Polymerase Chain Reaction Sequencing Research Articles

Occurrence and Multiplex PCR Detection of Citrus Yellow Vein Clearing Virus in Korea.

Citrus yellow vein clearing virus (CYVCV) is a member of the Alphaflexiviridae family that causes yellow vein clearing symptoms on citrus leaves. A total of 118 leaf samples from nine regions of six provinces in Korea were collected from various citrus species in 2020 and 2021. Viral diagnosis using next-generation sequencing and reverse transcription polymerase chain reaction (RT-PCR) identified four viruses: citrus tristeza virus, citrus leaf blotch virus, citrus vein enation virus, and CYVCV. A CYVCV incidence of 9.3% was observed in six host plants, including calamansi, kumquat, Persian lime, and Eureka lemon. Among the citrus infected by CYVCV, only three samples showed a single infection; the other showed a mixed infection with other viruses. Eureka lemon and Persian lime exhibited yellow vein clearing, leaf distortion, and water-soak symptom underside of the leaves, while the other hosts showed only yellowing symptoms on the leaves. The complete genome sequences were obtained from five CYVCV isolates. Comparison of the isolates reported from the different geographical regions and hosts revealed the high sequence identity (95.2% to 98.8%). Phylogenetic analysis indicated that all the five isolates from Korea were clustered into same clade but were not distinctly apart from isolates from China, Pakistan, India, and Türkiye. To develop an efficient diagnosis system for the four viruses, a simultaneous detection method was constructed using multiplex RT-PCR. Sensitivity evaluation, simplex RT-PCR, and stability testing were conducted to verify the multiplex RT-PCR system developed in this study. This information will be useful for developing effective disease management strategies for citrus growers in Korea.

Comparison between next-generation sequencing and multiplex polymerase chain reaction assays for nonsmall-cell lung cancer molecular diagnosis.

Genetic mutation detection has become an important step in nonsmall-cell lung cancer (NSCLC) treatment because of the increasing number of drugs that target genomic rearrangements. A multiplex test that can detect multiple gene mutations prior to treatment is thus necessary. Currently, either next-generation sequencing (NGS)-based or polymerase chain reaction (PCR)-based tests are used. We evaluated the performance of the Oncomine Dx Target Test (ODxTT), an NGS-based multiplex biomarker panel test, and the AmoyDx Pan Lung Cancer PCR Panel (AmoyDx PLC panel), a real-time PCR-based multiplex biomarker panel test. Patients with histologically diagnosed NSCLC and a sufficient sample volume to simultaneously perform the AmoyDx PLC panel and ODxTT-M were included in the study. The success and detection rates of both tests were evaluated. Biopsies revealed 116 cases of malignancies, 100 of which were NSCLC. Of these, 59 met the inclusion criteria and were eligible for analysis. The success rates were 100% and 98% for AmoyDx PLC panel and ODxTT-M, respectively. Nine driver mutations were detected in 35.9% and 37.3% of AmoyDx PLC and ODxTT-M panels, respectively. EGFR mutations were detected in 14% and 12% of samples using the AmoyDx PLC panel and ODxTT-M, respectively. Of the 58 cases in which both NGS and AmoyDx PLC panels were successful, discordant results were observed in seven cases. These differences were mainly due to different sensitivities of the detection methods used and the gene variants targeted in each test. The AmoyDx PLC panel, a PCR-based multiplex diagnostic test, exhibits a high success rate. The frequency of the nine genes targeted for treatment detected by the AmoyDx PLC panel was comparable to the frequency of mutations detected by ODxTT-M. Clinicians should understand and use the AmoyDx PLC panel and ODxTT-M with respect to their respective performances and limitations.

High Prevalence of Carbapenem-Resistant Enterobacterales Producing OXA-48 among Carbapenem-Resistant Isolates in a Regional Hospital in Central Taiwan.

In response to the increasing number of carbapenem-resistant Enterobacterales (CRE), we investigated carbapenemase-producing Klebsiella pneumoniae and non-K. pneumoniae epidemiology and genetics. We collected 76 clinical Enterobacterales and 4 stool surveillance Escherichia coli isolates resistant to ertapenem or imipenem. Using polymerase chain reaction (PCR) and DNA sequencing, we assessed carbapenemases, extended-spectrum β-lactamases, and AmpC β-lactamases. Molecular typing via pulsed-field gel electrophoresis (PFGE) and conjugation experiments were conducted to examine resistance gene transfer. Among the 80 isolates, 96.2% harbored at least one carbapenemase gene, with blaOXA-48 in 87.5%. KPC-2 and IMP-8 carbapenemases were found in 15.0 and 22.5% of the isolates, respectively, with 27.5% having 2 or more carbapenemase genes. The PFGE analysis revealed the presence of diverse genotypes. PCR-based plasmid replicon typing identified IncA/C as the most prevalent type among K. pneumoniae isolates (26/29), and IncF and IncFIB among E. coli isolates (22/28). Conjugal transfer was successful for plasmids encoding OXA-48, CTX-M-3, CTX-M-14, CMY-2, and other β-lactamases, except the KPC-2 gene. In conclusion, our study highlights high carbapenemase prevalence in CRE, primarily OXA-48. Multiple carbapenemases within strains were common, and PFGE showed diverse patterns in these carbapenem-resistant isolates.

Differential DNA methylation landscape of miRNAs genes in mice liver fibrosis.

Patients with chronic liver disease were found nearly all to have liver fibrosis, which is characterized by excess accumulation of extracellular matrix (ECM) proteins. While ECM accumulation can prevent liver infection and injury, it can destroy normal liver function and architecture. miRNA's own regulation was involved in DNA methylation change. The purpose of this study is to detect DNA methylation landscape of miRNAs genes in mice liver fibrosis tissues. Male mice (10-12 weeks) were injected CCl4 from abdominal cavity to induced liver fibrosis. 850K BeadChips were used to examine DNA methylation change in whole genome. The methylation change of 16 CpG dinucleotides located in promoter regions of 4 miRNA genes were detected by bisulfite sequencing polymerase chain reaction (BSP) to verify chip data accuracy, and these 4 miRNA genes' expressions were detected by RT-qPCR methods. There are 769 differential methylation sites (DMS) in total between fibrotic liver tissue and normal mice liver tissue, which were related with 148 different miRNA genes. Chips array data were confirmed by bisulfite sequencing polymerase chain reaction (R = 0.953; P < 0.01). GO analysis of the target genes of 2 miRNA revealed that protein binding, cytoplasm and chromatin binding activity were commonly enriched; KEGG pathway enrichment analysis displayed that TGF-beta signaling pathway was commonly enriched. The DNA of 148 miRNA genes was found to have methylation change in liver fibrosis tissue. These discoveries in miRNA genes are beneficial to future miRNA function research in liver fibrosis.

Comparison of polymerase chain reaction and next-generation sequencing with conventional urine culture for the diagnosis of urinary tract infections: A meta-analysis.

The limitations of conventional urine culture methods can be avoided by using culture-independent approaches like polymerase chain reaction (PCR) and next-generation sequencing (NGS). However, the efficacy of these approaches in this setting is still subject to contention. PRISMA-compliant searches were performed on MEDLINE/PubMed, EMBASE, Web of Sciences, and the Cochrane Database until March 2023. The included articles compared PCR or NGS to conventional urine culture for the detection of urinary tract infections (UTIs). RevMan performed meta-analysis, and the Cochrane Risk of Bias Assessment Tool assessed study quality. A total of 10 selected studies that involved 1,291 individuals were included in this meta-analysis. The study found that PCR has a 99% sensitivity and a 94% specificity for diagnosing UTIs. Furthermore, NGS was shown to have a sensitivity of 90% for identifying UTIs and a specificity of 86%. The odds ratio (OR) for PCR to detect Gram-positive bacteria is 0.50 (95% confidence interval [CI] 0.41-0.61), while the OR for NGS to detect Gram-negative bacteria is 0.23 [95% CI 0.09-0.59]. UTIs are typically caused by Gram-negative bacteria like Escherichia coli and Gram-positive bacteria like Staphylococci and Streptococci. PCR and NGS are reliable, culture-free molecular diagnostic methodsthat, despite being expensive, are essential for UTI diagnosis and prevention due to their high sensitivity and specificity.

Genotypic and Allelic Distribution of the CD36 rs1761667 Polymorphism in High-Level Moroccan Athletes: A Pilot Study.

Previous studies have shown that variations in the CD36 gene may affect phenotypes associated with fat metabolism as the CD36 protein facilitates the transport of fatty acids to the mitochondria for oxidation. However, no previous study has tested whether variations in the CD36 gene are associated with sports performance. We investigated the genotypic and allelic distribution of the single-nucleotide polymorphism (SNP) rs1761667 in the CD36 gene in elite Moroccan athletes (cyclists and hockey players) in comparison with healthy non-athletes of the same ethnic origin. Forty-three Moroccan elite male athletes (nineteen cyclists and twenty-four field hockey players) belonging to the national teams of their respective sports (athlete group) were compared to twenty-eight healthy, active, male university students (control group). Genotyping of the CD36 rs1761667 (G>A) SNP was performed via polymerase chain reaction (PCR) and Sanger sequencing. A chi-square (χ2) test was used to assess the Hardy-Weinberg equilibrium (HWE) and to compare allele and genotype frequencies in the "athlete" and "control" groups. The genotypic distribution of the CD36 rs1761667 polymorphism was similar in elite athletes (AA: 23.81, AG: 59.52, and GG: 16.67%) and controls (AA: 19.23, AG: 69.23, and GG: 11.54%; χ2 = 0.67, p = 0.71). However, the genotypic distribution of the CD36 rs1761667 polymorphism was different between cyclists (AA: 0.00, AG: 72.22, and GG: 27.78%) and hockey players (AA: 41.67, AG: 50.00, and GG: 8.33%; χ2 = 10.69, p = 0.004). Specifically, the frequency of the AA genotype was significantly lower in cyclists than in hockey players (p = 0.02). In terms of allele frequency, a significant difference was found between cyclists versus field hockey players (χ2 = 7.72, p = 0.005). Additionally, there was a predominance of the recessive model in cyclists over field hockey players (OR: 0.00, 95% CI: 0.00-0.35, p = 0.002). Our study shows a significant difference between cyclists and field hockey players in terms of the genotypic and allelic frequency of the SNP rs1761667 of the CD36 gene. This divergence suggests a probable association between genetic variations in the CD36 gene and the type of sport in elite Moroccan athletes.

Comparative Transcriptomics Analysis Reveals Unique Immune Response to Grass Carp Reovirus Infection in Barbel Chub (Squaliobarbus curriculus).

Grass carp (Ctenopharyngodon idella) and barbel chub (Squaliobarbus curriculus)-both Leuciscinae subfamily species-demonstrate differences in grass carp reovirus (GCRV) infection resistance. We infected barbel chubs with type II GCRV and subjected their liver, spleen, head kidney, and trunk kidney samples to investigate anti-GCRV immune mechanisms via RNA sequencing and quantitative real-time polymerase chain reaction (qRT-PCR). We identified 139, 970, 867, and 2374 differentially expressed genes (DEGs) in the liver, spleen, head kidney, and trunk kidney, respectively. Across all four tissues, gene ontology analysis revealed significant immune response-related DEG enrichment, and the Kyoto Encyclopedia of Genes and Genomes analysis revealed pattern recognition receptor (PRR) and cytokine-related pathway enrichment. We noted autophagy pathway enrichment in the spleen, head kidney, and trunk kidney; apoptosis pathway enrichment in the spleen and trunk kidney; and complement- and coagulation-cascade pathway enrichment in only the spleen. Comparative transcriptome analysis between GCRV-infected barbel chubs and uninfected barbel chubs comprehensively revealed that PRR, cytokine-related, complement- and coagulation-cascade, apoptosis, and autophagy pathways are potential key factors influencing barbel chub resistance to GCRV infection. qRT-PCR validation of 11 immune-related DEGs confirmed our RNA-seq data's accuracy. These findings provide a theoretical foundation and empirical evidence for the understanding of GCRV infection resistance in barbel chub and hybrid grass carp-barbel chub breeding.

Use of CRISPR/Cas9 with Homology-Directed Repair to Gene-Edit Topoisomerase IIβ in Human Leukemia K562 Cells: Generation of a Resistance Phenotype.

DNA topoisomerase IIβ (TOP2β/180; 180 kDa) is a nuclear enzyme that regulates DNA topology by generation of short-lived DNA double-strand breaks, primarily during transcription. TOP2β/180 can be a target for DNA damage-stabilizing anticancer drugs, whose efficacy is often limited by chemoresistance. Our laboratory previously demonstrated reduced levels of TOP2β/180 (and the paralog TOP2α/170) in an acquired etoposide-resistant human leukemia (K562) clonal cell line, K/VP.5, in part due to overexpression of microRNA-9-3p/5p impacting post-transcriptional events. To evaluate the effect on drug sensitivity upon reduction/elimination of TOP2β/180, a premature stop codon was generated at the TOP2β/180 gene exon 19/intron 19 boundary (AGAA//GTAA→ATAG//GTAA) in parental K562 cells (which contain four TOP2β/180 alleles) by CRISPR/Cas9 editing with homology-directed repair to disrupt production of full-length TOP2β/180. Gene-edited clones were identified and verified by quantitative polymerase chain reaction and Sanger sequencing, respectively. Characterization of TOP2β/180 gene-edited clones, with one or all four TOP2β/180 alleles mutated, revealed partial or complete loss of TOP2β mRNA/protein, respectively. The loss of TOP2β/180 protein correlated with decreased (2-{4-[(7-chloro-2-quinoxalinyl)oxy]phenoxy}propionic acid)-induced DNA damage and partial resistance in growth inhibition assays. Partial resistance to mitoxantrone was also noted in the gene-edited clone with all four TOP2β/180 alleles modified. No cross-resistance to etoposide or mAMSA was noted in the gene-edited clones. Results demonstrated the role of TOP2β/180 in drug sensitivity/resistance in K562 cells and revealed differential paralog activity of TOP2-targeted agents. SIGNIFICANCE STATEMENT: Data indicated that CRISPR/Cas9 editing of the exon 19/intron 19 boundary in the TOP2β/180 gene to introduce a premature stop codon resulted in partial to complete disruption of TOP2β/180 expression in human leukemia (K562) cells depending on the number of edited alleles. Edited clones were partially resistant to mitoxantrone and XK469, while lacking resistance to etoposide and mAMSA. Results demonstrated the import of TOP2β/180 in drug sensitivity/resistance in K562 cells and revealed differential paralog activity of TOP2-targeted agents.

Assessing microbiome population dynamics using wild-type isogenic standardized hybrid (WISH)-tags

Microbiomes feature recurrent compositional structures under given environmental conditions. However, these patterns may conceal diverse underlying population dynamics that require intrastrain resolution. Here we developed a genomic tagging system, termed wild-type isogenic standardized hybrid (WISH)-tags, that can be combined with quantitative polymerase chain reaction and next-generation sequencing for microbial strain enumeration. We experimentally validated the performance of 62 tags and showed that they can be differentiated with high precision. WISH-tags were introduced into model and non-model bacterial members of the mouse and plant microbiota. Intrastrain priority effects were tested using one species of isogenic barcoded bacteria in the murine gut and the Arabidopsis phyllosphere, both with and without microbiota context. We observed colonization resistance against late-arriving strains of Salmonella Typhimurium in the mouse gut, whereas the phyllosphere accommodated Sphingomonas latecomers in a manner proportional to their presence at the late inoculation timepoint. This demonstrates that WISH-tags are a resource for deciphering population dynamics underlying microbiome assembly across biological systems.

Enterocytozoon bieneusi and Cryptosporidium bat genotype XXI and bat genotype XXII in fruit bats (Rousettus leschenaultii) inhabiting a tropical park in Hainan Province, China

Bats stand as one of the most diverse groups in the animal kingdom and are key players in the global transmission of emerging pathogens. However, their role in transmitting Enterocytozoon bieneusi and Cryptosporidium spp. remains unclear. This study aimed to evaluate the occurrence and genetic diversity of the two pathogens in fruit bats (Rousettus leschenaultii) in Hainan, China. Ten fresh fecal specimens of fruit bats were collected from Wanlvyuan Gardens, Haikou, China. The fecal samples were tested for E. bieneusi and Cryptosporidium spp. using Polymerase Chain Reaction (PCR) analysis and sequencing the internal transcribed spacer (ITS) region and partial small subunit of ribosomal RNA (SSU rRNA) gene, respectively. Genetic heterogeneity across Cryptosporidium spp. isolates was assessed by sequencing 4 microsatellite/minisatellite loci (MS1, MS2, MS3, and MS16). The findings showed that out of the ten specimens analyzed, 2 (20 %) and seven (70.0 %) were tested positive for E. bieneusi and Cryptosporidium spp., respectively. DNA sequence analysis revealed the presence of two novel Cryptosporidium genotypes with 94.4 to 98.6 % sequence similarity to C. andersoni, named as Cryptosporidium bat-genotype-XXI and bat-genotype-XXII. Three novel sequences of MS1, MS2 and MS16 loci identified here had 95.4 to 96.9 % similarity to the known sequences, which were deposited in the GenBank. Two genotypes of E. bieneusi were identified, including a novel genotype named HNB-I and a zoonotic genotype PigEbITS7. The discovery of these novel sequences provides meaningful data for epidemiological studies of the both pathogens. Meanwhile our results are also presented that the fruit bats infected with E. bieneusi, but not with Cryptosporidium, should be considered potential public health threats.

Risk prediction and function evaluation by T-cell epitope model and expression model of HLA-DPB1 mismatching in unrelated-donor hematopoietic stem cell transplantations

Objective: To evaluate the risk prediction and assessment function of HLA-DPB1 T-cell epitope (TCE) model and expression model in human leukocyte antigen (HLA)-matched unrelated hematopoietic stem cell transplantation (MUD-HSCT) with HLA-DPB1 mismatching. Methods: A total of 364 (182 pairs) potential MUD-HSCT donors and recipients confirmed by HLA high-resolution typing in Shaanxi Blood Center from 2016 to 2019 were analyzed retrospectively. Of the 182 recipients, there were 121 males and 61 females with an average age of (26.3±14.2) years. Of the 182 donors, there were 148 males and 34 females with an average age of (33.7±7.5) years. Polymerase chain reaction-sequence-based typing (PCR-SBT), next-generation sequencing (NGS) and polymerase chain reaction-sequence specific oligonucleotide probe (PCR-SSO) based on LABScan®3D platform were used for high-resolution typing of HLA-A, B, C, DRB1, DQB1, DPB1 gene, and PCR-SBT was used for single nucleotide polymorphism (SNP) typing. TCE model and expression model were used to predict and evaluate the HLA-DPB1 mismatch pattern and acute graft-versus-host-disease (aGVHD) risk. Results: A total of 26 HLA-DPB1 alleles and their 3'-UTR rs9277534 SNP genotypes were detected in this study population, and two new alleles HLA-DPB1*1052∶01 and HLA-DPB1*1119∶01 were found and officially named. The overall mismatch rate of HLA-DPB1 in MUD-HSCT donors and recipients was 90.66% (165/182). In TCE model, the HLA-DPB1 mismatch rates of permissible mismatch (PM) and non-permissible mismatch (non-PM) were 47.80% (87/182) and 42.86% (78/182), respectively. The non-PM in GvH direction was 13.73% (25/182), and which in HvG direction was 29.12% (53/182). A total of 73 pairs of donors and recipients in TCE model met the evaluation criteria of expression model. Among of TCE PM group, recipient DP5 mismatches accounted for 34.25% (25/73) were predicted as aGVHD high risk according to expression model. For the TCE non-PM group, both the recipient DP2 mismatches of 6.85% (5/73) and recipient DP5 mismatches of 10.86% (8/73) were predicted to be at high risk for aGVHD. Risk prediction by TCE model and expression model was 27.27% concordant and 16.97% unconcordant. Conclusions: TCE model and expression model are effective tools to predict aGVHD risk of MUD-HSCT. Comprehensive application of the two models is helpful to the hierarchical assessment of HSCT risk.

CircALMS1 Alleviates Pulmonary Microvascular Endothelial Cell Dysfunction in Pulmonary Hypertension.

Circular RNAs can serve as regulators influencing the development of pulmonary hypertension (PH). However, their function in pulmonary vascular intimal injury remains undefined. Thus, we aimed to identify specifically expressed circular RNAs in pulmonary microvascular endothelial cells (PMECs) under hypoxia and PH. Deep RNA sequencing and quantitative real-time polymerase chain reaction revealed that circALMS1 (circular RNA Alstrom syndrome protein 1) was reduced in human PMECs under hypoxia (P<0.0001). Molecular biology and histopathology experiments were used to elucidate the roles of circALMS1 in regulating PMEC dysfunction among patients with PH. The circALMS1 expression was decreased in the plasma of patients with PH (P=0.0315). Patients with lower circALMS1 levels had higher risk of death (P=0.0006). Moreover, the circALMS1 overexpression of adeno-associated viruses improved right ventricular function and reduced pulmonary vascular remodeling in monocrotaline-PH and sugen/hypoxia-PH rats (P<0.05). Furthermore, circALMS1 overexpression promoted apoptosis and inhibited PMEC proliferation and migration under hypoxia by directly downregulating miR-17-3p (P<0.05). Dual luciferase assay confirmed the direct binding of circALMS1 to miR-17-3p and miR-17-3p binding to its target gene YT521-B homology domain-containing family protein 2 (YTHDF2) (P<0.05). The YTHDF2 levels were also downregulated in hypoxic PMECs (P<0.01). The small interfering RNA YTHDF2 reversed the effects of miR-17-3p inhibitors on PMEC proliferation, migration, and apoptosis. Finally, the results indicated that, although YTHDF2, as an N(6)-methyladenosine reader protein, contributes to the degradation of many circular RNAs, it could not regulate the circALMS1 levels in PMECs (P=0.9721). Our study sheds new light on circALMS1-regulated dysfunction of PMECs by the miR-17-3p/YTHDF2 pathway under hypoxia and provides insights into the underlying pathogenesis of PH.

Analysis of clinical characteristics and histopathological transcription in 40 patients afflicted by epilepsy stemming from focal cortical dysplasia.

This study aims to comprehensively analyze the clinical characteristics and identify the differentially expressed genes associated with drug-resistant epilepsy (DRE) in patients with focal cortical dysplasia (FCD). A retrospective investigation was conducted from July 2019 to June 2022, involving 40 pediatric cases of DRE linked to FCD. Subsequent follow-ups were done to assess post-surgical outcomes. Transcriptomic sequencing and quantitative reverse transcription polymerase chain reaction (qRT-PCR) were used to examine differential gene expression between the FCD and control groups. Among the 40 patients included in the study, focal to bilateral tonic-clonic seizures (13/40, 32.50%) and epileptic spasms (9/40, 22.50%) were the predominant seizure types. Magnetic resonance imaging (MRI) showed frequent involvement of the frontal (22/40, 55%) and temporal lobes (12/40, 30%). In cases with negative MRI results (13/13, 100%), positron emission tomography/computed tomography (PET-CT) scans revealed hypometabolic lesions. Fused MRI/PET-CT images demonstrated lesion reduction in 40.74% (11/27) of cases compared with PET-CT alone, while 59.26% (16/27) yielded results consistent with PET-CT findings. FCD type II was identified in 26 cases, and FCD type I in 13 cases. At the last follow-up, 38 patients were prescribed an average of 1.27 ± 1.05 anti-seizure medications (ASMs), with two patients discontinuing treatment. After a postoperative follow-up period of 23.50 months, 75% (30/40) of patients achieved Engel class I outcome. Transcriptomic sequencing and qRT-PCR analysis identified several genes primarily associated with cilia, including CFAP47, CFAP126, JHY, RSPH4A, and SPAG1. This study highlights focal to bilateral tonic-clonic seizures as the most common seizure type in patients with DRE due to FCD. Surgical intervention primarily targeted lesions in the frontal and temporal lobes. Patients with FCD-related DRE showed a promising prognosis for seizure control post-surgery. The identified genes, including CFAP47, CFAP126, JHY, RSPH4A, and SPAG1, could serve as potential biomarkers for FCD. This study aimed to comprehensively evaluate the clinical data of individuals affected by focal cortical dysplasia and analyze transcriptomic data from brain tissues. We found that focal to bilateral tonic-clonic seizures were the most prevalent seizure type in patients with drug-resistant epilepsy. In cases treated surgically, the frontal and temporal lobes were the primary sites of the lesions. Moreover, patients with focal cortical dysplasia-induced drug-resistant epilepsy exhibited a favorable prognosis for seizure control after surgery. CFAP47, CFAP126, JHY, RSPH4A, and SPAG1 have emerged as potential pathogenic genes for the development of focal cortical dysplasia.

Preliminary Study on Yeasts Associated with the Production of "Tostado"-a Traditional Sweet Wine from Galicia (NW Spain).

Tostado is a traditional sweet wine from the Designations of Origins (DOs) of Ribeiro and Valdeorras in Galicia (NW Spain). The harvested grapes are air-dried and pressed to increase the concentrations of sugars, acids, and flavour compounds. Therefore, knowledge of the yeasts involved in fermentation under these conditions is essential to guarantee the quality and uniqueness of the valuable, distinctive, and expensive Tostado wines. Saccharomyces and non-Saccharomyces yeasts were identified using Wallerstein Laboratory (WL) Nutrient Agar and lysine plating, followed by polymerase chain reaction (PCR) amplification, enzymatic digestion, and sequencing. Saccharomyces cerevisiae isolates were further characterised at the strain level using mitochondrial DNA (mtDNA) restriction fragment length polymorphism (RFLP). Statistical analyses were also performed, including different diversity indices, Similarity Percentage (SIMPER) analysis, principal component analysis (PCA), neighbor-joining clustering, parsimony-phylogram, and network plot. In addition, the total acidity, volatile acidity, reducing sugars, and alcoholic strength by volume of the Tostado wines were analysed. A wide diversity of autochthonous yeasts was found, which were predominantly species of oenological relevance, such as Lachancea thermotolerans, Starmerella bacillaris, Hanseniaspora uvarum, Debaryomyces hansenii, Torulaspora delbrueckii, Pichia spp., and Saccharomyces cerevisiae from the must and paste stages of Tostado wine. In addition, 19 different S. cerevisiae strains were identified. This high yeast diversity, which changed from the early stages of fermentation, could contribute to the distinctive characteristics observed in Tostado wine. Characteristic and differentiating chemical and microbiological profiles were found as early as the pre-fermentation stages, which adds value to these special wines that have rarely been studied.

Potassium transporter OsHAK9 regulates seed germination under salt stress by preventing gibberellin degradation through mediating OsGA2ox7 in rice.

Soil salinity has a major impact on rice seed germination, severely limiting rice production. Herein, a rice germination defective mutant under salt stress (gdss) was identified by using chemical mutagenesis. The GDSS gene was detected via MutMap and shown to encode potassium transporter OsHAK9. Phenotypic analysis of complementation and mutant lines demonstrated that OsHAK9 was an essential regulator responsible for seed germination under salt stress. OsHAK9 is highly expressed in germinating seed embryos. Ion contents and non-invasive micro-test technology results showed that OsHAK9 restricted K+ efflux in salt-exposed germinating seeds for the balance of K+/Na+. Disruption of OsHAK9 significantly reduced gibberellin 4 (GA4) levels, and the germination defective phenotype of oshak9a was partly rescued by exogenous GA3 treatment under salt stress. RNA sequencing (RNA-seq) and real-time quantitative polymerase chain reaction analysis demonstrated that the disruption of OsHAK9 improved the GA-deactivated gene OsGA2ox7 expression in germinating seeds under salt stress, and the expression of OsGA2ox7 was significantly inhibited by salt stress. Null mutants of OsGA2ox7 created using clustered, regularly interspaced, short palindromic repeat (CRISPR)/CRISPR-associated nuclease 9 approach displayed a dramatically increased seed germination ability under salt stress. Overall, our results highlight that OsHAK9 regulates seed germination performance under salt stress involving preventing GA degradation by mediating OsGA2ox7, which provides a novel clue about the relationship between GA and OsHAKs in rice.

Whole F8 gene sequencing identified pathogenic structural variants in the remaining unsolved patients with severe hemophilia A

BackgroundNo F8 genetic abnormality is detected in approximately 1% to 2% of patients with severe hemophilia A (HA) using conventional genetic approaches. In these patients, deep intronic variation or F8 disrupting genomic rearrangement could be causal. ObjectivesThe study aimed to identify the causal variation in families with a history of severe HA for whom genetic investigations failed. MethodsWe performed whole F8 gene sequencing in 8 propositi. Genomic rearrangements were confirmed by Sanger sequencing of breakpoint junctions and/or quantitative polymerase chain reaction. ResultsA structural variant disrupting F8 was found in each propositus, so that all the 815 families with a history of severe HA registered in our laboratory received a conclusive genetic diagnosis. These structural variants consisted of 3 balanced inversions, 3 large insertions of gained regions, and 1 retrotransposition of a mobile element. The 3 inversions were 105 Mb, 1.97 Mb, and 0.362 Mb in size. Among the insertions of gained regions, one corresponded to the insertion of a 34 kb gained region from chromosome 6q27 in F8 intron 6, another was the insertion of a 447 kb duplicated region from chromosome 9p22.1 in F8 intron 14, and the last one was the insertion of an Xq28 349 kb gained in F8 intron 5. ConclusionAll the genetically unsolved cases of severe HA in this cohort were due to structural variants disrupting F8. This study highlights the effectiveness of whole F8 sequencing to improve the molecular diagnosis of HA when the conventional approach fails.

Circulating Tumor DNA (ctDNA) and Its Role in Gynecologic Malignancies.

Circulating tumor DNA (ctDNA) refers to small fragments of DNA released into the bloodstream by cancer cells. It is obtained through "liquid biopsy;" which most commonly refers to plasma or blood samples, but can be obtained from a number of bodily fluids including ascitic fluid, saliva, and even urine and stool. ctDNA is detected via polymerase chain reaction (PCR) or next-generation sequencing (NGS). The DNA from these samples is analyzed for the detection of point mutations, copy-number alterations, gene fusion, and DNA methylation. These results have the potential for use in cancer diagnosis, determining prognosis, targeting gene-specific therapies, and monitoring for/predicting disease recurrence and response to treatment. ctDNA offers an alternative to tissue biopsy; it is less invasive and can be monitored serially over time without multiple procedures. Moreover it may have the ability to detect disease recurrence or predict behavior in a way that solid tissue biopsies, tumor marker surveillance, and imaging cannot. Recent explosion in interest in ctDNA shows promising developments for widespread adoption of these techniques in cancer care. However, the use of ctDNA in diagnosis and treatment of gynecologic malignancies is currently limited, compared to adoption in other solid-organ tumors such as breast and colorectal cancers. Compared to other cancer types, there appear to be fewer comprehensive studies and clinical validations specifically focusing on the use of ctDNA in gynecologic cancers. More research is needed in this area to advance the potential for use of ctDNA in ovarian, endometrial, and cervical cancers before this can be routinely adopted to improve care for patients with gynecologic malignancies.

The alternation of flood and ebb tide induced arsenic release and migration from coastal tidal flat sediments in Yellow Sea wetlands: An ex-situ study

Periodic floods and ebb tides driven by seawater affect arsenic (As) behavior in coastal wetlands, which is not fully understood. In this study, we combined DGT, high-throughput sequencing, and real-time quantitative polymerase chain reaction (qPCR) techniques for the first time to investigate the effects of flood and ebb tide on As transformation and mobility in a typical coastal tidal flat wetland by laboratory experiments. In the flood-tide stage, more As was released into the water solution, and As(III) concentration in water solution was notably higher (51 μg/L) than in the ebb-tide stage, especially in SA (sodium acetate addition) treatment. As-methylation also occurred in this stage. The release of As was attributed to the reductive dissolution of amorphous and crystalline Fe oxides bound As induced by microbes. Fusibacter, Sva1033, Bradyrhizobium, Ralstonia, and Desulfobacter were primarily involved in the reduction of As, Fe, and S. The copy numbers of arsC were 20–148 times higher than arrA, suggesting that the detoxification reduction pathway was the primary mechanism of direct As-reduction in the flood-tide stage. However, 2-D high-resolution DGT techniques revealed that the concentrations of As(III) and total As in pore water of sediments in the ebb-tide stage were higher than those in the flood-tide stage. The detected aioA gene indicated that As was oxidized in the sediment surface, resulting in As stabilization by crystalline iron minerals, whereas more As(III) were accumulated in pore water. The reduction pathway mediated by arrA may play a prominent role in the ebb-tide stage. This study provides new insights into As release processes in coastal wetlands in seawater environments, which further deepens our understanding of As biogeochemical cycling behavior in different natural environments.

Geographic distribution of a missense mutation in the KRT38 gene in Chinese indigenous cattle breeds

Context China has a vast area across many temperature zones and a variety of cattle breeds. These cattle resources are ideal models to research their adaptability to the environment. The KRT38 gene is an acidic protein, and its coding product can be used as a component of hair production. Aims The objective of this study was to investigate the diversity of the KRT38 gene in Chinese local cattle and the association of different genotypes with mean temperature, relative humidity and temperature humidity index. Methods A missense mutation g.41650738 A &gt; G in the KRT38 gene was screened from the database of bovine genomic variation (BGVD) and was genotyped in a total of 246 samples from 15 local cattle breeds in China by polymerase chain reaction amplification and sequencing. Finally, the correlation between the locus and the three climatic factors was analysed. Key results We successfully obtained the frequency of this single-nucelotide polymorphism in three groups of cattle in northern, central and southern China. The frequency of allele A gradually declined from north to south, whereas the frequency of allele G showed the opposite trend with a clear geographic distribution. Conclusions Our results indicate that KRT38 variation in Chinese indigenous cattle might be linked to heat tolerance. Implications Our analysis may assist in determining the importance of the variation as a genetic signal for heat tolerance in cattle reproduction and genetics.

Cloning and characterization of luciferase from an Asian firefly Pygoluciola qingyu and its comparison with other beetle luciferases.

The bioluminescence system of luminescent beetles has extensive applications in biological imaging, protein labeling and drug screening. To explore wild luciferases with excellent catalytic activity and thermal stability, we cloned the luciferase of Pygoluciola qingyu, one species living in areas of high temperature and with strong bioluminescence, by combining transcriptomic sequencing and reverse transcription polymerase chain reaction (RT-PCR). The total length of luciferase gene is 1638bp and the luciferase consists 544 amino acids. The recombinant P. qingyu luciferase was produced in vitro and its characteristics were compared with those of eight luciferases from China firefly species and two commercial luciferases. Compared with these luciferases, the P. qingyu luciferase shows the highest luminescence activity at room temperature (about 25-28℃) with similar KM value for D-luciferin and ATP to the Photinus pyralis luciferase. The P. qingyu luciferase activity was highest at 35℃ and can keep high activity at 30-40℃, which suggests the potential of P. qingyu luciferase for in vivo and cell application. Our results provide new insights into P. qingyu luciferase and give a new resource for the application of luciferases.