DNA Sequencing Results Research Articles

Extracting and analyzing micronuclei from mouse two-cell embryos fertilized with freeze-dried spermatozoa

Abnormal chromosome segregation (ACS) in preimplantation embryos causes miscarriages. For a normal pregnancy, it is necessary to reduce ACS occurrences in embryos. However, the causes of such abnormalities are unclear because no method to extract the segregated chromosomes from the blastomeres for detailed analysis. This study attempted to extract micronuclei derived from segregated chromosomes of mouse embryos. Some micronuclei in blastomeres were bound to the nucleus by DNA cross-links, some were bound to tubulin, and about half of the micronuclei had major satellite regions. By depolymerizing the cytoskeleton of blastomeres with cytochalasin B and colcemid, some micronuclei could be extracted from blastomeres of ACS embryos using a glass needle of a micromanipulator. DNA-sequencing results of each extracted micronucleus revealed that chromosomes in micronuclei were randomly selected, usually only one, and often contained a portion rather than the full length of the chromosome. This study allows a detailed analysis of micronuclei and facilitates the mechanism of the causes of ACS in embryos.

Unveiling non-coding DMD variants: synergising RNA sequencing and DNA sequencing for enhanced molecular diagnosis

BackgroundPathogenic variants in the DMD gene are associated with dystrophinopathy including Duchenne and Becker muscular dystrophy (DMD/BMD). Targeted DMD gene, gene panels, exomes and genome sequencing have advanced genetic diagnostics,...

New Refined Experimental Analysis of Fungal Growth in Degraded Bio-Based Materials

When exposed to different building environmental conditions, bio-composite materials, such as hemp mortars, represent a risk of mold proliferation. This later plays a critical role in the biodeterioration of the materials when their physical properties are locally modified by the natural aging process. The primary objectives of the present work are first to assess the evolution of the surface of contaminated mortar; second, to investigate an accurate DNA extraction method that could be used for both bio-composite mortars and their fiber sources collected in situ; then, to understand the process of the proliferation of mold strains on both hemp shives and hemp mortar; and finally, to compare mold strains present in these phases to show their relationship to mold contamination and their impact on human health. In situ hemp mortar contamination behavior was investigated in the region of Pau (France) two months after hemp mortar application in extreme conditions (high humidity, low temperature, no aeration), which did not match the standard conditions under which hemp mortar must be used. The SEM observations and FTIR and pH analyses highlighted the decrease in pH level and the presence of organic matter on the mortar surface. DNA sequencing results showed that hemp shives were the main source of fungal contamination of hemp mortar. A mold population analysis showed that the most dominant phylum was Ophistokonta, which represented 83.6% in hemp shives and 99.97% in hemp mortar. The Acrostalagmus genus representatives were the most abundant, with 42% in hemp shives and 96% in hemp mortar. The interconnection between the mold strain characteristics (particularly the ability to grow in extreme environments) and the presence of hemp mortar was emphasized.

3D genome contributes to MHC-II neoantigen prediction.

Reliable and ultra-fast DNA and RNA sequencing have been achieved with the emergence of high-throughput sequencing technology. When combining the results of DNA and RNA sequencing for tumor cells of cancer patients, neoantigens that potentially stimulate the immune response of either CD4+ or CD8+ T cells can be identified. However, due to the abundance of somatic mutations and the high polymorphic nature of human leukocyte antigen (HLA) it is challenging to accurately predict the neoantigens. Moreover, comparing to HLA-I presented peptides, the HLA-II presented peptides are more variable in length, making the prediction of HLA-II loaded neoantigens even harder. A number of computational approaches have been proposed to address this issue but none of them considers the DNA origin of the neoantigens from the perspective of 3D genome. Here we investigate the DNA origins of the immune-positive and non-negative HLA-II neoantigens in the context of 3D genome and discovered that the chromatin 3D architecture plays an important role in more effective HLA-II neoantigen prediction. We believe that the 3D genome information will help to increase the precision of HLA-II neoantigen discovery and eventually benefit precision and personalized medicine in cancer immunotherapy.

Fluorescence in-situ hybridization assessment of spindle cell-rich testicular sex cord stromal tumors demonstrates multiple chromosomal gains across histologic subtypes

Spindle cell-rich testicular sex cord-stromal tumors (TSCSTs) comprise a group that includes mostly (but not exclusively): myoid gonadal stromal tumor (MGST), adult granulosa cell tumor (AGCT), and unclassified TSCST. These entities demonstrate histopathologic overlap, and prior genomic studies have failed to identify specific oncogenic drivers. Results of DNA sequencing suggest that different types of spindle cell-rich TSCSTs harbor a recurrent pattern of chromosomal gains. However, these results have not been validated by alternative methods and the extent of these changes within individual tumors remains unknown. We used a combination of commercially available fluorescence in-situ hybridization (FISH) probes (3q11.2, 6p24.3, 6q11.1, 6q23, 7q11.21-q11.22, 9p21.3, 11q13.3, 17p11.2) to enumerate a subset of chromosomes identified as altered (gained) in prior studies. We analyzed 10 cases (3 MGST, 4 unclassified TSCST, 3 AGCT), including 7 that had been previously sequenced. FISH demonstrated gains of chromosomes 3, 6, 7, 9, and 11 above the pre-established threshold (25%) in 50%, 80%, 70%, 20%, and 40% of cases, respectively, with gains of chromosome 17 being present in only 1 unclassified TSCST. The proportion of cells with chromosomal gains ranged from 26% to 60%. Tumors with available copy number data from prior genomic analyses showed a partial discordance between FISH and sequencing results. This study demonstrates that spindle-cell rich TSCSTs harbor a recurrent pattern of chromosomal gains, which are present in variable subsets of neoplastic cells. Further studies are needed to determine if these chromosomal changes represent a mechanism relevant for oncogenesis or a secondary event.

Assessing the impact of HBB gene polymorphisms on the risk of beta thalassemia in the population of Diyala Governorate, Iraq

Objective The current research aims to investigate the genetic polymorphisms of the HBB gene and their relationship to the incidence of beta-thalassemia. Methods The study included 30 patients with beta-thalassemia major. Samples were collected during the period between August 2023 and February 2024. Their ages ranged from 2–31 years, in addition to 30 healthy people who did not suffer from hereditary blood diseases as a control group. Their ages ranged from 1–35 years. The exon region of the HBB gene was sequenced using nucleotide sequencing in order to identify the kind and location of mutations that alter the amino acid sequence that makes up the hemoglobin protein. Results The results of DNA sequencing of the exon region of the HBB gene, which consists of 382 base pairs, showed the presence of the rs713040 variant. This variant revealed that the GG genotype and the G allele, and the AA genotype and the A allele, were more frequent in the patient group compared to the healthy group and may be associated with risk. When comparing some physiological variables with the genotypes of the HBB gene between the group of patients and healthy controls at the rs713040 variant site, the results showed that there were significant differences (P<0.001, P<0.05) in the levels of WBC, HB, PLT, PCT, ALP, and Vitamin D in the three genotypes of the variant GG, GA, and AA. Conclusion The findings suggest that the rs713040 variant of the HBB gene may be associated with an increased risk of beta-thalassemia and that there are significant physiological differences between the genotypes of this variant in patients and healthy controls. Further research is needed to understand the underlying mechanisms and the potential clinical implications of these genetic differences.

Genetic Polymorphisms of GP1BA, PEAR1, and PAI-1 may be Associated with Serum sIgE and Blood Eosinophil Levels in Chinese Patients with Allergic Diseases.

It has been suggested that genetic factors may be substantially linked to allergy disorders. This study aims to investigate the relationship between the serum specific Immunoglobulin E (sIgE), blood eosinophil, and the polymorphisms of glycoprotein Ib alpha gene (GP1BA) rs6065, platelet endothelial aggregation receptor 1 gene (PEAR1) rs12041331, and plasminogen activator inhibitor 1 gene (PAI-1) rs1799762. From the Peking Union Medical College Hospital, this study enrolled 60 healthy participants and 283 participants with allergic diseases. TaqMan-minor groove binder (MGB) quantitative polymerase chain reaction (qPCR) was used to examine the gene polymorphisms in each group. The TaqMan-MGB qPCR results were completely consistent with the DNA sequencing results, according to other studies in this medical center (Kappa =1, p <0.001). The GP1BA rs6065, PEAR1 rs12041331, and PAI-1 rs1799762 polymorphisms did not show different distribution between allergy patients and healthy individuals. Concerning allergy patients, the CT (n=33) genotype of GP1BA rs6065 had higher blood eosinophil level than the CC (n=250) genotype (0.59, IQR 0.32-0.72 vs 0.31, IQR 0.15-0.61, *109/L, p =0.005). The serum sIgE of AA (n=46) genotype of PEAR1 rs12041331 was lower (median 3.7, interquartile quartiles (IQR) 0.2-16.8, kU/L) than the GA (n=136) and GG (n=101) genotypes (GA median 16.3, IQR 3.1-46.3, kU/L, p = 0.002; GG median 12.9, IQR 3.0-46.9, kU/L, p =0.003). The GA genotypes of PEAR1 rs12041331were with higher blood eosinophil levels (median 0.42, IQR 0.17-0.74 *109/L) than the AA genotype (median 0.25, IQR 0.15-0.41*109/L, p =0.012). The sIgE of the 5G5G (n=44) genotype of PAI-1 rs1799762 was lower (median 5.0, IQR 0.1-22.8, kU/L) than the 4G5G (n=144) (median 17.3, IQR 3.7-46.0, kU/L, p = 0.012). The GP1BA rs6065, PEAR1 rs12041331, and PAI-1 rs1799762 polymorphisms may be associated with the genetic susceptibility of serum sIgE or blood eosinophil in Chinese allergic disease patients.

Investigation of Ivermectin Susceptibility in Kangal and Akbaş Dogs via MDR1 Gene Mutation

This research aimed to investigate the sensitivity to the drug (ivermectin) in Kangal and Akbaş breed dogs, which are dog breeds native to Turkey, via the MDR1 gene mutation. For the research, blood, hair and intraoral swap samples were taken from 30 Kangal and 20 Akbaş breed dogs (male-female mixed, adult) with ethical permission and approval forms. Kangal dog samples were taken from the dogs bred in the farms in Sivas center and Kangal village, and Akbaş dog samples were taken from the dogs bred in the farms in the Sivrihisar center, Ankara and Eskişehir regions. The samples taken were evaluated based on polymerase chain reaction (PCR) from the wool and subsequent two-way dideoxy chain termination reaction in the presence of 4 base farm deletions (c.296-299delAGAT) in the 4th exon of the ABCB1 gene that encodes the P-glycoprotein (P-gp) drug transporter protein. According to the obtained DNA sequence results, the deletion of “AGAT” was not determined in any of the individuals screened. The results of this study, which is preliminary research, showed that Kangal and Akbaş breed dogs are safe in terms of sensitivity to drugs that are set to be absorbed and excreted by the P-gp pump, especially ivermectin. However, it would be useful to repeat the analysis of both breeds with more examples.

A snapshot of bacterial endophytes isolated from the roots of Vetiver grass (Chrysopogon zizanioides) grown at Bien Hoa airbase, Dong Nai province

Vetiver grass (Chrysopogon zizanioides) with a strong root system has been widely used for environmental pollution treatment. Recently, this grass has been cultivated at Bien Hoa airbase to mitigate dioxin concentration in the contaminated soil. Bacterial endophytes have been recognized to play key roles in plant growth promotion and responses of plant hosts to environmental factors. However, a beneficial association between endophytic bacteria and Vetiver grass root system grown in dioxin-contaminated soil has not been elucidated. In the present study, we isolated sixteen endophytic bacterial strains from the roots of Vetiver grass. The results of DNA sequencing of the 16S rRNA gene revealed that these culturable bacterial endophytes belong to 9 species of different genera. Four strains, including Klebsiella variicola B1, Enterobacter cloacae B4, Enterobacter kobei B6, and Enterobacter cloacae B11, produce high amounts of indol acetic acid (IAA). Strains K. variicola B1 and E. cloacae B4 also produce phytase and phosphatase to dissolve phytate and phosphate, respectively. These strains were selected to evaluate their abilities in promoting the growth of tomato plants as a model. After 28 days of inoculation with the tested strains through the roots, tomato plants grew 19% to 22% faster than the control plants. Conclusively, this study shows for the first time the successful isolation of endophytic bacteria from the roots of Vetiver grass cultivated in dioxin-contaminated soil, and some culturable bacterial strains exhibit as promising candidates for plant growth promotion.

Niche conservatism and spread explain introgression between native and invasive fish.

Hybridisation can be an important driver of evolutionary change, but hybridisation with invasive species can have adverse effects on native biodiversity. While hybridisation has been documented across taxa, there is limited understanding of ecological factors promoting patterns of hybridisation and the spatial distribution of hybrid individuals. We combined the results of ecological niche modelling (ENM) and restriction site-associated DNA sequencing to test theories of niche conservatism and biotic resistance on the success of invasion, admixture, and extent of introgression between native and non-native fishes. We related Maxent predictions of habitat suitability based on the native ranges of invasive Eastern Banded Killifish (Fundulus diaphanus diaphanus Lesueur 1817) and native Western Banded Killifish (Fundulus diaphanus menona Jordan and Copeland 1877) to admixture indices of individual Banded Killifish. We found that Eastern Banded Killifish predominated at sites predicted as suitable from their ENM, consistent with niche conservatism. Admixed individuals were more common as Eastern Banded Killifish habitat suitability declined. We also found that Eastern Banded Killifish were most common at sites closest to the presumed source of this invasion, whereas the proportion of admixed individuals increased with distance from the source of invasion. Lastly, we found little evidence that habitat suitability for Western Banded Killifish provides biotic resistance from either displacement by, or admixture with, invasive Eastern Banded Killifish. Our study demonstrates that ENMs can inform conservation-relevant outcomes between native and invasive taxa while emphasising the importance of protecting isolated Western Banded Killifish populations from invasive conspecifics.

Multi-omics panoramic analysis ofHBV integration, transcriptional regulation, and epigenetic modifications in PLC/PRF/5 cell line.

The clearance or transcriptional silencing of integrated HBV DNA is crucial for achieving a functional cure in patients with chronic hepatitis Band reducing the risk of hepatocellular carcinoma development. The PLC/PRF/5cell line is commonly used as an in vitro model for studying HBV integration. In this study, we employed a range of multi-omics techniques to gain a panoramic understanding of the characteristics of HBV integration in PLC/PRF/5cells and to reveal the transcriptional regulatory mechanisms of integrated HBV DNA. Transcriptome long-read sequencing (ONT)was conducted to analyze and characterize the transcriptional activity of different HBV DNA integration sites in PLC/PRF/5cells. Additionally, we collected data related to epigenetic regulation, including whole-genome bisulfite sequencing (WGBS),histone chromatin immunoprecipitation sequencing (ChIP-seq),and assays for transposase-accessible chromatin using sequencing (ATAC-seq),to explore the potential mechanisms involved in the transcriptional regulation of integrated HBV DNA. Long-read RNA sequencing analysis revealed significant transcriptional differences at various integration sites in the PLC/PRF/5cell line, with higher HBV DNA transcription levels at integration sites on chr11, chr13, and the chr13/chr5 fusion chromosome t (13:5). Combining long-read DNA and RNA sequencing results, we found that transcription of integrated HBV DNA generally starts downstream of the SP1, SP2, or XP promoters. ATAC-seq data confirmed that chromatin accessibility has limited influence on the transcription of integrated HBV DNA in the PLC/PRF/5cell line. Analysis ofWGBS data showed that the methylation intensity of integrated HBV DNA was highly negatively correlated with its transcription level (r = -0.8929, p = 0.0123). After AzaD treatment, the transcription level of integrated HBV DNA significantly increased, especially for the integration chr17, which had the highest level of methylation. Through ChIP-seqdata, we observed the association between histone modification of H3K4me3 and H3K9me3 with the transcription of integrated HBV DNA. Our findings suggest that the SP1, SP2 and XP in integrated HBV DNA, methylation level of surrounding host chromosome, and histone modifications affect the transcription of integrated HBV DNA in PLC/PRF/5cells. This provides important clues for future studies on the expression and regulatory mechanisms of integrated HBV.

Abstract 4665: USP18 promotes anlotinib resistance in medullary thyroid carcinoma by stabilizing aurora B kinase

Abstract Targeted therapy resistance is the main cause of disease progression in advanced medullary thyroid carcinoma (MTC). However, the key molecules and mechanisms remain unclear. We previously performed a genome-wide in vitro screen of Anlotinib resistance-related genes in human MTC cell line TT using CRISPR-dCas9-SAM system and identified ubiquitin-specific protease 18 (USP18) as a key molecule mediating Anlotinib resistance in MTC. However, its downstream mechanism needs further clarification. Based on the high-throughput DNA sequencing results of the control group and the Anlotinib screening group, a group of key genes related to Anlotinib resistance in MTC was screened through the MAGeCK algorithm and the enrichment ratio of sgRNA in the Anlotinib screening group. Among them, USP18 ranked first in the enrichment results. Analysis of the correlation between the expression level of USP18 in MTC patient specimens and survival revealed that patients with high USP18 expression in tumor cells had poorer prognosis. Overexpression of USP18 in MTC tumor cells significantly promoted the growth of subcutaneous xenograft tumors in NOD/SCID mice treated with Anlotinib. In vitro cell experiments also showed that overexpression of USP18 promoted the proliferation of MTC tumor cells and significantly inhibited the proportion of cell apoptosis after Anlotinib treatment. Further research found that USP18, as a deubiquitinating enzyme that can specifically remove ISG15-like ubiquitin-like proteins from substrate proteins, can improve the kinase stability of Aurora B by removing ISG15-like ubiquitin modification and promote MTC tumor cell proliferation through the activation of PI3K-Akt signaling regulated by Aurora B kinase. Application of Aurora B kinase inhibitor in Anlotinib-resistant MTC cell lines significantly inhibited tumor cell proliferation, and Aurora B kinase inhibitor significantly increased the proportion of cell apoptosis in MTC tumor cells overexpressing USP18. In the subcutaneous xenograft tumor model of MTC in NOD/SCID mice, the growth of xenograft tumors in the Anlotinib combined with Aurora B kinase inhibitor treatment group was also significantly inhibited. In summary, overexpressed USP18 can activate PI3K-Akt signaling and promote tumor cell proliferation by increasing the stability of Aurora B kinase, ultimately leading to the formation of Anlotinib resistance in MTC patients. Combined use of Aurora B kinase inhibitor is a potentially effective combined treatment strategy for Anlotinib-resistant MTC patients. Citation Format: Xuan Qin, Xing Wan, Dapeng Li, Yimeng Liu, Xianhui Ruan, Fangkun Luan, Kejia Xu, Jia Li, Rong Xiang, Yongjun Piao, Yi Shi, Xiangqian Zheng. USP18 promotes anlotinib resistance in medullary thyroid carcinoma by stabilizing aurora B kinase [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4665.

A 28-year-old male patient with asymptomatic and multi-drug-resistant HBV infection: a case report

Chronic hepatitis B virus (HBV) infection poses a significant global health challenge, impacting millions of individuals and elevating the risk of morbidity and mortality. Antiviral therapies are the primary treatment for chronic HBV infection, but treatment resistance can occur, leading to poor clinical outcomes and an increased risk of liver complications. This case report presents the clinical trajectory of a 28-year-old male diagnosed with asymptomatic HBV infection in 2016 under the auspices of the Hepatitis Control Program, Government of Azad Jammu and Kashmir, Pakistan. Over 6 years, persistent HBsAg, HBV, and HBeAg were observed, with absent acute markers and co-infections. Initial HBV DNA viral load was 1 × 104 copies/mL in 2016, escalating despite entecavir and pegylated interferons therapy, indicating multi-drug resistance. Tenofovir therapy initially reduced viral load but later exacerbated it, reaching 1.86 × 106 copies/mL in 2022. Liver function abnormalities and lipid profile irregularities persisted. Urine examination consistently showed abnormalities. Pending HBV DNA sequencing results may offer insights into treatment resistance. This case underscores the need for an adaptive approach in managing chronic HBV infections within public health programs. Continuous monitoring, integration of virological and biochemical data, and a tailored treatment strategy are essential for optimizing outcomes in similar cases, stressing the importance of refining therapeutic approaches against chronic HBV infection.

Untargeted metabolomics analysis of probiotic jujube juice and its anti-obesity effects on high-fat-diet-induced obese mice.

Dietary intervention, including polyphenol consumption, is recognized as an effective strategy to prevent obesity. Although fermented jujube juice (FJJ) with lactic acid bacteria has been shown to be rich in polyphenols and have strong antioxidant properties, little is known about its anti-obesity properties. Untargeted metabolomics was employed to identify and analyze the differential metabolites between FJJ and raw jujube juice. A total of 431 metabolites belonging to diverse classes and with various functional active ingredients were quantitatively identified. The animal experiments results showed that FJJ administration for 13 weeks significantly inhibited high-fat-diet-induced body and epididymal adipose weight gain, and improved the serum lipid parameters in obese mice. Additionally, DNA-sequencing results revealed that FJJ treatment increased Akkermansia abundance in the gut and changed the composition of fecal microbiota by decreasing the Firmicutes/Bacteroidota ratio and Helicobacter pylori abundance. These findings suggest that FJJ contributes to regulating lipid accumulation and gut microbiota composition in high-fat-diet-induced obese mice, which helps to prevent obesity. Hence, FJJ has the potential to be a beneficial beverage for controlling obesity. © 2024 Society of Chemical Industry.

An easy-to-use pipeline to analyze amplicon-based Next Generation Sequencing results of human mitochondrial DNA from degraded samples.

Genome and transcriptome examinations have become more common due to Next-Generation Sequencing (NGS), which significantly increases throughput and depth coverage while reducing costs and time. Mitochondrial DNA (mtDNA) is often the marker of choice in degraded samples from archaeological and forensic contexts, as its higher number of copies can improve the success of the experiment. Among other sequencing strategies, amplicon-based NGS techniques are currently being used to obtain enough data to be analyzed. There are some pipelines designed for the analysis of ancient mtDNA samples and others for the analysis of amplicon data. However, these pipelines pose a challenge for non-expert users and cannot often address both ancient and forensic DNA particularities and amplicon-based sequencing simultaneously. To overcome these challenges, a user-friendly bioinformatic tool was developed to analyze the non-coding region of human mtDNA from degraded samples recovered in archaeological and forensic contexts. The tool can be easily modified to fit the specifications of other amplicon-based NGS experiments. A comparative analysis between two tools, MarkDuplicates from Picard and dedup parameter from fastp, both designed for duplicate removal was conducted. Additionally, various thresholds of PMDtools, a specialized tool designed for extracting reads affected by post-mortem damage, were used. Finally, the depth coverage of each amplicon was correlated with its level of damage. The results obtained indicated that, for removing duplicates, dedup is a better tool since retains more non-repeated reads, that are removed by MarkDuplicates. On the other hand, a PMDS = 1 in PMDtools was the threshold that allowed better differentiation between present-day and ancient samples, in terms of damage, without losing too many reads in the process. These two bioinformatic tools were added to a pipeline designed to obtain both haplotype and haplogroup of mtDNA. Furthermore, the pipeline presented in the present study generates information about the quality and possible contamination of the sample. This pipeline is designed to automatize mtDNA analysis, however, particularly for ancient samples, some manual analyses may be required to fully validate results since the amplicons that used to be more easily recovered were the ones that had fewer reads with damage, indicating that special care must be taken for poor recovered samples.

Monitoring the prevalence of Pseudomonas fluorescens as a spoilage indicator in cow raw milk, teat surfaces, and milk tanks.

Milk and its products are very sensitive to spoilage if they are kept under unsuitable conditions which may provide favorable circumstances for the growth of specific spoilage organisms, Pseudomonas fluorescens accounted as the most dominant indicator for milk spoilage. This study highlights monitoring the prevalence of P. fluorescens as a spoilage indicator organism in cow raw milk and its contact surfaces represented by teat surfaces and milk tanks in Nineveh province. A total of 150 samples from cows' raw milk, teat surfaces, and milk tank swabs were collected from different locations in Nineveh province from October 2023 till February 2024. The Pseudomonas fluorescens were detected by using conventional cultivation methods supported by molecular detection of the target pathogen using the polymerase chain reaction technique. Out of 150 samples, 48 (32%) were positive for the prevalence of P. fluorescens by traditional methods, and 39 (26%) were positive using PCR assay according to the 16SPflu gene yielded a band at 850 bp. The P. fluorescens was recovered at 19 (38%) from raw milk. Teat surfaces revealed a higher isolation rate 11 (22%) compared to milk tanks 9 (18%). The mean counts of Pseudomonas in cows raw milk revealed 4.38, 6.29, and 7.37 log CFU/ml for the 0, 3, and 6 days of storage at chilling temperature. Results of DNA sequencing of the 16SrRNA gene revealed 12 strains recorded in the GenBank nucleotide sequence database. Our results shed light on the risk of P. fluorescens prevalence as a spoilage indicator in raw milk and surrounding surfaces which is inevitable to apply hygienic procedures during milk collecting, processing, and preservation to increase the shelf life of the products and ensure milk safety and consumer health.

Molecular Identification and Phylogenetic Analysis of Salmonella‎ species ‎Isolated from Diarrheal Children and Dogs in Baghdad Governorate, ‎Iraq‎

This work aimed to use conventional PCR to identify Salmonella‎ spp. that ‎were isolated from diarrheal children and healthy and diarrheic dogs based on four ‎virulence genes, hilA, stn, spvR‎, and marT. Sixteen Salmonella‎ isolates including: 9 ‎isolated from children's diarrhea from three species (S. Typhimurium, S. Enteritidis, S. ‎Typhi) and seven isolated from dogs including (S. Typhimurium, S. Enteritidis, S. ‎Muenchen), were identified primarily by several methods. The PCR products of the 16S ‎rRNA gene were sequenced and examined using BLAST analysis to find differences and ‎similarities between these Iraqi isolates and already-known global strains in order to ‎construct the phylogenetic tree of S. Muenchen which was detected for the first time in ‎dogs in Iraq. The results of the study revealed that all isolates of Salmonella‎ obtained ‎from children possess the hilA and stn genes. The marT gene was detected in 88% of the ‎Salmonella‎ serovars, and the spvR‎ gene was carried in 55% of the isolates. Among dog ‎Salmonella‎ isolates, the hilA gene was detected at 100%, the stn gene was at 85.7%, the ‎marT gene was present at 71.4%, while the spvR‎ gene was found at 57.1%. The result of ‎DNA sequencing and phylogenetic tree indicated that the local Iraqi S. Muenchen was ‎extremely close to the national strain and share the same ‎16S rRNA gene sequence, the ‎isolate was registered at NCBI and became a global reference with the accession number OQ999043.1. In conclusion, the presence of these important virulence genes among ‎Salmonella‎ serovars isolated from children and dogs alerted on the potential risk of ‎contamination of the environment and may lead to a community health crisis‎.

Comparison of HIV-1 DNA load measurements in blood and in relation to successful proviral sequencing

ObjectiveHIV DNA sequencing is now routinely used for HIV-infected individuals on antiretroviral therapy (ART) with or without partial genotypic history. Successful amplification of HIV pol gene has yet to be correlated with HIV DNA levels. Here, we assessed the relationship between HIV DNA load and sequencing results. MethodsWe analyzed three different qPCR measurements of total (LTR and LTR-gag) and integrated (Alu-LTR) HIV DNA in blood samples collected from viremic as well as virally suppressed HIV-infected individuals on ART. HIV DNA levels were compared to HIV DNA Sanger sequencing and clinical and therapeutic parameters. ResultsAmong the 135 individuals analyzed for HIV DNA measurements and sequencing, all three HIV DNA measurements were associated with HIV DNA Sanger sequencing results. A threshold of around 2 and 1.5 log copies/million leukocytes of total HIV DNA was identified for LTR and LTR-gag qPCRs, respectively. Integrated HIV DNA positivity was also associated with successful sequencing. We further compared HIV DNA measurement techniques in an extended cohort of 312 individuals and showed that all measurements correlated between the different techniques, regardless of the HIV-1 subtypes analyzed. However, higher detection rates were observed with LTR (96%) compared to LTR-gag (86%) and Alu-LTR (59%) qPCRs. Duration of virological control on ART and CD4 nadir were the main determinants of HIV reservoir size. ConclusionsHIV DNA measurement is associated with Sanger sequencing success, regardless of the technique used. In a clinical setting, Application of HIV DNA quantification before sequencing should be further evaluated.

Molecular Characterization of Liver Fluke Isolated from Sheep, Goat and Cattle in Sulaymaniyah, Iraq.

We aimed to determine species of liver fluke that predominately cause fascioliasis in sheep, goats, and cattle in the Sulaymaniyah Province, Iraq using the molecular technique of DNA sequencing and restriction fragment length polymorphism (RFLP). The samples were collected from November 2021 to May 2022. The flukes were collected from infected livers of livestock at the slaughterhouse of Sulaymaniyah Governorate, Iraq. A total of 205 flukes were collected from 56 hosts, cattle (n=22), sheep (n=28), and goats (n=6). The specific primers for FCOX1 and 28S rDNA gene amplification were used. The PCR products were subjected to restriction fragment polymorphism (RFLP) assay using Hpy188III and Dra II restriction enzymes, besides DNA sequencing. The results showed the genetic polymorphisms among the flukes. Three patterns of RFLP were observed Fasciola hepatica, F. gigantica, and F. intermediate, where 28 of them displayed F. hepatica (sheep, n=14, goat, n=3 and cattle, n= 11), whereas 24 samples displayed the F. gigantica (sheep, n=12, goat, n=3 and cattle, n= 9), and only four samples belonged to F. intermediate (sheep n=3 and cattle, n=1). In addition, the result of the ribosomal DNA (28S rDNA) sequencing confirmed that the isolated flukes belonged to F. hepatica, F. gigantica and F. intermediate. All three main species are present in the study area and F. hepatica predominated among the animal species in this area also, our results concluded that PCR-RFLP is a rapid and reliable method for liver fluke species identification.

Docking of vaginal Lactobacillus isolates as a potential bactericidal agent in Kirkuk 2022

Abstract Background: Because the female genital tract is a complex microbial community that plays various roles in disease development. Alternatively, Lactobacilli in the vaginal environment play an important role in maintaining a healthy vaginal ecosystem by preventing pathogenic overgrowth. Objective: Therefore, this study was suggested to isolate vaginal Lactobacilli with antibacterial activity. Materials and Methods: Two hundred and sixty women participated in this study aged between 15 and 50 years divided into two cohorts of bacterial vaginosis (BV): infected and healthy individuals. BV-isolates isolated and identified according to microbiological and biochemical assays. Accordingly, Lactobacillus isolates were further identified through genomic DNA sequencing of the 16S rRNA, and the results were statistically analyzed. Results: A significant increase (P &lt; 0.05) in the BV-infected women in the age range of 21–30 and 31–40 was recorded, with the percentages of 40.62% and 31.88%, respectively. Alternatively, a significant decrease (P &gt; 0.05) in the percentages of vaginal Lactobacillus species between women of Cohort 1 and Cohort 2 recorded the ratios of 3.125% and 31% validating Lactobacilli presence as a sign of a healthy vaginal environment. Accordingly, four Lactobacillus isolates were assigned for their broad spectrum of antibacterial activity against several pathogenic bacteria. Moreover, results of the partial DNA sequencing revealed the isolation of four Limosilactobacillus fermentum strains registered at the National Center for Biotechnology Information (NCBI) under specific gene bank accession numbers. Conclusion: Effective Lactobacillus species with potential bactericidal activity validates vaginal health.