Genotyping Results Research Articles

CO-INFECTION WITH COVID-19 AND GENETIC DIVERSITY OF SARS-COV-2 IN CHILDREN

In the pediatric population, coronavirus infection caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is more commonly observed in asymptomatic and moderately symptomatic cases. However, when COVID-19 coexists with other infections, it can result in a more severe disease progression. Various subtypes of the SARS-CoV-2 virus are associated with different clinical presentations, with certain subtypes exhibiting a predominance of specific symptoms or manifestations. Clinical manifestations of COVID-19 in children, both during the acute phase of the disease and in the post-COVID-19 period, are not fully understood and different compared to adults. This article presents four clinical cases in which PCR-confirmed COVID-19 was observed in combination with infections caused by viruses such as Ebstein-Barr, varicella, and rotavirus. The severity of the disease in these cases was attributed to symptoms related to intoxication, as well as skin and intestinal syndromes. To understand important genomic signatures of SARS-CoV-2 virus circulating in the area, the genome-wide sequencing was performed for the SARS-CoV-2 samples isolated from nasopharyngeal swabs collected from four patients. Based on the genotyping results, all four samples were found belonging to the SARS-CoV-2 virus Omicron lineage. Three months after the initial testing, follow-up evaluations of the children indicated a higher likelihood of complete recovery. However, some underlying disease manifestations, including anemia and atopic dermatitis, persisted despite the resolution of COVID-19 symptoms.

Bacillus changyiensis sp. nov., isolated from coastal sediment.

Two Gram-stain-positive, facultatively anaerobic, motile, endospore-forming, rod-shaped bacteria, designated CLL-3-40T and CLL-7-23, were isolated from coastal sediment sampled in Changyi, Shandong Province, PR China. Phylogenetic analysis based on 16S rRNA gene sequences indicated that these strains were related to the genus Bacillus and close to six type strains of species within the Bacillus licheniformis group. In phenotypic characterization tests, strain CLL-3-40T could grow at 15-50 °C (optimum, 37 °C) and in media with pH 5-9 (optimum pH 7.0), and tolerate up to 12 % (w/v) NaCl. The fermentation broth supernatant extracted by ethyl acetate of strain CLL-3-40T could inhibit aquaculture pathogenic vibrios. The predominant cellular fatty acids of strain CLL-3-40T were anteiso-C15 : 0 (30.7 %) and iso-C15 : 0 (31.5 %); the peptidoglycan from cell-wall contained meso-diaminopimelic acid; the predominant quinone was menaquinone 7; and the major polar lipids were diphosphatidylglycerol, phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine, an unidentified glycolipid and two unidentified phospholipids. The digital DNA-DNA hybridization values and average nucleotide identities among strains CLL-3-40T and CLL-7-23 and their close type strains were less than 21.9 and 48.4 %, respectively, thereby indicating that strain CLL-3-40T should represent a novel species of the genus Bacillus. The genomic DNA G+C contents were 38.4 mol% in strain CLL-3-40T and 38.3 mol% in strain CLL-7-23. The 12 biosynthetic gene clusters of strain CLL-3-40T were predicted based on results from the online server antiSMASH. Based upon the consensus of phenotypic and genotypic results, strain CLL-3-40T should be classified as representing a novel species of the genus Bacillus, for which the name Bacillus changyiensis sp. nov. is proposed. The type strain is CLL-3-40T (= MCCC 1A14857T=JCM 35755T).

Genome-wide association studies reveal the genetic basis of amino acid content variation in tea plants

Tea is one of the most popular non-alcoholic beverages in the world, and free amino acids, especially theanine, make a major contribution to the umami taste of tea. However, the genetic basis of the variation in amino acid content in tea plants remains largely unknown. Here, we measured the free amino acid content in fresh leaves of 174 tea accessions over two years using a targeted metabolomics approach and obtained genotype data via RNA sequencing. Genome-wide association studies were conducted to investigate loci affecting the content of free amino acids. A total of 69 quantitative trait loci (–log10(P-value)>5) were identified. Functional annotation revealed that branched-chain amino acid aminotransferase, glutamine synthetase, nitrate transporter, and glutamate decarboxylase might be important for amino acid metabolism. Two significant loci, glutamine synthetase (Glu1, P=3.71×10−4; Arg1, P=4.61×10−5) and branched-chain amino acid aminotransferase (Val1, P=4.67×10−5; I_Leu1, P=3.56×10−6), were identified, respectively. Based on the genotyping result, two alleles of CsGS (CsGS-L and CsGS-H) and CsBCAT (CsBCAT-L and CsBCAT-H) were selected to perform function verification. Overexpression of CsGS-L and CsGS-H enhanced the contents of glutamate and arginine in transgenic plants, and overexpression of CsBCAT-L and CsBCAT-H promoted the accumulation of valine, isoleucine and leucine. Enzyme activity assay uncovered that SNP1054 is important for CsGS catalyzing glutamate into glutamine. Furthermore, CsGS-L and CsGS-H differentially regulated the accumulation of glutamine, and CsBCAT-L and CsBCAT-H differentially regulated the accumulation of branched-chain amino acids. In summary, the findings in our study would provide new insights into the genetic basis of amino acids contents variation in tea plants and facilitate the identification of elite genes to enhance amino acids content.

Concordance study on Y-STRs typing between SeqStudio™ genetic analyzer for HID and MiSeq™ FGx forensic genomics system

BackgroundMassively Parallel Sequencing (MPS) allowed an increased number of information to be retrieved from short tandem repeat (STR) analysis, expanding them not only to the size, as already performed in Capillary Electrophoresis (CE), but also to the sequence. MPS requires constant development and validation of the analytical parameters to ensure that the genotyping results of STRs correspond to those obtained by CE. Given the increased frequency of usage of Y-STRs as supplementary markers to the autosomal STRs analysis, it is urgent to validate the concordance of the typing results between CE and MPS analyses.Methods and resultsDNA extracted from 125 saliva samples of unrelated males was genotyped using Yfiler™ Plus PCR Amplification Kit and ForenSeq™ DNA Signature Prep Kit, which were analyzed by SeqStudio™ Genetic Analyzer for HID and MiSeq™ FGx Forensic Genomics System, respectively. For each shared Y-STR, allele designation, number of length- and sequence-based alleles per locus, stutter percentage, and the intra-locus balance of multicopy Y-STRs were screened.ConclusionsAlthough the number of forensic genetics laboratories that are applying the MPS technique in routine analysis is small and does not allow a global assessment of MPS limitations, this comparative study highlights the ability of MPS to produce reliable profiles despite the generation of large amounts of raw data.

The Accuracy of Diagnosis and Genotyping of Leishmania Species Based on Spliced Leader Mini-Exon Gene by Nuclear Magnetic Resonance and Sequencing Assays.

We aimed to evaluate the accuracy of genotyping of Leishmania species by the spliced leader mini-exon gene. Suspected leishmaniasis patients, referred to Masieh Daneshvary Hospital, Tehran, Iran were included from May 2017 to September 2021. The Leishmania species were genotyped by PCRRFLP based on the SL mini-exon gene and the ITS1 region of SSU-rRNA gene and compared with the sequencing results. The expressed metabolites of metacyclic promastigotes were evaluated by Proton nuclear magnetic resonance (1H-NMR). Out of 66 suspected cases, 36 (54.4%) were positive for Leishmania species based on the PCR assays. In 21 (31.8%) cases, promastigotes grew on culture tubes. Based on the RFLP of SL RNA profile, 13 (19.7%) L. tropica, 9 (13.6%) L. major, 3 (4.5%) L. infantum, and 8 (12.1%) C. fasciculata isolates, isolated from culture media, were identified; however, 3 (4.5%) cases were unidentifiable due to the low number of parasites. Seventeen metabolites were expressed by the metacyclic forms of L. major, L. tropica and C. fasciculata isolates. The top differential metabolites expressed more in C. fasciculata were FAD, p-Methoxybenzyl alcohol and S-b-G-5, 5-G-b-S (A = CH2) (P<0.005) whereas Veratryl glycerols and D-(+)-Mannose were significantly increased in L. major and Betulin, LTyrosine in L. tropica (P<0.01). The invaluable techniques such as sequencing and 1H-NMR confirmed the results of genotyping of Leishmania species based on the SL mini-exon gene. SL mini exon gene can be used as a diagnostic tool to differentiate various Leishmania genotypes and detect contamination of culture media with C. fasciculata.

Impact of pretreatment low-abundance HIV-1 drug resistance on virological failure after 1 year of antiretroviral therapy in China.

To assess the impact of pretreatment low-abundance HIV drug-resistant variants (LA-DRVs) on virological outcomes among ART-naive HIV-1-infected Chinese people who initiated ART. A nested case-control study was conducted among HIV-1-infected individuals who had pretreatment drug resistance (PDR) genotypic results. Cases were defined as individuals with virological failure (HIV-1 RNA viral load ≥1000 copies/mL) after 1 year of ART, and controls were individuals from the same cohort whose viral load was less than 1000 copies/mL. Next-generation sequencing was used to identify low-abundance PDR mutations at detection thresholds of 10%, 2% and 1%. The mutant load was calculated by multiplying the abundance of HIV-1 drug-resistant variants by the pretreatment viral load. The impact of pretreatment low-abundance mutations on virological failure was estimated in logistic regression models. Participants (43 cases and 100 controls) were included in this study for the analysis. The proportion of participants with PDR was higher in cases than in controls at different detection thresholds (44.2% versus 22.0%, P = 0.007 at 10% threshold; 58.1% versus 31.0%, P = 0.002 at 2% threshold; 90.7% versus 69.0%, P = 0.006 at 1% threshold). Compared with participants without PDR, participants with ≥10% detectable PDR mutations were associated with an increased risk of virological failure (adjusted OR 8.0, 95% CI 2.4-26.3, P = 0.001). Besides this, individuals with pretreatment LA-DRVs (2%-9% abundance range) had 5-fold higher odds of virological failure (adjusted OR 5.0, 95% CI 1.3-19.6, P = 0.021). Furthermore, LA-DRVs at 2%-9% abundance resistant to NRTIs and mutants with abundance of ≥10% resistant to NNRTIs had a 4-fold and 8-fold risk of experiencing virological failure, respectively. It was also found that a mutant load of more than 1000 copies/mL was predictive of virological failure (adjusted OR 7.2, 95% CI 2.5-21.1, P = 0.0003). Low-abundance PDR mutations ranging from 2% to 9% of abundance can increase the risk of virological failure. Further studies are warranted to define a clinically relevant threshold of LA-DRVs and the role of NRTI LA-DRVs.

Genotypic and phenotypic comparison of drug resistance profiles of clinical multidrug-resistant Mycobacterium tuberculosis isolates using whole genome sequencing in Latvia

BackgroundMultidrug-resistant tuberculosis (MDR–TB) remains a major public health problem in many high tuberculosis (TB) burden countries. Phenotypic drug susceptibility testing (DST) take several weeks or months to result, but line probe assays and Xpert/Rif Ultra assay detect a limited number of resistance conferring gene mutations. Whole genome sequencing (WGS) is an advanced molecular testing method which theoretically can predict the resistance of M. tuberculosis (Mtb) isolates to all anti-TB agents through a single analysis.MethodsHere, we aimed to identify the level of concordance between the phenotypic and WGS-based genotypic drug susceptibility (DS) patterns of MDR–TB isolates. Overall, data for 12 anti-TB medications were analyzed.ResultsIn total, 63 MDR–TB Mtb isolates were included in the analysis, representing 27.4% of the total number of MDR–TB cases in Latvia in 2012–2014. Among them, five different sublineages were detected, and 2.2.1 (Beijing group) and 4.3.3 (Latin American-Mediterranean group) were the most abundant. There were 100% agreement between phenotypic and genotypic DS pattern for isoniazid, rifampicin, and linezolid. High concordance rate (> 90%) between phenotypic and genotypic DST results was detected for ofloxacin (93.7%), pyrazinamide (93.7%) and streptomycin (95.4%). Phenotypic and genotypic DS patterns were poorly correlated for ethionamide (agreement 56.4%), ethambutol (85.7%), amikacin (82.5%), capreomycin (81.0%), kanamycin (85.4%), and moxifloxacin (77.8%). For capreomycin, resistance conferring mutations were not identified in several phenotypically resistant isolates, and, in contrary, for ethionamide, ethambutol, amikacin, kanamycin, and moxifloxacin the resistance-related mutations were identified in several phenotypically sensitive isolates.ConclusionsWGS is a valuable tool for rapid genotypic DST for all anti-TB agents. For isoniazid and rifampicin phenotypic DST potentially can be replaced by genotypic DST based on 100% agreement between the tests. However, discrepant results for other anti-TB agents limit their prescription based solely on WGS data. For clinical decision, at the current level of knowledge, there is a need for combination of genotypic DST with modern, validated phenotypic DST methodologies for those medications which did not showed 100% agreement between the methods.

Candidate genes related to acute cerebral circulatory disorders in Preeclampsia in the Kazakh Population

BackgroundThe purpose of this study is to conduct a comparative analysis of the population frequencies of alleles and genotypes of polymorphic variants of coagulation and fibrinolysis genes SERPINE1 rs1799889, ITGA2 rs1126643, THBD rs1042580, FII rs1799963, FV rs6025, FVII rs6046, angiogenesis and endothelial dysfunction PGF rs12411, FLT1 rs4769612, KDR rs2071559, ACE rs4340, GWAS associated with the development of acute cerebral circulatory disorders in preeclampsia, in an ethnically homogeneous population of Kazakhs with previously studied populations of the world. MethodsThe genomic database was analysed based on the results of genotyping of 1800 conditionally healthy individuals of Kazakh nationality ∼2.5 million SNPs using OmniChip 2.5 M Illumina chips at the DECODE Iceland Genomic Center as part of the joint implementation of the project “Genetic Studies of Preeclampsia in Populations of Central Asia and Europe” (InterPregGen) within the 7th Framework Programme of the European Commission under Grant Agreement No. 282540. ResultThe study discovered a significantly higher population frequency of carrying the unfavorable rs1126643 allele of the ITGA2 gene polymorphism when compared with European populations. The population frequencies of carrying minor alleles of the SERPINE1 (rs179988) and KDR (rs2071559) genes in the Kazakh population were significantly lower when compared with the previously studied populations of Europe and Asia. An intermediate frequency of unfavorable minor alleles between European and Asian populations was found in Kazakhs for gene polymorphisms: FV rs6025, PGF rs12411, and ACE rs4340. The genomic analysis determined the choice of polymorphisms for their further replicative genotyping in patients with ACCD in PE in the Kazakh population. ConclusionThe obtained results will serve as a basis for the development of effective methods of early diagnosis and treatment of PE in pregnant women, carriers of unfavorable genotypes.

B-070 Performance Assessment of an Immunoassay for Detection of APOE4 Carriers

Abstract Background The APOE gene corresponds to apolipoprotein E, a glycoprotein involved in lipid metabolism. There are three different versions of the APOE gene, e2, e3, and e4 where e3 is the most common allele. APOE4 has been associated with Alzheimer’s Disease (AD) and other neurological diseases. The presence of a single APOE4 allele increases the risk of AD by 3–5 fold and two copies, increase the risk by 8–12 fold. Detection of the APOE4 allele is commonly performed using genetic test methods. Studies were undertaken to evaluate the analytical performance of an immunoassay that detects the presence of APOE4 protein in and assess the discrimination of APOE4 gene carriers from non-APOE4 gene carriers. Methods The Biocross e4Risk® test is a latex agglutination immunoassay used for the quantitative determination of isoform E4 of the apolipoprotein E (APOE4) in human EDTA plasma samples. The test consists of one reagent with latex particles and another reagent with a mouse anti-ApoE4, which induces the latex particle agglutination when ApoE4 is present in the plasma sample. This agglutination is measured photometrically. The turbidity is proportional to APOE4 content of the sample, which can be used to infer the APOE4 genotype responsible. The Biocross kit reagents were run on the Siemens Healthineers Atellica® Chemistry Analyzer (open channel). Stored clinical samples from Eisai’s elenbecestat Phase 3 MissionAD Studies were used in the analysis. Results: Conclusion The Biocross e4Risk® test on the Atellica® Chemistry Analyzer (open channel) demonstrated good analytical performance, with sensitivity and precision comparable to manufacturer’s claims. The assay also had very good sensitivity and specificity when compared to known APOE4 genotype results. A simple blood-based robust APOE4 immunoassay test that runs on a routine clinical chemistry instrument may be a more convenient and cost-effective tool to help recruit patients for AD clinical trial programs.

The protective association of HLA-C*12:02 and HLA-DQB1*06:01 with severe acute hepatitis of unknown origin in the Japanese population.

Global surveillance has been conducted to elucidate the pathogenesis of acute hepatitis of unknown origin (AHUO), However, the factors associated with the aggravation of this serious disease are unclear. Therefore, we conducted a HLA association study to identify HLA alleles or haplotypes predisposing or protective against Japanese AHUO. The HLA 5 locus (HLA-A, HLA-B, C, DRB1, and DQB1) 4-digit genotyping results of 72 AHUO patients who underwent liver transplantation at our institution between 2000 and 2021 were compared to those of 873 healthy Japanese controls. Protective associations of HLA-B*52:01 (p-corrected (pc) = 3.15 × 10-3 ), HLA-C*12:02 (pc = 1.66 ×10-3 ), HLA-DQB1*06:01 (pc = 1.42 × 10-2 ), and HLA-DRB1*15:02 (pc = 1.36 × 10-2 ) with severe AHUO in Japanese patients were observed. The amino acid residues of tryptophan at position 156, which are located in the antigen-binding grooves of the HLA-C protein, showed a protective association with AHUO, showing a significant difference from other amino acid variations (pc = 9.0 × 10-4 ). Furthermore, 5 amino acid residues of the HLA-DQB1 protein were also protectively associated with AHUO with a significant difference from other amino acid variations (pc = 1.42 × 10-2 to 2.89 × 10-2 ). These alleles have a protective association with the aggravation of AHUO in the Japanese population.

Development of a 20 K SNP array for the leopard coral grouper, Plectropomus leopardus

Single nucleotide polymorphism (SNP) genotyping array is a powerful tool for providing information on polymorphic variations of species of interest. These genomic variations can be used to investigate the genetic architecture of complex traits, population structures, and genomic selection of breeding programs. The aquaculture of the leopard coral grouper (Plectropomus leopardus), is rapidly developing in South China and Southeast Asia. However, the lack of a high-efficient genotyping platform has hampered the progress of both genetic studies and selective breeding practices of this fish. Here we developed and evaluated a 20 K SNP array namely “DongXin-I” for P. leopardus. Using the genome re-sequencing data of 730 leopard coral groupers, we identified over 3.67 million SNPs, from which 20,737 loci, with even distribution across the genome, were finally involved in the SNP array. Evaluation of the array performance with 232 fish showed a high overall SNP call rates (> 99%) and an average minor allele frequency (MAF) of 0.312. Comparison of genotyping results of 87 fish from both the “DongXin-I” array and genome resequencing demonstrated a high genotyping repeatability and accurate performance of the genotyping array. Furthermore, using the gentyping results generated by the “DongXin-I” array, we revealed the genetic structure of breeding populaitons currently used the in Hainan Province. To summarize, the “DongXin-I” array should be a useful tool for the genetic studies and genomic selection breeding in the aquaculture industry of the coral leopard grouper.

Visual single nucleotide polymorphism (SNP) detection for ALDH2 genotyping based on multiplex ligation probe amplification (MLPA) and lateral flow assay

Current techniques for single nucleotide polymorphism (SNP) detection require laborious experimental procedures, expensive and sophisticated instruments, as well as complex data analysis, which make it difficult to meet the demand for routine clinical laboratories or point of care testing (POCT). In this study, the authors propose a reliable and cost-effective method by integrating multiplex ligation probe amplification (MLPA) and gold nanoparticles (AuNPs)-based lateral flow assay (LFA) for visual detection of the ALDH2 gene rs671(G > A) polymorphism. The genotyping result can be easily observed with the naked eye within 5–10 min after loading the MLPA products on the LFA device. This system demonstrates high sensitivity with LOD down to 1 ng, meeting the requirement in clinical diagnostic. Furthermore, this method has been implemented in clinical practices to identify the genotypes of the ALDH2 gene. The results obtained from the MLPA-LFA, Sanger sequencing, and high-resolution melting (HRM) curve analysis demonstrate a perfect agreement rate of 100%. Compared to conventional genotyping assays, this system exhibits the following advantages, including visual interpretation of results, minimal requirements for sophisticated instruments and highly skilled technicians, time efficiency, as well as low cost with a price approximately at 5 dollars. We believe that the MLPA-LFA system is a robust and versatile method with the potential to detect other SNPs related to disease susceptibility and risk, as well as drug metabolism and therapeutic effects.

Resistome-based surveillance identifies ESKAPE pathogens as the predominant gram-negative organisms circulating in veterinary hospitals.

Healthcare-associated infections (HCAIs) associated with extended-spectrum cephalosporin-resistant gram-negative (ESC-R GN) bacteria are an emerging concern in veterinary hospitals, especially in companion animal intensive care units (ICUs). To understand the molecular epidemiology of ESC-R GN isolates in two veterinary hospitals (equine and small animal), a 6-month pilot study was performed during which fecal and environmental samples were obtained twice from selected patients, upon ICU admission and after 48 h of hospitalization. In total, 295 ESC-R GNs were analyzed using the Acuitas Resistome® Test (OpGen, Maryland, US), a PCR-based assay screening for 50 antimicrobial resistance gene families encoding for production of extended-spectrum beta-lactamase (ESBLs), TEM/SHV/OXA or AmpC beta-lactamases and carbapenemases. Combining organism identification and antimicrobial susceptibility data to genotyping results, unique "Acuitas profiles" were generated that can be used for fast typing the isolates and tracking transmission events. ESKAPE GN pathogens were the most prevalent ESC-R GN isolates circulating in both the small animal and equine hospitals, consisting of Enterobacter cloacae complex (21.7%), Pseudomonas aeruginosa (20%), Klebsiella pneumoniae (15.9%), and Acinetobacter baumannii complex (13.6%) followed by Escherichia coli (12.2%), most harboring a combination of genes encoding for beta-lactamases and ESBLs. Some ESKAPE genotypes showed likely intra-hospital transmission, including E. cloacae (two genotypes, one carrying SHV4, SHV5, and TEM7 and the other TEM1, TEM3, and TEM7 enzymes) in the equine and K. pneumoniae (SHV1, SHV5, and DHA1-positive) in the small animal ICUs, respectively. Furthermore, P. aeruginosa (carrying OXA-50), A. baumannii complex (OXA-51), and E. coli (CTX-M-1) genotypes were isolated across both hospitals, suggesting possible transfer mediated via movement of staff and students. Importantly, isolates carrying transmissible resistance to last-resort antimicrobials (i.e. carbapenems) were identified within the hospital environments, consisting of three environmental Acinetobacter spp. harboring blaOXA - 23 and one clinical E. coli with blaOXA - 48. We describe the widespread occurrence of ESKAPE gram-negative organisms in veterinary ICU patients and hospital environments. Findings from this project provide baseline data on the epidemiology of ESKAPE pathogens in veterinary settings, which can inform infection control policies to aid in patient management and prevent transmission of nosocomial infections associated with these pathogens.

Longitudinal Assessment of Virological Failure and Antiretroviral Drug Resistance among Treatment-naive Subjects Living with HIV.

Human immunodeficiency virus (HIV) infection, the etiological agent of acquired immunodeficiency syndrome (AIDS), is a serious public health issue. Therapeutic measures have been successful in increasing the survival and improving the quality of life. However, some treatment-naive subjects living with HIV present resistance-associated mutations as a result of late diagnosis and/or mutant strain infections. The objective of this study was to identify the virus genotype and assess the antiretroviral resistance profile based on the results of HIV genotyping in treatment-naive subjects living with HIV, after six months of taking antiretroviral therapy. This was a prospective cohort study on treatment-naive adults living with HIV attending a specialized outpatient clinic in southern Santa Catarina State, Brazil. The participants were interviewed and had blood samples drawn. The genotypic antiretroviral drug resistance profile was examined in patients with detectable viral loads. 65 treatment-naive subjects living with HIV were recruited for this study. After six months of taking antiretroviral therapy, resistance-associated mutations were observed in 3 (4.6%) subjects living with HIV. Subtype C was identified as the circulating subtype in southern Santa Catarina State, and L10V, K103N, A98G, and Y179D were the most common mutations found in treatment-naive subjects.

Genetic diversity and phylogenetic relationship of Angus herds in Hungary and analyses of their production traits.

This study aims to investigate the genetic structure and characteristics of the Angus cattle population in Hungary. The survey was performed with the assistance of the Hungarian Hereford, Angus, Galloway Association (HHAGA). Genetic parameters of 1,369 animals from 16 Angus herds were analyzed using the genotyping results of 12 microsatellite markers with the aid of PowerMarker, Genalex, GDA-NT2021, and STRUCTURE software. Genotyping of DNA was performed using an automated genetic analyzer. Based on pairwise identity by state values of animals, the Python networkx 2.3 library was used for network analysis of the breed and to identify the central animals. The observed numbers of alleles on the 12 loci under investigation ranged from 11 to 18. The average effective number of alleles was 3.201. The overall expected heterozygosity was 0.659 and the observed heterozygosity was 0.710. Four groups were detected among the 16 Angus herds. The breeders' information validated the grouping results and facilitated the comparison of birth weight, age at first calving, number of calves born and productive lifespan data between the four groups, revealing significant differences. We identified the central animals/herd of the Angus population in Hungary. The match of our group descriptions with the phenotypic data provided by the breeders further underscores the value of cooperation between breeders and researchers. The observation that significant differences in the measured traits occurred among the identified groups paves the way to further enhancement of breeding efficiency. Our findings have the potential to aid the development of new breeding strategies and help breeders keep the Angus populations in Hungary under genetic supervision. Based on our results the efficient use of an upcoming genomic selection can, in some cases, significantly improve birth weight, age at first calving, number of calves born and the productive lifespan of animals.

Genetical Signature-An Example of a Personalized Skin Aging Investigation with Possible Implementation in Clinical Practice.

We conducted a research study to create the groundwork for personalized solutions within a skin aging segment. This test utilizes genetic and general laboratory data to predict individual susceptibility to weak skin characteristics, leveraging the research on genetic polymorphisms related to skin functional properties. A cross-sectional study was conducted in a collaboration between the Private Clinic Medicina Practica Laboratory (Vilnius, Lithuania) and the Public Institution Lithuanian University of Health Sciences (Kaunas, Lithuania). A total of 370 participants agreed to participate in the project. The median age of the respondents was 40, with a range of 19 to 74 years. After the literature search, we selected 15 polymorphisms of the genes related to skin aging, which were subsequently categorized in terms of different skin functions: SOD2 (rs4880), GPX1 (rs1050450), NQO1 (rs1800566), CAT (rs1001179), TYR (rs1126809), SLC45A2 (rs26722), SLC45A2 (rs16891982), MMP1 (rs1799750), ELN (rs7787362), COL1A1 (rs1800012), AHR (rs2066853), IL6 (rs1800795), IL1Beta (rs1143634), TNF-α (rs1800629), and AQP3 (rs17553719). RT genotyping, blood count, and immunochemistry results were analyzed using statistical methods. The obtained results show significant associations between genotyping models and routine blood screens. These findings demonstrate the personalized medicine approach for the aging segment and further add to the growing literature. Further investigation is warranted to fully understand the complex interplay between genetic factors, environmental influences, and skin aging.

CsSE59 Encoding Invertase/Pectin Methyl Esterase Inhibitor Is a Candidate Gene Conferring the Virescent True Leaf Phenotype in Cucumber

Leaf color mutants are the ideal materials to study the regulation mechanisms of chlorophyll biosynthesis and chloroplast development or as markers for crop breeding. In this study, we identified a virescent true leaf mutant se59 from the ethyl methane sulfonate (EMS)-induced mutant lines of cucumber (Cucumis sativus L.). The se59 mutant showed normal cotyledons, but the true leaf displays light green at early growth stage, which can recover normal green later. The se59 locus was controlled by a single recessive nuclear gene. The grana stacking in the chloroplasts of se59 decreased significantly, and both the photosynthetic ability and the photosynthetic pigment contents of the se59 were significantly lower than those of wild type. The results of BSA-seq and genotyping showed that an Invertase/Pectin Methyl Esterase Inhibitor (INV/PMEI) protein encoded by CsSE59 is a candidate gene for the virescent true leaf mutant. The expression level of CsSE59 in stem, leaf and root is high. Based on the transcriptome analysis of the first true leaf of se59 mutant, the expression levels of 17 leaf color related genes changed significantly, suggesting CsSE59 may regulate virescent true leaf by interacting with some of these genes in cucumber. The identification of CsSE59 is helpful to clarify the role of INV/PMEI in chloroplast development and to understand the mechanisms of leaf color variation in cucumber.

Single and Multiple High-Risk Human Papillomavirus Infections in Histopathologically Confirmed Cervical Squamous Lesions: Incidences, Distribution, and Associated Detection Rates for Precancerous and Cancerous Lesions

Coinfection with multiple high-risk human papillomavirus (hrHPV) is frequently observed in cervical specimens; however, the clinical significance of concomitant multiple hrHPV infections is poorly understood, and the published results remain inconsistent. A retrospective study at a tertiary care institution was performed, evaluating Tellgenplex human papillomavirus (HPV) 27 genotyping or YanengBio HPV 23 genotyping results and immediate cervical histologic diagnosis (within 6 months after HPV genotyping), between November 2015 and October 2022. Among 49,299 cases with hrHPV genotyping and histologic diagnosis, 24,361 cases were diagnosed as cervical intraepithelial neoplasia (CIN) and squamous cell carcinoma. Among women with cervical squamous lesions, 86.5% (21,070/24,361) had hrHPV infections, and concomitant multiple hrHPV infections accounted for 24.7% of hrHPV-positive cases (5210/21,070). The hrHPV-positive rates in these cervical squamous lesions increased progressively with disease severity; however, the percentages of concomitant multiple hrHPV infection rates among hrHPV-positive cases decreased significantly with increasing degree of squamous abnormalities. There was no increased detection rate of CIN3+ (CIN3 and squamous cell carcinoma) in cases with concomitant 2 or 3 hrHPV genotype infections when compared with those with corresponding single hrHPV infections. Conversely, some combinations of multiple hrHPV infections demonstrated a decrease in the detection rates of CIN3+ lesions. In this large cohort, our results demonstrated that multiple hrHPV infections do not carry an increased risk for developing CIN3+ lesions when compared to the corresponding single-genotype infection. The reduced risk of CIN3+ in women infected with some combinations of hrHPV genotypes compared to those with single-genotype infections supports the concept of intergenotypic competition of hrHPV genotypes in cervical squamous lesions.

Variable Cre Recombination Efficiency in Placentas of Cyp19-Cre ROSAmT/mG Transgenic Mice.

The aromatase-Cre recombinase (Cyp19-Cre) transgenic mouse model has been extensively used for placenta-specific gene inactivation. In a pilot study, we observed unexpected phenotypes using this mouse strain, which prompted an extensive characterization of Cyp19-Cre placental phenotypes using ROSAmT/mG transgenic reporter mice. The two strains were mated to generate bi-transgenic Cyp19-Cre;ROSAmT/mG mice following a standard transgenic breeding scheme, and placental and fetal tissues were analyzed on embryonic day 17.5. Both maternal and paternal Cre inheritance were analyzed by mating the respective Cyp19-Cre and ROSAmT/mG males and females. The genotype results showed the expected percentage of Cyp19-Cre;ROSAmT/mG fetuses (73%) and Cre mRNA was expressed in all of the Cyp19-Cre placentas. However, surprisingly, only about 50% of the Cyp19-Cre;ROSAmT/mG placentas showed Cre-mediated recombinase activity as demonstrated by placental enhanced green fluorescent protein (EGFP) expression. Further genetic excision analysis of the placentas revealed consistent results showing the absence of excision of the tdTomato in all of the Cyp19-Cre;ROSAmT/mG placentas lacking EGFP expression. Moreover, among the EGFP-expressing placentas, there was wide variability in recombination efficiency, even in placentas from the same litter, leading to a mosaic pattern of EGFP expression in different zones and cell types of the placentas. In addition, we observed a significantly higher percentage of Cre recombination activity in placentas with maternal Cre inheritance. Our results show frequent mosaicism, inconsistent recombination activity, and parent-of-origin effects in placentas from Cyp19-Cre;ROSAmT/mG mice, suggesting that tail-biopsy genotype results may not necessarily indicate the excision of floxed genes in Cyp19-Cre positive placentas. Thus, placenta-specific mutagenesis studies using the Cyp19-Cre model require extensive characterization and careful interpretation of the placental phenotypes for each floxed allele.

Evaluation of allelic frequencies distribution of the BoLA-DRB3 gene polymorphisms within exon 2 using PCR-RFLP

BoLA-DRB3 is part of the major histocompatibility complex, which has more than 100 polymorphic sites in exon 2. This gene is associated with immunological traits, resistance or susceptibility to various diseases and productive traits and milk quality in cattle. The aim of our paper was to conduct a primary assessment of the allelic polymorphism of exon 2 of the BoLA-DRB3 gene in the Ural region using nested PCR and PCR-RFLP. The object of research were Holstein cattle (n=55). Restriction fragment lengths were determined using electrophoresis in an agarose gel. For the BoLA-DRB3.2 gene the most common genotypes were identi ed: *23/*24, *24/*24, *16/*16, *24/*28, *9/*38 with a prevalence from 8.9 % to 26.5 %; genotypes with a lower distribution frequency - *24/*38, *24/*9, *2/*37, *16/*26. In studied samples the majority of identified alleles (*2, *9, *26, *37, *38) were previously proven to be neutral. The results of genotyping of Holstein cattle according to the polymorphism of the BoLA-DRB3.2 gene were obtained: in the studied population, 56 % of individuals had a genotype associated with susceptibility to the leukemia virus, 29 % associated with resistance and 15 % had neutral genotype. Based on the obtained results, it can be concluded that there is a need for genetic screening of cattle populations for the BoLA-DRB3 polymorphism, especially in epidemiologically disadvantaged regions for the incidence of leukemia. The obtained data on the genotypes of animals will be used in breeding programs to saturate herds of farm animals with alleles of resistance to the bovine leukemia virus.