Genotyping Techniques Research Articles

Evaluation of two phenotypic techniques and a commercial multiplex molecular panel for the detection of different carbapenemases

The prevalence of carbapenemase-producing Enterobacterales has increased in recent years and is considered an important public health problem. A total of 106 clinical samples were analyzed by different carbapenemase detection techniques: inhibition discs (ID), immunochromatographic test (ICT) and a genotypic method, comparing them with a multiplex RT-PCR as a reference method. Overall, all 3 techniques exceeded 90% sensitivity, although with differences in the performance of some of them by carbapenemase type. DI had low specificity (62%) for OXA-48, while with TIC the sensitivity for NDM-type metallo-beta-lactamase (93%) was slightly lower than for OXA-48 (95%). The best results were obtained with the genotypic technique (100% overall performance). Despite the lower sensitivity of TICs (especially in NDM carbapenemases) compared to molecular techniques, with the modification of the protocol we managed to increase this sensitivity and, together with the lower price, simplicity and speed, it makes this technique a good screening option.

Association Analysis of LCORL Genetic Variant (rs657074013) with Wither-Height in Pakistani Goats

The goat stands as one of the most valued and economically efficient domestic animals, contributing significantly to human welfare through the provision of meat, milk, fiber, skin, and manure. Notably, the initial two production traits are contingent upon the physical attributes of the animals, exemplified by characteristics such as large body size and skeletal frame. Earlier (GWA) studies, employing high-density arrays, have revealed a noteworthy association of various variants located at the boundaries of NACPG and LCORL genes across diverse livestock species. Objective: To investigate the genetic variability/association of rs657074013A>AT variant of LCORL gene within diverse goats from Pakistan. Methods: ARMS-PCR genotyping technique was used where, a total of 51 goats belongs to diverse breeds were screened with allele-specific set of primers. Results: Current study showed that 27% sampled population is homozygous wild-type (A/A), 24% is homozygous-mutant (AT/AT) and 49% is heterozygous (A/AT) with a significant genetic association X2 p- value = 9.60 x 10-5 using PLINK. Hardy-Weinberg Equilibrium revealed that overall sampled population obeys the principle with X2 (2, N=51) = 0.046, p = 0.9730. Furthermore, alternative allele frequencies (AAF) of 0.68 and 0.28 were also observed within cases and control cohorts respectively along with an odds-ratio of 5.242 which depicts the AAF is ~5 times higher in cases vs controls. Conclusions: In summary, this pilot-scale study has advanced our genomic understanding by examining the variability and association of this LCORL variant (c.828_829insA) within the Pakistani goat population. The insights gained hold promise for enhancing this economically crucial trait through the implementation of marker-assisted breeding strategies in this particular species and warrant further exploration in other livestock species to broaden our understanding and potential applications

A Comprehensive Study of Phenotypic and Genotypic Techniques to Detect Metallo-β-lactamases in Carbapenem-resistant Escherichia coli (E. coli) and Klebsiella pneumoniae (KP) Strains Derived through Numerous Clinical Specimens in Advanced Health care Facilities

In recent years, an increase in Escherichia coli and Klebsiella pneumoniae resistant to carbapenem was reported globally. Due to their high prevalence and extensive range of medical conditions, Escherichia coli and Klebsiella pneumoniae are both confirmed to be major public health concerns. Furthermore, carbapenem resistance restricts treatment options for individuals infected with these bacteria. Consequently, early detection of carbapenem resistance is essential for starting effective therapy, achieving successful management, and avoiding the infection from spreading further in the future. This study’s objective was to identify the phenotypic and genotypic identification of Metallo-β-lactamases (MBL) in carbapenem-resistant E. coli and K. pneumoniae in advanced healthcare facilities. Meropenem resistance was tested in E. coli and K. pneumoniae using the Kirby-Bauer disc diffusion technique. MBL was discovered using a combination of Disc diffusion testing and the Modified Hodge Test. The Polymerase Chain Reaction was used to determine the genotypes of the bla NDM-1 genes that express these enzymes. Out of 427 strains, including 223 E. coli and 204 K. pneumoniae, 35 (8.2%) consisted of carbapenem-resistant, and 29 (82.85%) showed phenotypically verified as metallo-beta-lactamase producers by using the Combined disc test and 20 (57.14%) using the Modified Hodge test. Polymerase Chain Reaction tests for genes detect those three different strains all showed the bla NDM-1 gene. Carbapenemase production and MBL can be recognized with the help of phenotypic combination disc and MHT tests in labs. Since both tests showed 100% concordance, laboratories may use the less expensive CDT instead of the MHT. The current study supports institutional antibiotic stewardship programmes to manage antibiotic use and prevent CRE worldwide.

Sour Beer as Bioreservoir of Novel Craft Ale Yeast Cultures.

The increasing demand for craft beer is driving the search for novel ale yeast cultures from brewing-related wild environments. The focus of bioprospecting for craft cultures is to identify feral yeasts suitable to imprint unique sensorial attributes onto the final product. Here, we integrated phylogenetic, genotypic, genetic, and metabolomic techniques to demonstrate that sour beer during aging in wooden barrels is a source of suitable craft ale yeast candidates. In contrast to the traditional lambic beer maturation phase, during the aging of sour-matured production-style beer, different biotypes of Saccharomyces cerevisiae dominated the cultivable in-house mycobiota, which were followed by Pichia membranifaciens, Brettanomyces bruxellensis, and Brettanomyces anomalus. In addition, three putative S. cerevisiae × Saccharomyces uvarum hybrids were identified. S. cerevisiae feral strains sporulated, produced viable monosporic progenies, and had the STA1 gene downstream as a full-length promoter. During hopped wort fermentation, four S. cerevisiae strains and the S. cerevisiae × S. uvarum hybrid WY213 exceeded non-Saccharomyces strains in fermentative rate and ethanol production except for P. membranifaciens WY122. This strain consumed maltose after a long lag phase, in contrast to the phenotypic profile described for the species. According to the STA1+ genotype, S. cerevisiae partially consumed dextrin. Among the volatile organic compounds (VOCs) produced by S. cerevisiae and the S. cerevisiae × S. uvarum hybrid, phenylethyl alcohol, which has a fruit-like aroma, was the most prevalent. In conclusion, the strains characterized here have relevant brewing properties and are exploitable as indigenous craft beer starters.

Genome-wide association study as a powerful tool for dissecting competitive traits in legumes.

Legumes are extremely valuable because of their high protein content and several other nutritional components. The major challenge lies in maintaining the quantity and quality of protein and other nutritional compounds in view of climate change conditions. The global need for plant-based proteins has increased the demand for seeds with a high protein content that includes essential amino acids. Genome-wide association studies (GWAS) have evolved as a standard approach in agricultural genetics for examining such intricate characters. Recent development in machine learning methods shows promising applications for dimensionality reduction, which is a major challenge in GWAS. With the advancement in biotechnology, sequencing, and bioinformatics tools, estimation of linkage disequilibrium (LD) based associations between a genome-wide collection of single-nucleotide polymorphisms (SNPs) and desired phenotypic traits has become accessible. The markers from GWAS could be utilized for genomic selection (GS) to predict superior lines by calculating genomic estimated breeding values (GEBVs). For prediction accuracy, an assortment of statistical models could be utilized, such as ridge regression best linear unbiased prediction (rrBLUP), genomic best linear unbiased predictor (gBLUP), Bayesian, and random forest (RF). Both naturally diverse germplasm panels and family-based breeding populations can be used for association mapping based on the nature of the breeding system (inbred or outbred) in the plant species. MAGIC, MCILs, RIAILs, NAM, and ROAM are being used for association mapping in several crops. Several modifications of NAM, such as doubled haploid NAM (DH-NAM), backcross NAM (BC-NAM), and advanced backcross NAM (AB-NAM), have also been used in crops like rice, wheat, maize, barley mustard, etc. for reliable marker-trait associations (MTAs), phenotyping accuracy is equally important as genotyping. Highthroughput genotyping, phenomics, and computational techniques have advanced during the past few years, making it possible to explore such enormous datasets. Each population has unique virtues and flaws at the genomics and phenomics levels, which will be covered in more detail in this review study. The current investigation includes utilizing elite breeding lines as association mapping population, optimizing the choice of GWAS selection, population size, and hurdles in phenotyping, and statistical methods which will analyze competitive traits in legume breeding.

Genetic Risk Score Constructed from Polymorphisms in the VEGFA, TBX5, and SMAD7 Genes Provides Novel Insights into the Molecular Mechanisms of the Tetralogy of Fallot and Ventricular Septal Defect (Case-Control Study from the Pakistani Population)

Congenital heart defects are common and complex birth-defect malformations in developed and developing countries. It is a multifactorial disease that involves the interaction of either gene-gene or gene-environment. This comparative study was the first report on the genotypic-phenotypic correlation in the Pakistani population. The single nucleotide polymorphisms (SNPs) were further tested for association with maternal diabetes mellitus or hypertension. In addition, the cumulative genetic risk score (GRS) for low to moderately-associated SNPs was calculated for each study subject, which can ultimately guide us for better therapeutic options and prevention strategies. According to the predefined selection criteria, 376 subjects were recruited. The multiplex mini-sequencing genotyping technique opted for the cost-effective genotyping of selected loci. The association of variants with the disease was examined using logistic regression analysis. The statistical and graphical analysis was conducted using SPSS, Haploview, SNPStats, and GraphPad Prism. The results for all SNPs analysis suggested a nonsignificant association with overall congenital heart defect risk except rs3809923. However, interestingly on stratified analysis variants, rs3809923 and rs3809922 showed an association only with tetralogy of Fallot. The remaining risk factor analysis for maternal hypertension and diabetes mellitus association with SNPs were nonsignificant. The GRS was the first time constructed for this low to moderately-associated variants. Interestingly, the cumulative GRS was significantly different from the control group revealing the cumulative effect of these polymorphisms panel in patients. In conclusion, the use of GRS in the clinical setting can predict better risk association and patient outcomes.

Role of Oxacillin Susceptibility Testing Strategy in Changing Scenario of mecA Positive Staphylococcus aureus Isolates (OS-MRSA) Detection

Staphylococcus aureus strains that are mecA and PBP2a positive but phenotypically susceptible to oxacillin are becoming more and more abundant, according to research from all around the world. The oxacillin susceptibility of Staphylococcus aureus (OS-MRSA) contributes to consequent treatment-failure due to misidentification by conventional susceptibility tests. Therefore, the objective of the current study was to ascertain the prevalence of OSMRSA in a tertiary care facility located in Mysore, South India. 395 MRSA isolates collected from diverse clinical samples were included in this lab-based prospective investigation. These isolates were tested using an oxacillin 1μg disc phenotypically by standard disc diffusion test, and simultaneously MIC to Oxacillin was determined from Vitek2 systems. Additionally, MRSA specific mecA gene detection was applied to these isolates in order to confirm their MRSA status genotypically. PCR findings demonstrate that 65% of the isolates were MRSA. The vitek2 system detected 4.06% OS-MRSA isolates with an oxacillin MIC of ≤2µg/ml. The disc diffusion method identified a total of 13.75% isolates as oxacillin sensitive and 10% isolates were oxacillin intermediately sensitive. Oxacillin sensitivity was shown for 1.87% of the mecA-positive MRSA isolates using the VITEK2 and disc diffusion techniques. This analysis found isolates with lower oxacillin MICs but relatively reduced OS-MRSA incidence. Using an oxacillin disc for routine laboratory MRSA detection might occasionally produce false negative results, which can result in improper antibiotic administration and treatment failure. In order to distinguish OS-MRSA from MRSA, it is crucial to combine phenotypic and genotypic techniques.

Методы генотипирования крупного рогатого скота для post-GWAS аннотирования SNPs

Abstract. Genome-wide association analysis is one of the key tools for elucidating the genetic etiology of various phenotypes, including diseases and the degree of predisposition to them. Subsequently, for statistically significant genetic markers, it is necessary to conduct validation studies on independent cohorts. These post-GWAS validation studies test genetic markers that are strongly associated with the phenotype, regardless of sample size, allowing to identify false-positive results from the initial association analysis. When choosing a genotyping technique for post-GWAS validation studies, consideration should be given to the sample size and the number of genetic markers planned to be studied, since genotyping techniques differ in throughput und cost. The aim of this paper is to describe modern methods of genotyping depending on their performance and to carry out genotyping of cattle for the SNPs rs137396952 and rs134055603, for which a high degree of association with the development of ketosis was shown in previous GWAS studies. Utilized genotyping methods include TaqMan and High-Resolution Melt Analysis; genotype analysis was performed using the SNPStats web tool. When comparing the results of genotyping using these technologies, the specifity of allelic discrimination carried out using these methods was demonstrated. Testing of the genotyping results had shown that rs134055603 does not obey the Hardy-Weinberg equilibrium in the studied cohort of animals. Scientific novelty. Obtained genotyping results will be used in further association tests with physiologically valuable parameters of dairy cattle, including resistance to diseases.

Exploring the Association Between PRC2 Genes Variants and Lung Cancer Risk in Chinese Han Population.

Genetic susceptibilities play a large role in the pathogenesis of lung cancer (LC). The polycomb repressive complex 2 (PRC2) is a conserved chromatin-associated complex that represses gene expression and is crucial for proper organismal development and gene expression patterns. Despite PRC2 dysregulation has been observed in various human cancers, the relationship between PRC2 genes variants and lung cancer risk remains largely unexplored. To investigate the association between single nucleotide polymorphisms (SNPs) in PRC2 genes and the risk of developing LC, we genotyped blood genomic DNA from 270 LC patients and 452 healthy individuals of Chinese Han ethnicity using the TaqMan™ genotyping technique. We found that rs17171119T>G(adjusted odds ratio (OR) = 0.662, 95% CI: 0.467-0.938, P < 0.05), rs10898459 T>C(adjusted OR = 0.615, 95% CI: 0.4-0.947, P < 0.05), and rs1136258 C>T(adjusted OR = 0.273, 95% CI: 0.186-0.401, P < 0.001) were significantly associated with a reduced risk of LC. Stratified analysis revealed a protective effect of rs17171119 in both male and female patients, specifically those with lung adenocarcinoma (LUAD). Additionally, rs1391221 showed a protective effect in both the LUAD and lung squamous cell carcinoma (LUSC) groups, while rs1136258 exhibited a protective effect in both females and males, as well as in both LUAD and LUSC groups. Furthermore, analysis of The Cancer Genome Atlas (TCGA) dataset revealed expression levels of EED and RBBP4 in both LUAD and LUSC. This study provides evidence that allelic variants in EZH2, EED, and RBBP4 may act as protective factors against LC development and could serve as genetic markers associated with susceptibility to LC.

Comparison of Brucella abortus population structure based on genotyping methods with different levels of resolution

Numerous genotyping techniques based on different principles and with different costs and levels of resolution are currently available for understanding the transmission dynamics of brucellosis worldwide. We aimed to compare the population structure of the genomes of 53 Brazilian Brucella abortus isolates using eight different genotyping methods: multiple-locus variable-number tandem-repeat analysis (MLVA8, MLVA11, MLVA16), multilocus sequence typing (MLST9, MLST21), core genome MLST (cgMLST) and two techniques based on single nucleotide polymorphism (SNP) detection (parSNP and NASP) from whole genomes. The strains were isolated from six different Brazilian states between 1977 and 2008 and had previously been analyzed using MLVA8, MLVA11, and MLVA16. Their whole genomes were sequenced, assembled, and subjected to MLST9 MLST21, cgMLST, and SNP analyses. All the genotypes were compared by hierarchical grouping method based on the average distances between the correlation matrices of each technique. MLST9 and MLST21 had the lowest level of resolution, both revealing only four genotypes. MLVA8, MLVA11, and MLVA16 had progressively increasing levels of resolution as more loci were analyzed, identifying 6, 16, and 44 genotypes, respectively. cgMLST showed the highest level of resolution, identifying 45 genotypes, followed by the SNP-based methods, both of which had 44 genotypes. In the assessed population, MLVA was more discriminatory than MLST and was easier and cheaper to perform. SNP techniques and cgMLST provided the highest levels of resolution and the results from the two methods were in close agreement. In conclusion, the choice of genotyping technique can strongly affect one's ability to make meaningful epidemiological conclusions but is dependent on available resources: while the VNTR based techniques are more indicated to high prevalence scenarios, the WGS methods are the ones with the best discriminative power and therefore recommended for outbreaks investigation.

Graft-Derived Cell-Free DNA Quantification following Liver Transplantation Using Tissue-Specific DNA Methylation and Donor-Specific Genotyping Techniques: An Orthogonal Comparison Study.

Background: Graft-derived cell-free DNA (gdcfDNA) analysis has shown promise as a non-invasive tool for monitoring organ health following solid organ transplantation. A number of gdcfDNA analysis techniques have been described; however, the majority rely on sequencing or prior genotyping to detect donor-recipient mis-matched genetic polymorphisms. Differentially methylated regions of DNA can be used to identify the tissue-of-origin of cell-free DNA (cfDNA) fragments. In this study, we aimed to directly compare the performance of gdcfDNA monitoring using graft-specific DNA methylation analysis and donor-recipient genotyping techniques in a pilot cohort of clinical samples from patients post-liver transplantation. Results: 7 patients were recruited prior to LT, 3 developed early, biopsy-proven TCMR in the first 6 weeks post-LT. gdcfDNA was successfully quantified in all samples using both approaches. There was a high level of technical correlation between results using the two techniques (Spearman testing, rs = 0.87, p < 0.0001). gdcfDNA levels quantified using the genotyping approach were significantly greater across all timepoints in comparison to the tissue-specific DNA methylation-based approach: e.g., day 1 post-LT median 31,350 copies/mL (IQR 6731-64,058) vs. 4133 copies/mL (IQR 1100-8422), respectively. Qualitative trends in gdcfDNA levels for each patient were concordant between the two assays. Acute TCMR was preceded by significant elevations in gdcfDNA as quantified by both techniques. Elevations in gdcfDNA, using both techniques, were suggestive of TCMR in this pilot study with a 6- and 3-day lead-time prior to histological diagnosis in patients 1 and 2. Conclusions: Both the graft-specific methylation and genotyping techniques successfully quantified gdcfDNA in patients post-LT with statistically significant concordance. A direct comparison of these two techniques is not only important from a technical perspective for orthogonal validation, but significantly adds weight to the evidence that gdcfDNA monitoring reflects the underlying biology. Both techniques identified LT recipients who developed acute TCMR, with several days lead-time in comparison to conventional diagnostic workflows. Whilst the two assays performed comparably, gdcfDNA monitoring based on graft-specific DNA methylation patterns in cfDNA offers major practical advantages over the donor-recipient genotyping, and hence enhances the potential to translate this emerging technology into clinical practice.

CESSAT: A chemical additive-enhanced single-step accurate CRISPR/Cas13 testing system for field-deployable ultrasensitive detection and genotyping of SARS-CoV-2 variants of concern.

The continued emergence of SARS-CoV-2 variants of concern (VOCs) has raised great challenges for epidemic prevention and control. A rapid, sensitive, and on-site SARS-CoV-2 genotyping technique is urgently needed for individual diagnosis and routine surveillance. Here, a field-deployable ultrasensitive CRISPR-based diagnostics system, called Chemical additive-Enhanced Single-Step Accurate CRISPR/Cas13 Testing system (CESSAT), for simultaneous screening of SARS-CoV-2 and its five VOCs (Alpha, Beta, Gamma, Delta, and Omicron) within 40min was reported. In this system, a single-step reverse transcription recombinase polymerase amplification-CRISPR/Cas13a assay was incorporated with optimized extraction-free viral lysis and reagent lyophilization, which could eliminate complicated sample processing steps and rigorous reagent storage conditions. Remarkably, 10% glycine as a chemical additive could improve the assay sensitivity by 10 times, making the limit of detection as low as 1 copy/μL (5 copies/reaction). A compact optic fiber-integrated smartphone-based device was developed for sample lysis, assay incubation, fluorescence imaging, and result interpretation. CESSAT could specifically differentiate the synthetic pseudovirus of SARS-CoV-2 and its five VOCs. The genotyping results for 40 clinical samples were in 100% concordance with standard method. We believe this simple but efficient enhancement strategy can be widely incorporated with existing Cas13a-based assays, thus leading a substantial progress in the development and application of rapid, ultrasensitive, and accurate nucleic acid analysis technology.

Genetic Susceptibility to Severe Forms of COVID-19: What we learned in 2022

Host genetics of COVID-19 patients is constantly evolving and may play an important role in the management of hospitalized patients and the identification of new biomarkers. In 2022, numerous studies have been published examining genetic factors that may be associated with severe outcomes of COVID-19 disease, as well as different genetic biomarkers have been suggested for early diagnosis of severe SARS-CoV-2 infection. In this literature review, we provide relevant updated analyses, examining studies published in 2022 in the literature that correlate with what was published in previous years and that focused on host genetics in patients with severe or fatal forms of COVID-19, Studies using different genotyping and sequencing techniques for the identification of genetic polymorphisms with promising results on different genes of the Renin angiotensin system, Interferon system, ABO system, Apolipoprotein E, Dipeptidyl petptidase, Leucine Zipper Transcription Factor-Like Protein 1(LZTFL1) and HLA system in diverse populations.

An Improved Method for Efficient DNA Extraction from Grapevine

Grapevine (Vitis vinifera L.) is one of the oldest and most important perennial crops worldwide which has been the subject of extensive genetic studies including gene mapping, genetic transformation, and DNA fingerprinting. Grapevines are rich in polysaccharides, polyphenolic compounds, and various secondary metabolites, many of which have significant importance in food, agrochemical, and pharmaceutical industries. While metabolites are one of the indicators of quality of grapevines, the presence of them makes grapevine one of the most difficult plants to extract DNA from. These metabolites not only affect DNA extraction procedures but also downstream reactions such as restriction digestion and PCR. Development of new genotyping techniques based on sequencing such as genotyping by sequencing (GBS) requires high-quality DNA for digestion and sequencing. To date, several protocols have been developed for DNA extraction from grapevine. In this study, three different protocols with modifications were compared for DNA extraction performance from grapevine leaves from four different cultivars. Efficiencies of these methods were determined by extracted DNA’s quantity and quality. To confirm the suitability for GBS, extracted DNA was digested with restriction enzymes. Although all protocols were based on the traditional CTAB method, they resulted in different DNA yield and restriction digestion efficiency. The modified protocol including PVP-40 and ß-mercaptoethanol was found to be the most efficient method to obtain high quality and quantity grapevine DNA that is amenable to restriction digestion.

The Keys to Controlling Wheat Rusts: Identification and Deployment of Genetic Resistance.

Rust diseases are among the major constraints for wheat production worldwide due to the emergence and spread of highly destructive races of Puccinia. The most common approach to minimise yield losses due to rust is to use cultivars that are genetically resistant. Modern wheat cultivars, landraces, and wild relatives can contain undiscovered resistance genes, which typically encode kinase or nucleotide-binding site leucine rich repeat domain containing receptor proteins. Recent research has shown these genes can provide either resistance in all growth stages (all-stage resistance; ASR) or specially in later growth stages (adult-plant resistance; APR). ASR genes are pathogen and race-specific, meaning they can function against selected races of the rust fungus due to the necessity to recognise specific avirulence molecules in the pathogen. APR genes are either pathogen specific or multipathogen resistant but often race-nonspecific. Prediction of resistance genes through rust infection screening alone remains complex when more than one resistance gene is present. However, breakthroughs during the past half century such as single nucleotide polymorphism (SNP) based genotyping techniques and resistance gene isolation strategies such as MutRenSeq (Mutagenesis, Resistance gene enrichment and Sequencing), MutChromSeq (Mutagenesis and Chromosome Sequencing), and AgRenSeq (Association genetics combined with RenSeq) enable rapid transfer of resistance from source to modern cultivars. There is a strong need for combining multiple genes for better efficacy and longer-lasting resistance. Hence techniques such gene cassette creation speed up the gene combination process, but their widespread adoption and commercial use is limited due to their transgenic nature.

Would Cutibacterium acnes Be the Villain for the Chronicity of Low Back Pain in Degenerative Disc Disease? Preliminary Results of an Analytical Cohort.

In the last decade, several studies have demonstrated Cutibacterium acnes colonization in intervertebral discs (IVDs) in patients with lumbar disc degeneration (LDD) and low back pain (LBP), but the meaning of these findings remains unclear. Being aware of this knowledge gap, we are currently conducting a prospective analytical cohort study with LBP and LDD patients undergoing lumbar microdiscectomy and posterior fusion. The IVDs samples collected during the surgeries are subjected to a stringent analytical protocol using microbiological, phenotypic, genotypic, and multiomic techniques. Additionally, pain-related scores and quality-of-life indexes are monitored during patient follow-up. Our preliminary results for 265 samples (53 discs from 23 patients) revealed a C. acnes prevalence of 34.8%, among which the phylotypes IB and II were the most commonly isolated. The incidence of neuropathic pain was significantly higher in the colonized patients, especially between the third and sixth postoperative months, which strongly suggests that the pathogen plays an important role in the chronicity of LBP. The future results of our protocol will help us to understand how C. acnes contributes to transforming inflammatory/nociceptive pain into neuropathic pain and, hopefully, will help us to find a biomarker capable of predicting the risk of chronic LBP in this scenario.

Microsatellites as Molecular Markers with Applications in Exploitation and Conservation of Aquatic Animal Populations.

A large number of species and taxa have been studied for genetic polymorphism. Microsatellites have been known as hypervariable neutral molecular markers with the highest resolution power in comparison with any other markers. However, the discovery of a new type of molecular marker-single nucleotide polymorphism (SNP) has put the existing applications of microsatellites to the test. To ensure good resolution power in studies of populations and individuals, a number of microsatellite loci from 14 to 20 was often used, which corresponds to about 200 independent alleles. Recently, these numbers have tended to be increased by the application of genomic sequencing of expressed sequence tags (ESTs) and the choice of the most informative loci for genotyping depends on the aims of research. Examples of successful applications of microsatellite molecular markers in aquaculture, fisheries, and conservation genetics in comparison to SNPs are summarized in this review. Microsatellites can be considered superior markers in such topics as kinship and parentage analysis in cultured and natural populations, the assessment of gynogenesis, androgenesis and ploidization. Microsatellites can be coupled with SNPs for mapping QTL. Microsatellites will continue to be used in research of genetic diversity in cultured stocks, and also in natural populations as an economically advantageous genotyping technique.

Brucella abortus in Kazakhstan, population structure and comparison with worldwide genetic diversity.

Brucella abortus is the main causative agent of brucellosis in cattle, leading to severe economic consequences in agriculture and affecting public health. The zoonotic nature of the infection increases the need to control the spread and dynamics of outbreaks in animals with the incorporation of high resolution genotyping techniques. Based on such methods, B. abortus is currently divided into three clades, A, B, and C. The latter includes subclades C1 and C2. This study presents the results of whole-genome sequencing of 49 B. abortus strains isolated in Kazakhstan between 1947 and 2015 and of 36 B. abortus strains of various geographic origins isolated from 1940 to 2004. In silico Multiple Locus Sequence Typing (MLST) allowed to assign strains from Kazakhstan to subclades C1 and to a much lower extend C2. Whole-genome Single-Nucleotide Polymorphism (wgSNP) analysis of the 46 strains of subclade C1 with strains of worldwide origins showed clustering with strains from neighboring countries, mostly North Caucasia, Western Russia, but also Siberia, China, and Mongolia. One of the three Kazakhstan strains assigned to subclade C2 matched the B. abortus S19 vaccine strain used in cattle, the other two were genetically close to the 104 M vaccine strain. Bayesian phylodynamic analysis dated the introduction of B. abortus subclade C1 into Kazakhstan to the 19th and early 20th centuries. We discuss this observation in view of the history of population migrations from Russia to the Kazakhstan steppes.

Learning Genotyping and Cell Culture Techniques to Perform Experiments to Study Cardiovascular Calcification

The St. Hilaire Lab at the University of Pittsburgh studies important cardiovascular diseases such as Calcific Aortic Valve Disease (CAVD), Arterial Calcification due to Deficiency of CD73 (ACDC) and Medial Arterial Calcification (MAC). The similarity between all these conditions is the calcification of cells, which leads to the main research question of the lab: how and why does a healthy cell transition into an osteogenic cell? To determine why a healthy cell becomes calcified, we perform experiments using both in vivo and in vitro environments. However, to begin in vitro experiments that involve valuable human cells, basic techniques in cell culture such as plating and splitting are needed, and in vivo experiments require knowing the genotype of the animals being used. With these fundamental techniques, biochemical analysis such as qPCR, western blots and staining can be performed in order to help answer research questions that pertain to the lab.

Colonization of intervertebral discs by Cutibacterium acnes in patients with low back pain: Protocol for an analytical study with microbiological, phenotypic, genotypic, and multiomic techniques.

Lumbar disc degeneration (LDD) and low back pain (LBP) are two conditions that are closely related. Several studies have shown Cutibacterium acnes colonization of degenerated discs, but whether and how these finding correlates with LBP is unknown. A prospective study was planned to identify molecules present in lumbar intervertebral discs (LLIVD) colonized by C. acnes in patients with LDD and LBP and correlate them with their clinical, radiological, and demographic profiles. The clinical manifestations, risk factors, and demographic characteristics of participants undergoing surgical microdiscectomy will be tracked. Samples will be isolated and pathogens found in LLIVD will be characterized phenotypically and genotypically. Whole genome sequencing (WGS) of isolated species will be used to phylotype and detect genes associated with virulence, resistance, and oxidative stress. Multiomic analyses of LLIVD colonized and non-colonized will be carried out to explain not only the pathogen's role in LDD, but also its involvement in the pathophysiology of LBP. This study was approved by the Institutional Review Board (CAAE 50077521.0.0000.5258). All patients who agree to participate in the study will sign an informed consent form. Regardless of the study's findings, the results will be published in a peer-reviewed medical journal. Trials registration number NCT05090553; pre-results.