limb were calculated using the Frisancho formula. All three polymorphisms met the Hardy-Weinberg equilibrium (p > 0.05) and their frequencies fell within the worldwide variability. A significant correlation between rs1544410 and the increase in calf muscle mass was observed. Individuals carrying the A allele showed higher calf muscular mass than those carrying the G allele (p = 0.034). Moreover, a haplotype analysis applied to the two SNPs in linkage disequilibrium (rs7975232 and rs1544410) showed that the AG haplotype appeared negatively correlated to the calf muscle area. In conclusion, we confirm an association between VDR polymorphisms and muscular mass that could encourage the genetic screening of the VDR gene to identify a potential risk of injury and for individual nutritional interventions.

A 96-Well Polyacrylamide Gel for Electrophoresis and Western Blotting.

Western blotting is a stalwart technique for analyzing specific proteins and/or their post-translational modifications. However, it remains challenging to accommodate more than ~10 samples per experiment without substantial departure from trusted, established protocols involving accessible instrumentation. Here, we describe a 96-sample western blot that conforms to standard 96-well plate dimensional constraints and has little operational deviation from standard western blotting. The main differences are that (i) submerged polyacrylamide gel electrophoresis is operated horizontally (similar to agarose gels) as opposed to vertically, and (ii) a 6 mm thick gel is used, with 2 mm most relevant for membrane transfer (vs ~1 mm typical). Results demonstrate both wet and semi-dry transfer are compatible with this gel thickness. The major tradeoff is reduced molecular weight resolution, due primarily to less available migration distance per sample. We demonstrate proof-of-principle using gels loaded with molecular weight ladder, recombinant protein, and cell lysates. We expect the 96-well western blot will increase reproducibility, efficiency (cost and time ~8-fold), and capacity for biological characterization relative to established western blots.

First Report of Burkholderia glumae Causing Bacterial Panicle Blight in Rice in Bangladesh.

Bacterial panicle blight (BPB) is one of the emerging diseases occurring in different Agro-Ecological Zones (AEZ) of Bangladesh and can cause up to 75% yield loss. In Bangladesh, the typical symptoms of BPB include sheath rot, panicle blight, grain spotting, and grain rot in both inbred and hybrid rice varieties, which resemble those reported by Zhou (2019). To confirm, 300 field samples of 20 panicles each with typical BPB symptoms from 20 districts (3 locations each district and 5 fields per location) were collected during mid-November 2022 for the causal pathogen(s) isolation. Nearly 70% of the panicles showed a dark brown chaffy appearance in the fields. For identification of the causal pathogen(s), 1 g of rice grains with typical BPB symptoms was surface sterilized by immersing for 15 seconds in 70% ethanol, 1 min in 3% sodium hypochlorite solution followed by rinsing the grains three times, and soaked in 1 mL sterile distilled water for 10 min (Mirghasempour et al. 2018). During grinding using mortar and pestle, 5 mL water was added (Islam et al. 2023) after which the suspension (20 μL) was then streaked onto the selective medium (S-PG) (Tsushima et al. 1986). Purple color colonies on the S-PG medium were selected and purified as candidate pathogens. For further confirmation, the genomic DNA of the bacterial isolates was extracted and amplified by PCR using 16SF (5'-AGAGTTTGATCCTGGCTCAG-3') and 16SR (5'-GGCTACCTTGTTACGACTT-3') (Nandakumar et al. 2009), and glu-FW (5'-GAAGTGTCGCCGATGGAG-3') and glu-RV (5'-CCTTCACCGACAGCACGCAT-3') primers (Maeda et al. 2006). The PCR products were visualized on 1% agarose gel resulting amplicons of 1494bp for 16S-rDNA and 529bp for gyrB. The PCR results revealed 529bp amplification for gyrB gene in one sample that was collected from a field in Natore (24°21'0.00" N 89°04'59.88" E) district cultivating Swarna (a local rice variety), primarily indicating the causal pathogen is Burkholderia glumae. The PCR products were sequenced using both primers and sequence data was analyzed by the BLAST nucleotide program. The obtained partial sequences of 16S rDNA and gyrB were deposited in Genbank (OR573691 and PP332812 respectively). The homology of 16S rDNA resulted over 98% with B. glumae (OK559611 and ON870618.1) and 100% with B. glumae (PP332812 and KX213523) for gyrB gene. To confirm B. glumae by pathogenicity test, 10 mL (108 UFC/ml) suspension of the representative strains, 0.5 mL was then injected into the panicles and sheaths of Horidhan (a susceptible local variety) in greenhouse condition and a control was inoculated with distilled water (Nandakumar et al. 2009). Typical BPB like symptoms were observed after 3 weeks post inoculation. The pathogen was again confirmed by reisolating from the infected spots as B. glumae to fulfill Koch's postulates. This report confirms the presence of B. glumae causing BPB of rice in Bangladesh. Future research for the investigation of BPB and the evolutionary origins of its causal bacteria is necessary to reduce the emergence of the disease and its management in Bangladesh.

Near-infrared irradiation study of honey-mediated Au nanoparticles for photothermal therapy

Gold nanoparticles synthesized via honey-mediated chemical reduction have desirable features that make them ideal candidates for nanoparticle-assisted photothermal therapy (N-PTT). However, their photothermal properties have not been previously explored. In this study, after synthesis and characterization (structural and optical) of the HM-AuNPs, we investigated their photothermal conversion efficiency (η) and absorption cross section (σabs) in aqueous solution, cytotoxic effects in in vitro MDA-MB-468 breast cancer cell culture, and temperature profiles in agarose gel under 810 nm NIR irradiation. The results showed that ≈15 nm and primarily spherical HM-AuNPs had η values of up to 40% and an average σabs of 2.15±0.08×10−18 m2. Furthermore, cell viability was reduced to about 52% and the temperature profile in agarose gel had the typical radially increasing topology. Collectively, the findings show that HM-AuNPs can be used in N-PTT.

Preparation of DNA nanoflower-modified capillary silica monoliths for chiral separation.

Innovative chiral capillary silica monoliths (CSMs) were developed based on DNA nanoflowers (DNFs). Baseline separation of enantiomers such as atenolol, tyrosine, histidine, and nefopam was achieved by using DNF-modified CSMs, and the obtained resolution value was higher than 1.78. To further explore the effect of DNFs on enantioseparation, different types of chiral columns including DNA strand containing the complementary sequence of the template (DCT)-modified CSMs, DNF2-modified CSMs, and DNF3-modified CSMs were prepared as well. It was observed that DNF-modified CSMs displayed better chiral separation ability compared with DCT-based columns. The intra-day and inter-day repeatability of model analytes' retention time and resolution kept desirable relative standard deviation values of less than 8.28%. DNF2/DNF3-modified CSMs were able to achieve baseline separation of atenolol, propranolol, 2'-deoxyadenosine, and nefopam enantiomers. Molecular docking simulations were performed to investigate enantioselectivity mechanisms of DNA sequences for enantiomers. To indicate the successful construction of DNFs and DNF-modified CSMs, various charaterization approaches including scanning electron microscopy, agarose gel electrophoresis, dynamic light scattering analysis, electroosmotic flow, and Fourier-transform infrared spectroscopy were utilized. Moreover, the enantioseparation performance of DNF-modified CSMs was characterized in terms of sample volume, applied voltage, and buffer concentration. This work paves the way to applying DNF-based capillary electrochromatography microsystems for chiral separation.

Identification of Insertion and Deletion (InDel) Markers for Chickpea (Cicer arietinum L.) Based on Double-Digest Restriction Site-Associated DNA Sequencing.

Enhancing the marker repository and the development of breeder-friendly markers in chickpeas is important in relation to chickpea genomics-assisted breeding applications. Insertion-deletion (InDel) markers are widely distributed across genomes and easily observed with specifically designed primers, leading to less time, cost, and labor requirements. In light of this, the present study focused on the identification and development of InDel markers through the use of double-digest restriction site-associated DNA sequencing (ddRADSeq) data from 20 chickpea accessions. Bioinformatic analysis identified 20,700 InDel sites, including 15,031 (72.61%) deletions and 5669 (27.39%) insertions, among the chickpea accessions. The InDel markers ranged from 1 to 25 bp in length, while single-nucleotide-length InDel markers were found to represent the majority of the InDel sites and account for 79% of the total InDel markers. However, we focused on InDel markers wherein the length was greater than a single nucleotide to avoid any read or alignment errors. Among all of the InDel markers, 96.1% were less than 10 bp, 3.6% were between 10 and 20 bp, and 0.3% were more than 20 bp in length. We examined the InDel markers that were 10 bp and longer for the development of InDel markers based on a consideration of the genomic distribution and low-cost genotyping with agarose gels. A total of 29 InDel regions were selected, and primers were successfully designed to evaluate their efficiency. Annotation analysis of the InDel markers revealed them to be found with the highest frequency in the intergenic regions (82.76%), followed by the introns (6.90%), coding sequences (6.90%), and exons (3.45%). Genetic diversity analysis demonstrated that the polymorphic information content of the markers varied from 0.09 to 0.37, with an average of 0.20. Taken together, these results showed the efficiency of InDel marker development for chickpea genetic and genomic studies using the ddRADSeq method. The identified markers might prove valuable for chickpea breeders.

Innovative Multiplex PCR Assay for Detection of tlh, trh, and tdh Genes in Vibrio parahaemolyticus with Reference to the U.S. FDA's Bacteriological Analytical Manual (BAM).

Vibrio parahaemolyticus is an important foodborne bacterium that causes severe gastroenteritis following the consumption of contaminated seafood. To identify V. parahaemolyticus and determine its pathogenicity, the U.S. Food and Drug Administration (FDA)'s Bacteriological Analytical Manual (BAM) recommends a multiplex polymerase chain reaction (PCR) protocol to simultaneously detect the species-specific thermolabile hemolysin (tlh) gene and the pathogenic thermostable-related hemolysin (trh) and thermostable-direct hemolysin (tdh) genes. However, this assay has shown two limitations: difficulty in separating the amplicons of the trh (486 bp) and tlh (450 bp) genes due to their highly similar sizes, and the weaker band exhibited by the tdh gene amplicon (270 bp). The present study aimed to improve the BAM's multiplex PCR assay by separating the three amplicons with similar intensity. A new primer set was applied for the tlh gene (369 bp) alongside the existing primers for the trh and tdh genes. The amplicons for the three genes were effectively separated by electrophoresis on a 2% tris-borate-EDTA (TBE) agarose gel within 45 min. Primer concentrations of 0.25 µM for three genes produced a significant amount of amplicons among various combinations of primer concentrations with 35 PCR cycles. This assay exhibited a detection limit of 10 pg of bacterial DNA, demonstrating its high sensitivity. It did not display amplicons from nine Vibrio species known to be human pathogens or from 18 well-documented foodborne pathogens. Therefore, the present multiplex PCR protocol could help overcome the limitations of existing assays and provide a more reliable method for detecting the three genes of V. parahaemolyticus.

Heat conduction simulation of chondrocyte-embedded agarose gels suggests negligible impact of viscoelastic dissipation on temperature change

Agarose is commonly used for 3D cell culture and to mimic the stiffness of the pericellular matrix of articular chondrocytes. Although it is known that both temperature and mechanical stimulation affect the metabolism of chondrocytes, little is known about the thermal properties of agarose hydrogels. Thermal properties of agarose are needed to analyze potential heat production by chondrocytes induced by various experimental stimuli (carbon source, cyclical compression, etc). Utilizing ASTM C177, a custom-built thermal conductivity measuring device was constructed and used to calculate the thermal conductivity of 4.5 % low gelling temperature agarose hydrogels. Additionally, Differential Scanning Calorimetry was used to calculate the specific heat capacity of the agarose hydrogels. Testing of chondrocyte-embedded agarose hydrogels commonly occurs in Phosphate-Buffered Saline (PBS), and thermal analysis requires the free convection coefficient of PBS. This was calculated using a 2D heat conduction simulation within MATLAB in tandem with experimental data collected for known boundary and initial conditions. The specific heat capacity and thermal conductivity of 4.5 % agarose hydrogels was calculated to be 2.85 J/g°C and 0.121 W/mK, respectively. The free convection coefficient of PBS was calculated to be 1000.1 W/m2K. The values of specific heat capacity and thermal conductivity for agarose are similar to the reported values for articular cartilage, which are 3.20 J/g°C and 0.21 W/mK (Moghadam, et al. 2014). These data show that cyclical loading of hydrogel samples with these thermal properties will result in negligible temperature increases. This suggests that in addition to 4.5 % agarose hydrogels mimicking the physiological stiffness of the cartilage PCM, they can also mimic the thermal properties of articular cartilage for in vitro studies.

Synthesis, crystal structure, DNA/protein interactions and cytotoxicity studies of tridentate ligand based Cu(II) complexes with various amine co-ligands

The present study utilizes a tridentate ligand H2L (1) derived as a condensation product of dehydroacetic acid and 2-furoic acid hydrazide for the synthesis of a series of copper complexes, namely [Cu(L)(bipy)] (3), [Cu(L)(4,4′-Me2-bipy)] (4), [Cu(L)(6,6′-Me2-bipy)] (5), [Cu(L)(phen)] (6), [Cu(L)(2,9-Me2-phen)] (7) [Cu(L)(pyrazino[2,3]phen)] (8) [Cu(L)(2,2′-dipyridylamine)] (9) [Cu(L)(diphenylmethanamine)] (10), obtained by reacting Cu(acetate)2 with the doubly deprotonated ligand and a range of co-ligand amines. All the synthesized compounds were fully structurally characterized using IR, 1H NMR, HRMS and single-crystal X-ray diffraction studies. The thermal stability and decomposition pattern was investigated using TGA studies. The bio-efficacy of the developed ligand and its complexes was evaluated in anti-cancer assay against SKOV3 cell lines. Copper complexes have been extensively studied for their potential to generate anticancer properties. Most complexes comprise mixed ligands, such as N-N-chelating heterocycles like 2,2’-bipyridine (bpy) and 1,10 phenanthroline (phen), chosen for their chelating and intercalative characteristics. Complex 7 displayed the highest anti-cancer activity with an IC50 value of 0.8 µM compared to standard drug doxorubicin. The impact of complex 7 on DNA fragmentation was also assessed through agarose gel electrophoresis, which indicated that complex 7 promotes observable DNA fragmentation. The serum binding affinity of the complex 7 was also investigated against BSA and HSA protein. The strong binding affinity of complex [Cu(L)(2,9-Me2-phen)] (7) with BSA/HSA and its better stability compared to the other investigated complexes were deduced based on its biological activity. The impact of varying concentrations of complex 7 on BSA and HSA was investigated using absorption spectroscopy, revealing the presence of a dynamic quenching mechanism. In silico molecular dynamics and docking, approaches have been utilized to validate the empirical data derived from serum binding studies.

Генетический потенциал токсигенных Escherichia coli, выделенных от телят и поросят

Abstract. The study aims to genetically characterize diarrhoeagenic Escherichia coli isolated from cattle and pigs. The main propose is genetic potential responsible for the production of exotoxins in pathogenic E. coli, the causative agents of escherichiosis in calves and piglets. The scientific novelty of the work consists in deciphering the genomes of diarrheogenic E. coli with the presence of nucleotide sequences of several exotoxins, including thermolabile, thermostable and shigap-like, as well as colicins, hemolysins and cyclomodulins, which have pathogenetic significance in the development of escherichia infection in calves and piglets. The study was carried out using microbiological and molecular genetic methods of research and mass spectrometric analysis. As a result, 135 E. coli isolates were subjected to genetic screening by polymerase chain reaction in agarose gel. It was found that 68 (50.36 %) escherichia had toxigenicity markers, while the thermolabile exotoxin gene was recorded more often than others (48.5 %), and the majority was recorded in E. coli isolated from piglets (29.4 %). In 19 (27.9 %) isolates, the presence of genes encoding the production of several exotoxins was established. According to the results of the polymerase-chain reaction, 4 E. coli isolates with a different set of nucleotide sequences responsible for the production of two or more exotoxins at the same time were subjected to genome-wide sequencing. The Escherichia genomes were assembled and annotated and deposited in the NCBI Prokaryotic Genome Annotation Pipeline database under the general number BioProject PRJNA887444. Further studies of E. coli genes and their role in the pathogenic potential of escherichiosis pathogens are needed for the subsequent development of effective means of preventing and controlling infection.

InDel mutations within the bovine PER2 gene are significantly associated with reproductive traits.

Functioning as a key regulator of circadian rhythms, the PER2 gene exerts a substantial impact on the reproductive traits of animals. However, the effect of the PER2 gene on ovarian development remains unclear. In order to examine the relationship between bovine reproductive trait and the PER2 gene, a total of 901 ovarian samples were collected, categorized into different oestrus cycles (proestrus, oestrus, post-oestrus, anoestrous), and subjected to analysis for two potential insertion/deletions (InDels) in the PER2 gene. Through agarose gel electrophoresis and DNA sequencing, two polymorphic deletion mutations (P2-D5-bp, P3-D13-bp) were identified. Furthermore, a significant association between mature follicle diameter and P2-D5-bp was found (P < 0.05). Additionally, several significant correlations with ovarian length, width, height, and white body diameter were found for P3-D13-bp (P < 0.05). These findings suggested that the bovine PER2 gene plays an important role in above-mentioned reproductive traits, offering new avenues for improving cow fertility through marker-assisted selection (MAS).

Characterisation and biological activities of synthesised copper oxide nanoparticles from the Pimpinella anisum L. aqueous extract

AbstractNanoparticles can be synthesised by several methods. Due to the long duration, high cost, and toxic by-products of chemical and physical methods, the biological method has become more preferred. Among various sources such as bacteria, fungi, or yeast, the use of plants in biological synthesis has proven to be the most ideal. Many metals can be used in the biological method, including copper oxide (CuO). In this study, copper oxide nanoparticles (CuONPs) were synthesised using Pimpinella anisum L. aqueous extract. For characterisation of the CuONPs, UV–Visible Spectroscopy (UV–Vis), Scanning Electron Microscopy (SEM), Energy Dispersion Spectroscopy (EDS), and Fourier Transform Infrared Spectroscopy (FT-IR) analyses were performed. The biological activity of the P. anisum extract and CuONPs was determined using DNA cleavage (agarose gel electrophoresis), antioxidant (1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging activity and hydrogen peroxide scavenging activity), mutagenic (Ames/Salmonella test), and catalytic (methylene blue degradation) activities. In DNA cleavage activity test, CuONPs completely denatured DNA at high concentrations (100 and 200 μg mL−1) due to their oxidative activity. The results showed that both the extract and CuONPs have antioxidant properties in DPPH and hydrogen peroxide scavenging activities. According to the mutagenicity, CuONPs did not have a mutagenic effect. In catalytic activity, CuONPs degraded methylene blue within 240 min by 99.45%.

Development of an effective method for purifying trypsin using a recombinant inhibitor

A trypsin affinity material was prepared by covalently immobilizing buckwheat trypsin inhibitor (BTI) on epichlorohydrin-activated cross-linked agarose gel (Selfinose CL 6 B). The optimal conditions for activating Selfinose CL 6 B were 15 % epichlorohydrin and 0.8 M NaOH at 40 °C for 2 h. The optimal pH for immobilizing BTI was 9.5. BTI-Sefinose CL 6 B showed a maximum adsorption capacity of 2.25 mg trypsin/(g support). The material also displayed good reusability, retaining over 90 % of its initial adsorption capacity after 30 cycles. High-purity trypsin was obtained from locust homogenate using BTI-Selfinose CL 6 B through one-step affinity chromatography. The molecular mass and Km value of locust trypsin were determined as 27 kDa and 0.241 mM using N-benzoyl-DL-arginine-nitroanilide as substrate. The optimal temperature and pH of trypsin activity were 55 °C and 9.0, respectively. The enzyme exhibited good stability in the temperature range of 30–50 °C and pH range of 4.0–10.0. BTI-Selfinose CL 6 B demonstrates potential application in the preparation of high-purity trypsin and the discovery of more novel trypsin from various species.

Identification of antigenic proteins from the venom of Malaysian snakes using immunoprecipitation assay and tandem mass spectrometry (LC-MS/MS)

Snake envenomation poses a significant risk to Malaysians and country visitors. Malaysia witnesses an estimated 650 snake bites per 100,000 population annually. The primary treatment for snake envenomation involves administering antivenom derived from horses, despite its drawbacks, such as anaphylactic reactions and serum sickness. Identifying the venom proteome is crucial for understanding and predicting the clinical implications of envenomation and developing effective treatments targeting specific venom proteins. In this study, we employ an immunoprecipitation assay followed by LC-MS/MS to identify antigenic proteins in five common venomous snakes in Malaysia compassing of two families which are pit vipers, (Calloselasma rhodostoma and Cryptelytrops purpureomaculatus) and cobras (Ophiophagus hannah, Naja kaouthia, and Naja sumatrana). The immunoprecipitation assay utilises a 2 % agarose gel, allowing antigenic proteins to diffuse and bind with antibodies in the antivenom. The antivenom utilised in this research was procured from the Queen Saovabha Memorial Institute (QSMI), Thailand, including king cobra antivenom (KCAV), cobra antivenom (CAV), Malayan pit viper antivenom (MPAV), Russell's viper antivenom (RPAV), hematopolyvalent antivenom (HPAV), neuropolyvalent antivenom (NPAV), banded krait antivenom (BKAV), and Malayan krait antivenom (MKAV). The protein identified through these interactions which are exclusive to the cobras are three-finger toxins (3FTXs) while snake C-type lectins (Snaclecs) are unique to the pit vipers. Common protein that are present in both families are L-amino acid oxidase (LAAO), Phospholipase A2 (PLA2), and snake venom metalloproteinase (SVMP). Identifying these proteins is vital for formulating a broad-spectrum antivenom applicable across multiple species.