DNA Sequence Research Articles

Lactococcus garvieae aggravates cholestatic liver disease by increasing intestinal permeability and enhancing bile acid reabsorption

BACKGROUND Although an association between gut microbiota and cholestatic liver disease (CLD) has been reported, the precise functional roles of these microbes in CLD pathogenesis remain largely unknown. AIM To explore the function of gut microbes in CLD pathogenesis and the effects of gut microbiota on intestinal barrier and bile acid (BA) metabolism in CLD. METHODS Male C57BL/6J mice were fed a 0.05% 3,5-diethoxycarbonyl-1,4-dihydrocollidine diet for 2 weeks to induce CLD. The sterile liver tissues of mice were then meticulously harvested, and bacteria in homogenates were identified through culture methods. Furthermore, 16S ribosomal DNA sequencing was employed to analyze sterile liver samples collected from eight patients with primary biliary cholangitis (PBC) and three control individuals with hepatic cysts. The functional roles of the identified bacteria in CLD pathogenesis were assessed through microbiota transfer experiments, involving the evaluation of changes in intestinal permeability and BA dynamics. RESULTS Ligilactobacillus murinus (L . murinus ) and Lactococcus garvieae (L. garvieae ) were isolated from the bacterial culture of livers from CLD mice. L . murinus was prevalently detected in PBC patients and controls, whereas L. garvieae was detected only in patients with PBC but not in controls. Mice inoculated with L. garvieae exhibited increased susceptibility to experimental CLD, with both in vitro and in vivo indicating that L. garvieae disrupted the intestinal barrier function by down-regulating the expression of occludin and zonula occludens-1. Moreover, L. garvieae administration significantly upregulated the expression of the apical sodium-dependent BA transporter in the terminal ileum and increased serum BA levels. CONCLUSION L. garvieae contributes to excessive BA-induced hepatobiliary injury and liver fibrosis by increasing intestinal permeability and enhancing BA reabsorption.

The eradication of Blastocystis spp. subtype 3 improves the course of chronic spontaneous urticaria.

The role of Blastocystis spp. subtypes (STs) in chronic spontaneous urticaria (CSU) and the impact of the eradication are unclear. To investigate the role of Blastocystis spp. STs in CSU and the impact of the eradication on disease activity. Stool samples from 295 CSU patients and 38 healthy controls (HCs) were searched for Blastocystis spp. by direct microscopy. Fifty-seven samples from patients and 12 samples from HCs were analyzed for ST distribution by DNA sequencing. DNA sequencing was only performed in samples detected by Real Time (RT)-polymerase chain reaction (PCR). The patients and HCs with Blastocystis spp. formed groups A and B, respectively. Anti-parasitic agents were used to eradicate the parasite in group A. Urticaria medication scores (MSs), urticaria activity score-7 (UAS7), blood eosinophil and basophil counts, total IgE values were compared between groups having different STs at the beginning and six months after the eradication. The subjects who were not taking any medication for the CSU such as antihistamine, montelukast, anti-IgE treatment or cyclosporin had no active urticarial lesions during a 6-month period were considered to be in remission. The parasite was detected in 45.7% (n=135) of the CSU patients and 34.2% (n=13) of the HCs. Fifty-seven samples (85.1%) in 67 CSU patients and 12 samples (92.3%) in 13 HCs revealed positive RT-PCR and formed groups A and B, respectively. ST3 was more frequent in group A (p<0.001) whereas ST1 was more common in group B (p<0.001). The patients having ST3 in group A formed subgroup A1 and other STs in group A formed subgroup A2. After the parasite eradication, the CSU remission rate was higher (p=0.031) and the control values of UAS7, MSs were lower (p=0.028, p=0.049) in subgroup A1 than A2. Although the presence of Blastocystis spp. ST3 does not worsen CSU, its eradication improves the CSU symptoms.

Enrichment of Microbial DNA in Plasma to Improve Pathogen Detection in Sepsis: A Pilot Study.

Diagnosis of sepsis and timely identification of pathogens in critically ill patients remains challenging. Plasma metagenomic sequencing to detect microbial cell-free DNA (mDNA) has shown promise, but low abundance of mDNA in plasma limits sensitivity and necessitates high sequencing depth. mDNA is shorter and more fragmented than human cell-free DNA. Here, we evaluated whether combining single-stranded DNA (ssDNA) sequencing library preparation and size selection can enrich mDNA and improve pathogen detection. We prospectively enrolled 48 trauma patients and collected daily blood samples during the first 10 days of intensive care unit (ICU) admission. For patients with culture-proven infections, we extracted plasma DNA, prepared double-stranded DNA (dsDNA) and ssDNA sequencing libraries, and applied size selection to exclude fragments >110 bp. Following sequencing, we performed taxonomic classification, and evaluated differences in mDNA fractions and in sensitivity for pathogen detection (compared to background noise). We analyzed 46 plasma samples from 5 patients who developed culture-proven infections, including 17 samples coincident with positive microbial cultures. Size-selected ssDNA libraries showed the total mDNA fraction 204-fold higher on average than conventional dsDNA libraries (P < 0.0001). However, for pathogen-specific DNA (at the genus level), the highest sensitivity was observed in size-selected dsDNA (82%), compared to dsDNA (41%), ssDNA (71%), and size-selected ssDNA (35%) library preparations. Our results demonstrate that combining ssDNA library preparation together with fragment size selection improves mDNA yield, potentially reducing sequencing requirements. However, at the genus level, this combination also increases background noise, which limits sensitivity for pathogen detection.

Re-evaluation of boundaries of Streptococcus mitis and Streptococcus oralis and demonstration of multiple later synonyms of Streptococcus mitis, Streptococcus oralis and Streptococcus thalassemiae: description of Streptococcus mitis subsp. carlssonii subsp. nov. and emended description of Streptococcus mitis.

The commensal species Streptococcus mitis and Streptococcus oralis are genetically diverse to a degree that challenges traditional definitions of species. This causes automatic identification based on DNA sequences or cellular extract profiles problematic. Based on an initial analysis of 266 genomes, we subjected a subset of 100 representative genomes to detailed phylogenetic, pairwise distance and gene pattern analyses. S. mitis and S. oralis constitute a continuum of clones that are genetically unique. To recognize most isolates as separate species is biologically and practically meaningless. We recommend bending the proposed similarity borders to accommodate the biological reality. Accordingly, we conclude that Streptococcus toyakuensis, Streptococcus chosunensis, Streptococcus gwangjuensis, Streptococcus humanilactis and Streptococcus hohhotensis are later heterotypic synonyms of S. mitis. Type strains of effectively but not validly published 'Streptococcus shenyangsis', 'Streptococcus symci' and 'Streptococcus vulneris' belong in S. mitis. Streptococcus parapneumoniae and Streptococcus nakanonensis are later synonyms of Streptococcus thalassemiae. Streptococcus downii is a later synonym of Streptococcus oralis subsp. dentisani, and the type of 'Streptococcus halitosis' belongs in Streptococcus oralis subsp. tigurinus. The genome sequence of the type of the recently proposed 'Streptococcus bouchesdurhonensis' is based on a mixed culture. Phylogenetic results and the pattern of presence/absence of accessory genes support the distinction of two subspecies of S. mitis, i.e. Sreptococcus mitis subsp. mitis subsp. nov. (type strain is NCTC 12261T) and Sreptococcus mitis subsp. carlssonii subsp. nov. (type strain is SK608=CCUG 55085T=LMG 33510T). The special population structure of the Streptococcus mitis-pneumoniae-pseudopneumoniae-thalassemiae complex renders automated classification of isolates based on average nucleotide identity or digital DNA-DNA hybridization values problematic. As an alternative, for initial taxonomic assignment, we present a whole-genome phylogeny-based method that enables phylogenetic comparison of new isolates in the context of a set of 117 well-characterized reference strains assigned to the Mitis/Sanguinis group.

Longitudinal Outcomes in Pi*MZ Alpha-1 Antitrypsin Deficient Individuals with Tobacco Smoking History from the SPIROMICS Cohort.

Reliable data about the natural history of lung function decline in alpha-1 antitrypsin (AAT) deficient Pi*MZ heterozygotes is largely missing. We hypothesized that, in adults with a tobacco smoking history, lung function deteriorates faster in Pi*MZ compared to Pi*MM genotype. We identified 1856 Pi*MM and 79 Pi*MZ participants with ≥20 pack-years tobacco smoking history from the SPIROMICS cohort by DNA sequencing and followed them over a median of 4.8 years, comparing radiographic and clinical characteristics between the two groups over time using regression models. Adjusted for age, sex, race, smoking pack-year history and smoking status, Pi*MZ participants had a lower baseline percent-predicted FEV1 (65.4% vs. 75.1%, difference 95% CI: -15.4%, -3.9%), more radiographic emphysema (<-950HU%: 12.9% vs. 7.8%, difference 95% CI: 2.8%, 7.5%) and non-significantly lower lung density. In the longitudinal analysis, the FEV1 annual rate of decline was similar in both groups over the course of the study (-34.5mL/year vs. -34.6mL/year for Pi*MZ and Pi*MM, difference 95%CI: -16.9,17.1mL/year). There were no significant differences between Pi*MZ and Pi*MM individuals in the annualized change in lung density, emphysema, patient-reported outcomes, exacerbations or survival. The proportion with faster FEV1 decline (annual loss ≥40mL) was similar in Pi*MZ and Pi*MM groups. In both groups, faster FEV1 decline was associated with more air trapping and small airway disease at baseline. In Pi*MZ only, faster decline was associated with higher blood eosinophil counts (310 vs. 220 cells/µL, difference 95% CI: 30, 140 cells/µL). In the subgroup analysis limited to a small number of, currently smoking participants, no significant differences in longitudinal outcomes were found. Despite a lower FEV1 and more emphysema at enrollment, the longitudinal analysis did not demonstrate significantly greater lung function decline or lung density loss in Pi*MZ compared to Pi*MM participants with tobacco smoking history. Limited sample size and duration of longitudinal follow up constrain generalizability of our findings, thus prohibiting the conclusion that longitudinal trajectories did not differ between these groups. However, our results may suggest that earlier life events could be responsible for more extensive lung disease at enrollment in Pi*MZ compared to Pi*MM tobacco-exposed individuals.

Quantitative Microbial Cell-Free DNA Sequencing from Plasma: A Potential Biomarker for the Diagnosis of Staphylococcal Infection of Cardiac Implantable Electronic Devices.

Staphylococcus aureus bacteremia in patients with cardiac implantable electronic devices (CIED) is often associated with infective endocarditis (CIED-IE). The CIED-IE diagnosis is syndromic. Diagnostic uncertainty is common. Frequently, these patients are classified possible CIED-IE, resulting in guideline non-compliant treatment and heterogeneous outcomes. Improved outcomes require accurate diagnoses. In these patients, we evaluated whether metagenomic sequencing of microbial cell-free DNA (mcfDNA) in serial plasma specimens could improve diagnostic precision. We studied 16 patients with staphylococcal bacteremia who were classified definite or possible CIED-IE and recommended for device removal, if there was a positive blood culture within 7 days and no concurrent deep infection. Plasma specimens obtained at consent, before extraction, and during 96 hours after extraction underwent metagenomic sequencing and quantification of staphylococcal mcfDNA. In 10 of 11 definite CIED-IE patients, mcfDNA persisted during antibiotic therapy for prolonged durations (median 11 days, IQR 7.5 days [7.5,15]). In these cases, mcfDNA concentration in plasma obtained early after lead extraction increased significantly and thereafter decreased rapidly. In 5 cases of possible CIED-IE, mcfDNA was undetectable after 6 days (IQR 2 days [5.5,7.5]) of antibiotic therapy and remained undetectable after CIED extraction. These mcfDNA patterns differ significantly (p=0.001), suggesting two distinct patient populations: one with definite CIED-IE and one without lead infection. If confirmed, these mcfDNA patterns can serve as biomarkers, together with clinical features, to improve precision in diagnosing or rejecting S. aureus CIED-IE. Strategically timed mcfDNA testing before and after CIED extraction may aid in planning therapy.

Arachnandra, a new monotypic genus in Tillandsioideae (Bromeliaceae) from the Atlantic Forest in Brazil

Arachnandra, a new monotypic genus of Bromeliaceae subtribe Vrieseinae, is described to accommodate a single intriguing species, Arachnandra drepanocarpa, formerly placed in either Tillandsia or Vriesea. This new genus is ecologically and morphologically distinct from the remaining genera of subfamily Tillandsioideae in its unique combination of characters including the lanceolate-acuminate sepals, petals distinctly appendaged, corolla with an open, fan blade-like shape, stamens prostrate on the corolla, pollen sulcus broad, smooth, without prominent ornamental elements, margins moderately distinct, stigma of the conduplicate-patent type, and seeds with a distinctly long basal appendaged and an inconspicuous apical one. Phylogenetic DNA sequence analyses indicate that the individuals of A. drepanocarpa form a highly supported monophyletic group in sister position to Vriesea s.str., with Vriesea and Arachnandra being sister to Stigmatodon, and all three being sister to the clade containing Alcantarea and Waltillia.

Altered KEGG pathways are associated with different immunologic responses to antiretroviral therapy in HIV-infected men who have sex with men

BackgroundThe association between Kyoto Encyclopedia of Genes and Genomes (KEGG) metabolic pathways and immunologic non-response among people living with HIV (PLHIV) on antiretroviral therapy (ART) is not well documented. This study aimed to characterize KEGG metabolic pathways among HIV-infected men who have sex with men (MSM) with different immunologic responses.MethodsWe recruited HIV-uninfected MSM (healthy controls, HC) and HIV-infected MSM on ART > 24 months in Guangzhou, June-October 2021. HIV-infected MSM with HIV viral load < 20 copies/mL were grouped into poor immunological responders (PIR) (CD4 + T cell count < 350 cells/µL) and good immunological responders (GIR) (CD4 + T cell count ≥ 350 cells/µL). Stool samples were collected. Bacterial 16S ribosomal DNA sequencing was performed on stool samples, and KEGG metabolic pathways of gut microbiota were imputed from 16S rDNA sequences.ResultsA total of 51 HC, 41 PIR, and 56 GIR were enrolled. The median time since HIV diagnosis for PIR and GIR was 4.6 and 4.2 years. KEGG metabolic pathways were significantly different among HC, PIR, and GIR groups. Retrograde endocannabinoid signaling, pantothenate and CoA biosynthesis, bile secretion, non-homologous end-joining, and gastric cancer were the top five important KEGG metabolic pathways associated with the immunologic responses. Specifically, retrograde endocannabinoid signaling performed best in discriminating PIR and GIR.ConclusionsThis study presented a comprehensive landscape of KEGG metabolic pathways in PLHIV with different treatment outcomes. These results suggest the potential of KEGG metabolic pathways as novel disease progression markers and therapeutic targets.

Non-invasive sampling reveals landscape genetic structure in the threatened ghost bat (Macroderma gigas) in an ore-rich region of Western Australia.

Bat species are expected to exhibit low genetic structuring due to their high mobility. Thus, habitat connectivity is important to maintain gene flow and genetic diversity to retain evolutionary potential. The ghost bat (Macroderma gigas) is a large carnivorous bat endemic to Australia. Listed as Vulnerable, the species has a disjunct distribution across northern Australia and is patchily distributed at local scales due to limited roost habitat availability and anthropogenic impacts. Here, we survey the genetic diversity and structure of M. gigas in the isolated, arid Pilbara bioregion in Western Australia, primarily using non-invasively collected faecal DNA samples obtained from roosts. Faecal and tissue samples, representing 399 individuals, were genotyped using an optimised autosomal marker panel, with a subset also being sequenced at the mitochondrial D-Loop region to investigate historical gene flow. Spatially-explicit Bayesian clustering analyses of autosomal markers revealed low genetic structure and high levels of gene flow amongst the two Pilbara subregions, with some further structuring evident within the Hamersley Ranges. Mitochondrial DNA sequencing showed strong geographic structuring of haplotypes between the subpopulations, with only a small number of shared haplotypes indicating low levels of maternal gene flow. Such patterns across the two marker types are consistent with maternal philopatry and male-mediated gene flow that has previously been described for this species. Conservation actions for the ghost bat in the Pilbara should therefore recognise maintenance of connectivity between roosts and subregions is important to maintain gene flow for this threatened species in the face of anthropogenic threats.

First Report of Plectosphaerella pauciseptata Associated with Tomato Root Rot Disease in China.

Tomato (Solanum lycopersicum) is widely grown worldwide, ranking first among vegetable crops. Root diseases of tomatoes can cause serious yield losses. In June 2023 and 2024, tomato root rot symptoms were observed in the greenhouse with 70%-90% incidence approximate number of plants (N=210) in Beizhen City (121°47 ' 30 ''E, 41°35' 45 ''N), Liaoning Province, China. In 2024, the same symptoms were observed in the greenhouse in Shunyi City (116°39 ' 41 ''E, 40°09' 00 ''N), Beijing Province. Initially tomato leaves turned yellow, followed by wilting and withering of the entire plant. Roots and stems of the plants exhibited brown necrosis. Infected root tissues from six individual plants in each of two distinct regions were cut into 24 small fragments (5 × 5 mm) at the interface between healthy and symptomatic root tissue and surface sterilized with 2% NaOCl for 2 min followed by 75% ethanol for 30 s, rinsed five times with sterile water, and placed on potato dextrose agar (PDA). After 3-6 days of incubation at 25℃, hyphal tips from the edges of colonies were transferred to new PDA plates. Nine morphologically similar fungal isolates (JZB3540002-JZB3540010) were obtained from diseased samples. Six days old colonies on PDA were dense, mycelium appressed, white and the reverse was white to light yellow-brown. Conidiophores were straight or flexuous, unbranched, smooth, and had an elliptical apex. Conidia were elliptical, apex rounded, aseptate, and multi-guttulate. Conidia were 6 to 12 × 2 to 5 µm (average 7.70 × 2.95 µm, n=30). Following 14 days of incubation, colonies were hyaline with white mucilaginous masses, and aerial hyphae appeared at the center of the colony (Carlucci et al. 2012; Usami et al. 2012; Yang et al. 2021). For molecular phylogenetic analysis, genomic DNA was extracted from the nine isolates, and the internal transcribed spacer (ITS) region, large subunit 28S rDNA (LSU) and translation elongation factor 1 alpha (TEF-1alpha) gene were amplified using ITS1/ITS4 (White et al. 1990), LROR/LR5 (Vilgalys and Hester 1990), and EF-688F/EF-1251R (Alves et al. 2008) primers respectively. BLAST results indicated that the ITS, LSU, and TEF sequences showed 98 to 100% identity with Plectosphaerella pauciseptata type specimens sequences at NCBI (GenBank KY399823, KY662250, and KY421321). Maximum likelihood (ML) inference phylogenetic tree was constructed to confirm the identity of the nine strains. ML tree reveals that the nine strains clustered with Plectosphaerella pauciseptata with 100% bootstrap support value. Sequence data were deposited in NCBI GenBank with accession numbers PP716848 and PQ621101-PQ621108 (ITS), PP717077 and PQ621369-PQ621376 (LSU), and PQ629447-PQ629455 (TEF). To confirm pathogenicity, roots of 18 three-leaf-stage cultivar 'Ying fen 8' tomato seedlings were wounded and dipped in 50 ml conidial suspension (107 conidia/ml) or sterile water (control) for 30 min. Tomato seedlings were transplanted into new pots and placed in the greenhouse at 22℃(night)/25℃ (day) with natural daylight. After 25 days, inoculated plants had symptoms similar to naturally infected plants, and no symptoms were observed in the control plants. Plectosphaerella pauciseptata was re-isolated from the base of the roots of inoculated tomatoes, and the identification was confirmed based on morphological characteristics and DNA sequence analysis, The experiment was repeated three times with similar results. Plectosphaerella pauciseptata has been reported causing tomato root rot in Italy (Raimondo and Carlucci 2018), however, to our knowledge, this is the first report of P. pauciseptata causing tomato root rot in China. This study provides insight into the root diseases of tomatoes and provides the basis for further investigations to assess and implement effective disease management strategies.

Development of quick and accurate methods for identifying human pathogenic yeast by targeting the phospholipase B gene, topoisomerase II gene, Candida drug resistance gene, and species-specific ITS 2

Development of quick and accurate methods for identifying human pathogenic yeast by targeting the phospholipase B gene, topoisomerase II gene, Candida drug resistance gene, and species-specific ITS 2

Genetic dissection of QTL for important agronomic traits and fine-mapping of qGL4 and qGW6 based on a short-width grain rice CSSL-Z691

Rice chromosome segment substitution lines (CSSLs) are ideal for creating natural variation and dissecting complex quantitative traits. In addition, it builds a bridge for molecular breeding and accurate identification of quantitative trait loci (QTLs). In this study, to construct an indica rice library of single-segment substitution lines (SSSLs) spanning the whole genome, a rice CSSL-Z691 carrying four substitution segments (4.07 Mb of average length) was identified by marker-assisted selection (MAS) from indica restorer line “Jinhui35” in the “Xihui18” genetic background. Compared with large panicle type Xihui18, seed setting ratio, grain width, and 1000-grain weight increased in Z691. In contrast, the number of primary branches, spikelet number per panicle, grain number per panicle, grain length, rate of length to width, and yield per plant decreased in Z691. Then, 11 QTLs were identified in the secondary F2 population from Xihui18/Z691. Again, four QTLs (qGW6, qGL4, qRLW4, and qGWT4) were validated by three SSSLs (S1–S3) developed in F3. In addition, 11 new QTLs were detected by the three SSSLs that were not identified in the F2 population. Moreover, the different QTLs in D1–D3 showed various genetic models. Some QTLs, e.g., qGWT6 (a = 0.96 g) and qGWT7 (a = −0.29 g), displayed independent inheritance, while others exhibited various epistatic interactions. Thus, it is vital to identify different QTLs and their genetic models. Resolving the epistasis effects among different QTLs is crucial for screening QTLs for breeding by design. Finally, qGL4 and qGW6 were fine-mapped to 160- and 240-kb intervals on chromosomes 4 and 6, and two candidate genes were determined by DNA sequencing. These results provide valuable genetic and breeding materials for cloning qGL4 and qGW6 and for future molecular breeding by design.

First Report of Brown Stripe Disease Caused by Paracidovorax oryzae on Rice in Guangxi (China)

Rice, being the most important food crop in China, is also the world's largest producer and consumer, accounting for 30% of global production and consumption. In 2023, brown stripes were observed on the leaf sheaths of diseased rice seedlings in a Jingxi rice field (22°51'N, 105°56'E) in Guangxi, China. The investigated area of rice fields, which was about 1.5 hectares, had an estimated disease incidence of 15 ± 5%. Leaf samples exhibiting symptoms of suspected brown stripe disease was collected and stored at 4 °C for bacterial isolation. The junction margins of lesions were surface-sterilized with 75% ethanol-saturated cotton swabs, and sterilized scissors were used to excise leaf sections (5 mm × 5 mm) from the diseased-healthy tissue interfaces. Within a sterile environment, these leaf fragments were soaked in 75% ethanol for three minutes, then ground and cultured on KB medium at a temperature of 28 °C for two days. A single colony (strain JX258) was then inoculated into sterile KB medium and incubated at 28 °C with a rotation speed of 220 rpm for 15 hours to obtain a bacterial suspension. Universal primers (27F/1492R) (Kinoshita et al., 2021) were used to amplify the 16S rRNA gene from strain JX258. A 1,394 bp fragment (GenBank PQ465223) amplified from strain JX258 was obtained and showed 97.80% sequence identity with Paracidovorax oryzae ATCC 19882 (Li, X et al. 2017). Specific primers (Aaaf3/ Aaar2, Aaaf3/ Aaar2) (Osdaghi E. 2023) successfully confirmed that this particular strain is indeed responsible for causing brown stripe disease in rice plants. Rice seeds were soaked in a bacterial suspension (1 × 10^6 CFU·mL⁻¹) for two days before undergoing germination followed by sowing in perlite under moist conditions (Supplemental Figures 1A-D). Brown lesions developed on leaves by 7 days post-inoculation (dpi), whereas mock-inoculated controls (10 mM MgCl₂) remained symptom-free. The seedlings soaked in a suspension of strain JX258 exhibited significantly reduced viability, germination, height, and growth rate, ultimately leading to seed mortality. Moreover, healthy rice leaves that were treated with a bacterial suspension (1 × 10^4 CFU·mL⁻¹) via spray inoculation, resulted in the manifestation of brown rice stripe disease symptoms within 7 days post-inoculation, P. oryzae was reisolated from the infected rice leaves, similar to those observed in the field, fulfilling Koch’s postulates (Supplemental Figures 1E-F). The whole genome of JX258 was sequenced using the Sanger method, assembled, and analyzed to identify the strain at the species level. Whole-genome sequencing of JX258 (GenBank JBHJCP000000000.1) revealed 91.20% digital DNA-DNA hybridization (dDDH) and 98.14% average nucleotide identity (ANI) with P. oryzae ATCC 19882 (Meier-Kolthoff et al., 2013; Yoon et al., 2017). In the Biolog Microbial Identification System, Version 4.2 (Biolog Inc., Hayward, CA), JX258 showed a similarity with a match probability of 73% to Paracidovorax avenae. The utilization of the unique carbon source on BIOLOG GEN III reagent strip by JX258 is shown in Supplemental Table 1. The pathogen was identified as Paracidovorax oryzae based on technological means of pathogenicity assessment, biochemical analysis, phylogenetic analysis (Supplemental Figure 1G), and molecular characterization. To our knowledge, this is the first report of Paracidovorax oryzae causing brown stripe disease on rice in Guangxi, China. Therefore, phytosanitary measures have to be implemented to prevent the introduction and spread of the pathogen in other areas.

Application of rapid clinical exome sequencing technology in the diagnosis of critically ill pediatric patients with suspected genetic diseases

PurposeThis study evaluates the efficacy of rapid clinical exome sequencing (CES) and mitochondrial DNA (mtDNA) sequencing for diagnosing genetic disorders in critically ill pediatric patients.MethodsA multi-centre investigation was conducted, enrolling critically ill pediatric patients suspected of having genetic disorders from March 2019 to December 2020. Peripheral blood samples from patients and their parents were analyzed using CES (proband-parent) and mtDNA sequencing (proband-mother) based on Next-Generation Sequencing (NGS) technology.ResultsThe study included 44 pediatric patients (24 males, 20 females) with a median age of 27 days. The median turnaround time for genetic tests was 9.5 days. Genetic disorders were diagnosed in 25 patients (56.8%): 5 with chromosome microduplication/deletion syndromes (11.3%), 1 with UPD-related disease (2.3%), and 19 with monogenic diseases (43.2%). De novo variants were identified in nine patients (36.0%). A neonate was diagnosed with two genetic disorders due to a homozygous SLC25A20 variant and an MT-TL1 gene variation.ConclusionRapid genetic diagnosis is crucial for critically ill pediatric patients with suspected genetic disorders. CES and mtDNA sequencing offer precise and timely results, guiding treatment and reducing mortality and disability, making them suitable primary diagnostic tools.

Single nucleotide polymorphisms: rs833061, rs699947, and rs35569394, and the expression of the vascular endothelial growth factor gene in Moroccan patients with lung cancer

Aim: Angiogenesis is a universal hallmark of all cancers involving a variety of proteins including vascular endothelial growth factor (VEGF). Extensive studies have explored the potential implications of single nucleotide polymorphisms (SNPs) within VEGF-A in lung cancer (LC) susceptibility, tumor growth, and their effect on the gene expression level. Accordingly, we have planned in the present study to evaluate the prevalence of the -460T/C (rs833061), the -2578C/A (rs699947), and the -2549I/D (rs35569394) SNPs and their association with clinicopathological parameters and to assess their impact on the expression of VEGF-A, VEGFR-1, and VEGFR-2 to be used in the accurate management of LC in Morocco. Methods: A total of 60 fresh biopsies were collected from patients with primary LC and were subjected to polymerase chain reaction (PCR)-DNA sequencing of VEGF-A to detect -460T/C (rs833061), -2578C/A (rs699947), and -2549I/D (rs35569394) SNPs. Reverse transcription (RT)-PCR was used to evaluate VEGF-A, VEGFR-1, and VEGFR-2 expression levels. Results: Sequencing analysis revealed the occurrence of -460T/C, -2578C/A, and -2549I/D polymorphisms with different frequencies. VEGF-2549I/D polymorphism was associated with cancer staging for both genotypes and alleles distributions (p &lt; 0.05). Overall, gene expression analysis revealed an overexpression of VEGF-A, VEGFR-1, and VEGFR-2. The expression of VEGFR-1 and VEGFR-2 was significantly associated with histological types (p = 0.0114). Of note, no significant correlation was obtained between VEGF-A expression and VEGF-A gene polymorphisms (p &gt; 0.05). Conclusions: This study is very informative providing the first insight into polymorphisms and expression of VEGF ligand and its receptors in LC patients from Morocco. Globally, -2549I/D SNP and VEGFR-1 and VEGFR-2 expressions appear to be promising prognostic biomarkers and are likely potential keys for better management of LC.

SNPeBoT: a tool for predicting transcription factor allele specific binding

BackgroundMutations in non-coding regulatory regions of DNA may lead to disease through the disruption of transcription factor binding. However, our understanding of binding patterns of transcription factors and the effects that changes to their binding sites have on their action remains limited.SummaryTo address this issue we trained a Deep learning model to predict the effects of Single Nucleotide Polymorphisms (SNP) on transcription factor binding. Allele specific binding (ASB) data from Chromatin Immunoprecipitation sequencing (ChIP-seq) experiments were paired with high sequence-identity DNA binding Domains assessed in Protein Binding Microarray (PBM) experiments. For each transcription factor a paired DNA binding Domain was selected from which we derived E-score profiles for reference and alternate DNA sequences of ASB events. A Convolutional Neural Network (CNN) was trained to predict whether these profiles were indicative of ASB gain/loss or no change in binding. 18211 E-score profiles from 113 transcription factors were split into train, validation and test data. We compared the performance of the trained model with other available platforms for predicting the effect of SNP on transcription factor binding. Our model demonstrated increased accuracy and ASB recall in comparison to the best scoring benchmark tools.ConclusionIn this paper we present our model SNPeBoT (Single Nucleotide Polymorphism effect on Binding of Transcription Factors) in its standalone and web server form. The increased recovery and prediction accuracy of allele specific binding events could prove useful in discovering non-coding mutations relevant to disease.

Phylogenetics, diversification, and biogeography of Azorella (Apiaceae), with a special focus on the Australasian clades

Abstract To expand on prior evolutionary studies conducted on the genus Azorella (Apiaceae), we here present analyses based on more intensive sampling from Australasian representatives of the genus, which had been less thoroughly represented in the prior studies, especially species from section Schizeilema, a group largely native to New Zealand. In this study, 144 specimens representing 56 of the 57 recognized species and putatively new species were sampled for nine DNA sequence regions from the plastid genome and two regions from the nuclear genome. The study also provides deeper analyses of biogeography (DEC model selected by BioGeoBears + ModelTest in RASP 4), divergence (BEAST), and diversification (RevBayes) compared to prior work. Our phylogenetic analyses corroborated earlier results regarding the relationships among the ten sections of Azorella and provided stronger evidence for other relationships that had been less well established. Incongruencies between phylogenetic trees based on plastid and nuclear data suggest reticulation events within the group, especially regarding the origin of section Schizeilema and among the subclades within that section, where speciation by hybridization and polyploidy appears common. The two nuclear datasets provide evidence of nonuniform concerted evolution and incongruence among sections Ranunculus, Schizeilema, and Stilbocarpa. These analyses also suggest that the origin and diversification of Azorella has been affected by events related to the cooling and break up of Antarctica during the Oligocene. Since that time, parallel dispersals to and radiations in Australasia (especially New Zealand) and the Andes have occurred, and that diversification rates accelerated during the Pliocene and Pleistocene concurrent with the opening of new high-altitude ecological niches in New Zealand’s Southern Alps and the Andes of South American.

Genomic insights into Mediterranean pepper diversity using ddRADSeq.

This work investigated the genetic diversity and population structure of 99 pepper lines (Capsicum annuum L.), acclimated to Mediterranean climate conditions, using double-digest restriction site-associated DNA sequencing (ddRADSeq). The aims were to understand the genetic relationships among these lines, correlate genetic clusters with botanical classifications, and provide insights into pepper domestication in the region. Obtained were 318.76 million raw sequence reads overall, averaging 3.21 million reads per sample. A total of 8475 high-quality SNPs were identified and used to assess genetic diversity and population structure. Chromosome NC_061113.1 displayed the highest amount and Chromosome NC_061118.1 the fewest of these SNPs, which were not equally spaced around the genome. Heterozygosity measures and a negative inbreeding coefficient point to the great genetic diversity seen, therefore highlighting the genetic health of the population. Different genetic clusters found by phylogenetic study and STRUCTURE analysis can be used in breeding programs to mix desired features from many genetic backgrounds. This work showed how well ddRADSeq generates high-quality SNPs for genomic research on peppers, therefore offering useful molecular tools for genomic selection and marker-assisted selection. The analysis identified significant genetic diversity and distinct genetic clusters which are valuable for breeding programs focused on crop improvement. These findings enhance our understanding of pepper domestication and provide valuable genetic resources for breeding programs aimed at improving pepper varieties.

Epidemiology of Helminthic Infections and Phylogenetic Tree of Strongyloides stercoralis in Rubber Tree Plantation in Lower Northern Part of Thailand

Background: Helminthic infections cause helminthiasis, including infections by Strongyloides stercoralis, a kind of helminths that cause reinfection and lead to severe infections, can be transmitted through the soil. We aimed to identify S. stercoralis and other helminthic infections in rubber tree plantations in Thailand's lower northern regions. The specific goals include assessing prevalence using Formalin Ethyl–acetate Concentration Technique (FECT) and Agar Plate Culture (APC) and constructing S. stercoralis phylogenetic tree. Methods: Overall, 646 fecal samples from rubber plantation workers in five provinces in northern Thailand were examined using FECT and APC under microscope. DNA from larvae confirmed as Strongyloides spp. by Polymerase Chain Reaction (PCR) was sequenced for phylogenetic analysis. The DNA sequences were also submitted to the GenBank database. Results: Prevalence of helminthic infections was 8.82%, with soil transmitted helminths (STH) prevalence at 6.81%; S. stercoralis accounted for 5.41%, with Ascaris lumbricoides at 0.62%, hookworm 0.46%, and T. trichiura 0.31%. PCR analysis successfully amplified the 18S rRNA gene in 26 out of 34 genomic DNAs, indicating a detection rate of 70.59%. Sequencing of these PCR products identified S. stercoralis strains closely related to those reported in the Republic of Lao, Myanmar, and Japan, suggesting genetic diversity within the species. Conclusion: STH prevalence, predominantly S. stercoralis, highlights public health concerns in rubber plantation areas, necessitating enhanced monitoring and intervention strategies. Phylogenetic analysis of S. stercoralis, revealing a close genetic relationship among strains from various Southeast Asian countries, which underscores potential patterns of transmission and evolutionary relationships in the regions.

Molecular confirmation of Cryptosporidium and Cyclospora species in children with acute diarrhoea in Quindio region, Colombia

BackgroundThere are no reports with molecular confirmation of Cryptosporidium spp. and Cyclospora spp. in children consulting the emergency service due to diarrhoea in Colombia.MethodsA descriptive study was performed on 137 children who visited the Hospital San Juan de Dios Emergency Service in Armenia between April 1 and 31, 2022. Questionnaires and sampling were performed to identify parasites in the faecal samples. Fresh preparations were prepared with 1% iodine, and a modified Ziehl-Neelsen stain was used to identify pathogenic intestinal protozoa (Cryptosporidium spp. and Cyclospora spp.). PCR and sequencing of positive samples were performed to confirm infection.ResultsThe prevalence of Cryptosporidium spp. infection in children was 19,7%, and that of Cyclospora spp. was 10,9%. 59,2% of the children with cryptosporidiosis and 66,6% of the children with cyclosporiasis were hospitalized. PCR for Cryptosporidium spp. was positive in six of 28 (21%) samples and for Cyclospora in 11 of 15 (73%) samples. Cyclospora spp. SSU rRNA DNA sequences clustered 10 samples nearest to lineage A, two with lineage B, and one with lineage C.ConclusionsCryptosporidiosis and cyclosporiasis are common in children with acute diarrhoea when consulting emergency services, and their search should be performed systematically.