Genomic DNA Research Articles

The effects of RT-qPCR standards on reproducibility and comparability in monitoring SARS-CoV-2 levels in wastewater

Reverse transcription-quantitative PCR (RT-qPCR) is widely used for monitoring viruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), in wastewater. Various materials, including plasmid DNA, synthetic nucleic acids, PCR amplicons, genomic DNA, and cDNA, are currently used for SARS-CoV-2 quantification by generating standard curves. We assessed three common standards on quantifying SARS-CoV-2 RNA across nine wastewater treatment plants in Finland, as part of the national wastewater surveillance effort. We pairwise compared RT-qPCR results from 148 wastewater samples, using both IDT (#10006625, IDT, USA) and CODEX standards (#SC2-RNAC-1100, CODEX DNA), and 179 samples using both IDT and EURM019 standards (#EURM-019, European Commission, Joint Research Centre) in our assessment. Amongst the tested standards, the CODEX standard consistently yielded more stable results than either the IDT or EURM019 standards. We found that SARS-CoV-2 levels were higher with the IDT standard (4.36 Log10 GC/100 mL) compared to the CODEX standard (4.05 Log10 GC/100 mL). Similarly, quantification using the IDT standard was higher (5.27 Log10 GC/100 mL) than values obtained with the EURM019 (4.81 Log10 GC/100 mL). SARS-CoV-2 RNA quantified with IDT and CODEX standards exhibited stronger concordance (Spearman’s correlation rho median of 0.79) compared to those quantified with IDT and EURM019 standards (rho median of 0.59). This study highlights the significant impact of standard material selection on SARS-CoV-2 RNA quantification, emphasizing the need for harmonization in standard material.

Accurate prenatal diagnosis of facioscapulohumeral muscular dystrophy 1 using nanopore sequencing

BackgroundFacioscapulohumeral muscular dystrophy 1 (FSHD1) is an autosomal dominant muscular disorder mainly caused by the contraction and hypomethylation of the D4Z4 repeat array in chromosome 4q35. Prenatal diagnosis of FSHD1...

First report of ergot caused by Claviceps clavispora in switchgrass (Panicum virgatum L.) in Georgia, USA.

Switchgrass (Panicum virgatum L.) is a North American grass species with biofuel potential. Claviceps spp. is known to infect the florets of various grass species, initially characterized by a sticky honeydew exudate and later as sclerotium replacing the seed in the infected ovary (Tanaka et al 2023). Since 2019, from July to October, switchgrass panicles in Georgia have been observed with honeydew and black sclerotia. The disease was first noted on some of the 285 accessions of the genome-wide association study (GWAS) panels at the University of Georgia's Iron Horse Farm in Watkinsville and Gibbs Farm in Tifton, GA. In Watkinsville, GA, ergot incidence was 5%, 6%, 65% and 54% in 2019, 2020, 2021 and 2022, respectively. Symptomatic panicles with honeydew and sclerotia were collected in 2021 (sample Scl) and 2023 (samples Cla_M and ATH20cl) from Watkinsville, GA. Under microcopy, panicles with honeydew symptoms had mycelium and conidia (9 μm long [range of 5-13 μm] and 4 μm wide [3-5 μm]) consistent with Claviceps spp. (Tooley et al. 2001). Sclerotia were 1.5 mm long (range 1-3 mm). Sclerotia were surface sterilized for 3 minutes in 5% NaOCl, followed by 70% ethanol then rinsed three times in distilled water. Sterilized sclerotia were plated on potato dextrose agar and placed on bench top with a 12-hour day/night cycle at room temperature (22°C) (Singh 1976). After 2 months, the sclerotia produced sterile apothecia. For molecular identification, genomic DNA was extracted from three honeydew samples following the protocol of Doyle and Doyle (1987). The internal transcribed spacer (ITS) region and RNA polymerase second largest subunit (RPB2) gene were amplified using ITS4/ITS5 (White et al. 1990) and 5F2/7CR (Liu et al. 1999) primer sets. The ITS region of ATH20cl, Cla_M, and Scl (GenBank nos. PP546317- PP546319) showed 92.83-97.48% identity to C. clavispora (NR_163506.1). The RPB2 region of ATH20cl, Cla_M, and Scl (GenBank nos. PP573916- PP573918) showed 97.46-97.72% identity to C. clavispora (LT216566.1). The maximum likelihood tree constructed in MEGA-X (Kumar et al. 2018) using concatenated ITS (539 bp) and RPB2 (792 bp) gene sequences from this study and eleven reference sequences from Tanaka et al. (2023), revealed close relatedness of ATH20cl, Cla_M, and Scl to C. clavispora under section Pusillae. The pathogenicity test for samples ATH20cl and Cla_M was conducted in the greenhouse on switchgrass cultivar 'Alamo' grown in injection molded pots containing Sungro professional growing mix. Three replicates plants at reproductive (R3) growth stage were inoculated by immersing panicles in 105 spores/ml suspension for 5 minutes and bagged for 3 days (Tooley et al. 2001). Control plants were immersed in distilled water. Honeydew symptoms and sclerotia appeared within 7- and 90-days post-inoculation, respectively, whereas control plants remained symptom-free. The honeydew collected from the infected Alamo panicles were reconfirmed to produce similar spores to those collected from the field. This is the first report of ergot caused by C. clavispora in switchgrass. Ergot was also observed since 2019 on some accessions from the same GWAS panel planted at the University of Tennessee Plant Sciences Farm in Knoxville, TN. This information will be beneficial to determine the effect of ergot on biomass production and seed quality of switchgrass in the southeastern U.S.

Epidemiological analysis of pediculosis and the distribution of kdr mutation frequency in head lice populations in Torbat Heydarieh city of Khorasan Razavi Province, Northeastern Iran

Head lice infestations are the most prominent ectoparasitic infection in the world, including Iran, particularly among school children. Recently, numerous cases of infestation have been reported in various provinces of the country. This study aimed to investigate the prevalence of head louse infestations and analyze kdr gene sequences in terms of resistance mutations in the Torbat-e Heydarieh, Mahvelat, and Zaveh cities of Khorasan Razavi Province, Northeast Iran. The data related to the epidemiological and demographic history of head lice populations were extracted from their medical records and analyzed in Excel software. After extracting the genomic DNA, the kdr fragment was amplified using specific primers. The sequences were also analyzed using bioinformatics software. The prevalence of head louse infestations was 1.59% and 1.7% during 2016 and 2017, respectively. Sequence analysis revealed the frequency distribution of two kdr haplotypes, I and V, in the study areas. The increase in head louse infestations in recent years and the simultaneous presence of kdr mutations indicate the need for new treatments and monitoring/controlling resistance to head louse insecticides.

Targeted nanopore sequencing using the Flongle device to identify mitochondrial DNA variants

Variants in mitochondrial genomes (mtDNA) can cause various neurological and mitochondrial diseases such as mitochondrial myopathy, encephalopathy, lactic acidosis, stroke-like episodes (MELAS). Given the 16 kb length of mtDNA, continuous sequencing is feasible using long-read sequencing (LRS). Herein, we aimed to show a simple and accessible method for comprehensive mtDNA sequencing with potential diagnostic applications for mitochondrial diseases using the compact and affordable LRS flow cell “Flongle.” Whole mtDNA amplification (WMA) was performed using genomic DNA samples derived from four patients with mitochondrial diseases, followed by LRS using Flongle. We compared these results to those obtained using Cas9 enrichment. Additionally, the accuracy of heteroplasmy rates was assessed by incorporating mtDNA variants at equimolar levels. Finally, mtDNA from 19 patients with Parkinson’s disease (PD) was sequenced using Flongle to identify disease risk-associated variants. mtDNA variants were detected in all four patients with mitochondrial diseases, with results comparable to those obtained from Cas9 enrichment. Heteroplasmy levels were accurately detected (r2 > 0.99) via WMA using Flongle. A reported variant was identified in three patients with PD. In conclusion, Flongle can simplify the traditionally cumbersome and expensive mtDNA sequencing process, offering a streamlined and accessible approach to diagnosing mitochondrial diseases.

Variations of Major Flavonoids, Nutritional Components, and Antioxidant Activities in Mung Beans (Vigna radiate L.) of Different Seed Weights

This study examined the levels of major flavonoids, nutritional components, total secondary metabolite contents, and antioxidant activities in 136 mung bean accessions and statistically analyzed the effect of seed weight difference on each. Vitexin and isovitexin were detected in all the mung bean accessions, with isovitexin being in a higher concentration regardless of seed weight difference. The contents of total protein and total starch were in the ranges of 22.01–28.96 and 32.62–49.03 g/100 g, respectively. Five fatty acids were detected by GC–FID analysis in all mung bean accessions, with linoleic acid being the most dominant (37.96–50.71 g/100 g). Total saponin content (TSC), total phenol content (TPC), DPPH• scavenging activity, ABTS•+ scavenging activity, and ferric reducing antioxidant power (FRAP) showed more than five-fold differences. Analysis of variance supported by multivariate analysis demonstrated that seed weight difference had a significant effect on total starch, all individual fatty acids except for stearic acid and oleic acid, TSC, and all antioxidant activities except for ABTS•+ scavenging activity. On the other hand, vitexin, isovitexin, total protein, total phenol, and total fatty acid contents remained unaffected by seed weight difference. Overall, this study showed the diversity of key flavonoids, nutritional components, total secondary metabolite contents, and antioxidant activities in mung bean genetic materials. Moreover, the study unveiled how seed weight affects the analyzed parameters in mung beans for the first time. These findings could maximize the use of mung beans in food industries and breeding programs as well as lead to more studies in metabolomics and genomics.

Hepatitis B virus entry, assembly, and egress.

SUMMARYHepatitis B virus (HBV) is an important human pathogen that chronically infects approximately 250 million people in the world, resulting in ~1 million deaths annually. This virus is a hepatotropic virus and can cause severe liver diseases including cirrhosis and hepatocellular carcinoma. The entry of HBV into hepatocytes is initiated by the interaction of its envelope proteins with its receptors. This is followed by the delivery of the viral nucleocapsid to the nucleus for the release of its genomic DNA and the transcription of viral RNAs. The assembly of the viral capsid particles may then take place in the nucleus or the cytoplasm and may involve cellular membranes. This is followed by the egress of the virus from infected cells. In recent years, significant research progresses had been made toward understanding the entry, the assembly, and the egress of HBV particles. In this review, we discuss the molecular pathways of these processes and compare them with those used by hepatitis delta virus and hepatitis C virus , two other hepatotropic viruses that are also enveloped. The understanding of these processes will help us to understand how HBV replicates and causes diseases, which will help to improve the treatments for HBV patients.

Comparative genomic analyses of the genus Robertmurraya and proposal of the novel species Robertmurraya mangrovi sp. nov., isolated from mangrove soil.

A Gram-positive, aerobic, motile, rod-shaped bacterial strain, designated 31A1RT, was isolated from the mangrove soil of Xilian village, Zhanjiang, China. Strain 31A1RT thrives at temperatures ranging from 15 to 45°C (optimum at 30°C), pH 6.5-10 (optimum at 8.5), and in the presence of 0-5% (w/v) NaCl (optimum at 1.5%). The strain shares the highest 16S rRNA gene sequence similarity with Robertmurraya crescens (97.24%) and Robertmurraya dakarensis (97.18%). The complete genome of strain 31A1RT spans 4.71 Mbp with a genomic DNA G + C content of 35.9 mol%. The average nucleotide identity and DNA-DNA hybridization values between strain 31A1RT and type strains of other species of the genus Robertmurraya were 71.24-72.11% and 19.90-21.40%, respectively. The amino acid identity values and percentage of conserved proteins ranged from 66.94 to 68.10% and from 58.34 to 61.62%, respectively, aligning with intrageneric cutoff values. The major fatty acids (≥ 5.0%) were iso-C14:0 (5.0%), iso-C15:0 (41.4%), iso-C16:0 (12.6%), C16:1ω7c alcohol (12.2%), and iso-C17:1 ω10c (6.5%). The polar lipids profile was mainly composed of diphosphatidyl glycerol, phosphatidyl glycerol, and phosphatidyl ethanolamine. We also profiled the pan-genome and metabolic features of genomic assemblies of strains belonging to the genus Robertmurraya, which indicated functional capacities and metabolic similarities. Consequently, we propose that strain 31A1RT represents a new species in the genus Robertmurraya, for which the name Robertmurraya mangrovi sp. nov. is proposed, with the type strain being 31A1RT (= GDMCC 1.4378T = JCM 36937T).

First Report of Leaf Blight on Paris polyphylla Caused by Ceratobasidium ramicola in China.

Paris polyphylla var. yunnanensisis a perennial herb with significant medicinal properties in anticancer, anti-inflammatory, antibacterial immunomodulatory and antispasmodic activities (Duan et al. 2018). In April 2022, leaf blight disease emerged in Xiangtan City (Hunan), affecting P. polyphylla plantings over an area of 3×104 m2 (27.904°N, 112.918°E). The disease incidence reached an average of 22% of the plants in the field, with infected plants initially displaying water-soaked chlorosis, followed by dry yellow shrinkage that gradually spread from the leaf tips to the entire plant. To identify the causal agent, 20 leaf lesions (4 mm2) collected from 20 plants were surface sterilized with 75% ethanol for 10 s, 5% NaOCl for 30 s, rinsed in sterile distilled water three times, transferred to potato dextrose agar (PDA) plates with lactic acid (0.125%) , and incubated at 28 °C in the dark. Four isolates (PP21 to PP24) with similar morphologies were obtained and purified by the hyphal-tip method. Colonies on PDA initially appeared white with cottony mycelium, later turning light-yellow on the underside. Septate hyphae were branched at right angles with a small constriction at the departure of the branch point and measured 4.16 to 7.93 µm in diameter. Binucleate cells within the septate hyphae were visualized using Giemsa staining (Servicebio, China). For molecular identification, the rDNA internal transcribed spacer (ITS), the second largest subunit of nuclear DNA-directed RNA polymerase II (RPB2), and ATP synthase subunit 6 (ATP6) were amplified from genomic DNA of the isolates extracted by Fungus Genomic DNA Extraction Kit (Bioflux, China) using primers ITS1/ITS4 (White et al. 1990), bRPB2-6F/bRPB2-7.1R (Matheny 2005; Reeb et al. 2004) and ATP61/ATP62 (Kretzer and Bruns 1999), respectively. The ITS, RBP2 and ATP6 of the four isolates were sequenced and deposited in GenBank. BLASTn search of sequenced ITS (PQ187050, PQ187051, PP728052, PQ187052), RBP2 (PQ202833, PQ202834, PP735921, PQ202835) and ATP6 (PQ202837, PQ202838, PP735922, PQ202839) revealed a >99% identity with the type strain of Ceratobasidium ramicola CBS133.82 (NR138368, DQ301708, and DQ301577). For phylogenetic analysis, concatenated sequences of ITS, RBP2, and ATP6 were employed using the maximum-likelihood method in MEGA-X. Based on the morphological and molecular analyses, the isolates were classified into the C. ramicola clade (Bandoni 1979; Samuels et al. 2012; Jeong et al. 2023). To test the pathogenicity of the isolate PP23, mycelial plugs (5 mm in diameter) were placed directly on 15 healthy leaves from 15 three-year-old plants after puncturing with sterile needles. Sterile PDA plugs served as controls. All plants were kept in a greenhouse with conditions of 25°C, 80% relative humidity and a photoperiod of 12 h. After 5 days, all infected plants developed leaf blight symptoms similar to those described above, whereas the control plants remained asymptomatic. The pathogenicity test was conducted three times. C. ramicolawas re-isolated from all symptomatic leaves and confirmed to be identical to the original isolate based on morphology and nucleotide sequences of ITS, RBP2, and ATP6. While C. ramicola is commonly reported to be isolated from diseased cacao, its pathogenicity to cacao remains unknown (Samuels et al. 2012). To our knowledge, this is the first report of C. ramicola causing leaf blight on P. polyphylla, a medicinal herb with significant economic importance in China.

DUTP pyrophosphatases from hyperthermophilic eubacterium and archaeon: Structural and functional examinations on the suitability for PCR application.

Deoxyuridine triphosphate pyrophosphatase (DUT) suppresses incorporation of uracil into genomic DNA during replication. Thermostable DUTs from hyperthermophilic archaea such as Thermococcus pacificus enhance PCR amplification by preventing misincorporation of dUTP generated by spontaneous deamination of dCTP. However, it is necessary to elucidate whether DUTs do not cause dNTP imbalances during PCR by unwanted side activity. Moreover, it has been unknown what structural features define the thermostability of those DUTs. Here, DUT from a hyperthermophilic eubacterium, Aquifex aeolicus (Aa-DUT), was characterized together with those from T. pacificus (Tp-DUT). Aa-DUT was as thermostable as Tp-DUT up to at least 95°C. The crystal structures of the two thermostable enzymes were determined, which revealed that the structures of Aa-DUT and Tp-DUT resembled those of monofunctional and bifunctional DUTs, respectively. Generally, bifunctional DUTs harbor the dCTP deaminase activity in addition to the DUT activity. However, not only Aa-DUT but also Tp-DUT showed poor activity towards dCTP, indicating both enzymes are monofunctional. We further examined eight types of parameters related to thermostability of protein structure and found that the thermostability of Aa-DUT and Tp-DUT might be accomplished by increased numbers of ion pairs on the protein surface. Finally, we verified that Aa-DUT promoted PCR amplification with Pfu DNA polymerase to the same extent as Tp-DUT. Collectively, we conclude that both DUTs from hyperthermophiles maintain the enzymatic activity at high temperatures without consuming dCTP due to the lack of the deaminate activity.

Towards the genomic sequence code of DNA fragility for machine learning.

Genomic DNA breakages and the subsequent insertion and deletion mutations are important contributors to genome instability and linked diseases. Unlike the research in point mutations, the relationship between DNA sequence context and the propensity for strand breaks remains elusive. Here, by analyzing the differences and commonalities across myriads of genomic breakage datasets, we extract the sequence-linked rules and patterns behind DNA fragility. We show the overall deconvolution of the sequence influence into short-, mid- and long-range effects, and the stressor-dependent differences in defining the range and compositional effects on DNA fragility. We summarize and release our feature compendium as a library that can be seamlessly incorporated into genomic machine learning procedures, where DNA fragility is of concern, and train a generalized DNA fragility model on cancer-associated breakages. Structural variants (SVs) tend to stabilize regions in which they emerge, with the effect most pronounced for pathogenic SVs. In contrast, the effects of chromothripsis are seen across regions less prone to breakages. We find that viral integration may bring genome fragility, particularly for cancer-associated viruses. Overall, this work offers novel insights into the genomic sequence basis of DNA fragility and presents a powerful machine learning resource to further enhance our understanding of genome (in)stability and evolution.

First Report of Alternaria alternata Causing Alternaria Leaf Spot of Black Nightshade in China.

Black nightshade (Solanum nigrum L.) belongs to Solanaceae family, which is distributed all over China and has become one of the malignant weeds in cotton field in Xinjiang (Zhang et al., 2020; Zhang et al., 2021). During July 2016 to August 2023, leaf spot was observed on black nightshade in Hami and Shihezi city, Xinjiang, China. The spots appeared as brown circular or irregular, with some small black dots surrounded with yellow halos. Twenty five black nightshade plants were collected from a cotton field (100 m2) for pathogen isolation in Shihezi, July 2023. Leaf samples (2×2 mm) from the boundary between diseased tissue and healthy tissue, sterilized in 70% ethanol for 45 s, dipped in 2% NaClO for 30 s, rinsed three times with sterile distilled water, placed on potato dextrose agar (PDA) after dried and cultivated at 25°C in the dark for 4 days. Twenty-five fungal isolates were obtained and purified by using the single-spore isolation method, and all isolates were found to be similar in morphology, appeared as dark brown and produced dark brown pigmentation on PDA. Conidiophores were black brown, 8 to 58 × 10 to 20 μm, with 1 to 5 transverse septa and 0 to 3 longitudinal septa, the results similar to those of Alternaria alternata isolated from buckwheat (Li et al., 2021). To confirm this identification, total genomic DNA of the isolates were extracted by CTAB method (Doyle et al., 1987). The internal transcribed spacer (rDNA-ITS), Alternaria major allergen gene (Alt α1) and glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH) of the A. alternata 26 were amplified using the primers ITS1/ITS4 (Glass et al., 1995), Altα1-F/Altα1-R (Li et al., 2024) and gpd-F/gpd-R (5'-CAACGGCTTCGGTCGCAT- TG-3' / 5'-GCCAAGCAGTTGGTTGTG-3'), respectively. The rDNA-ITS, Altα1 and GAPDH gene sequences were deposited in GenBank (KX904867, PP263361, PP263360), and showed 100% identity (rDNA-ITS: 572 out of 609 bp; Alt α1: 514 out of 516 bp; GAPDH: 619 out of 619 bp) to A. alternata (KU179665; MW522975; MK451977), respectively. The multi-gene phylogenetic tree showed that the representative isolate was grouped with A. alternata and identified as A. alternata. Black nightshade was used for pathogenicity assay. Fifteen plants at 3 to 4-leaf stage were selected and sprayed with the conidial suspension (1.0×106 spores/mL) of A. alternata 26 after wound the underside of leaves (2 mm length) with inoculating needle. Five plants were sprayed with sterilized water as control. After inoculation, placed the plants in a plastic box and covered with plastic wrap to keep 80% relative humidity for 3 days at 25℃ in the greenhouse, then removed the plastic box. Symptoms of leaf spots appeared within 12 - 15 days that were similar to the symptoms observed in the field, and the fungal pathogen that was re-isolated from symptomatic leaves was identical to original pathogen on the basis of morphological and molecular analysis to fulfill Koch's postulates. This experiment was repeated three times, and the disease incidence was above 80% after inoculation at each time. To our knowledge, this is the first report of A. alternata causing leaf spot of black nightshade in China. This report will help us to monitoring distribution of the disease and providing theoretical basis for disease prevention and control.

Enhancing CRISPR-Cas-based gene targeting in tomato using a dominant-negative ku80

Abstract The CRISPR-Cas-based gene targeting (GT) method has enabled precise modifications of genomic DNA ranging from single base to several kilobase scales through homologous recombination (HR). In plant somatic cells, canonical nonhomologous end-joining (cNHEJ) is the predominant mechanism for repairing double-stranded breaks (DSBs), thus limiting the HR-mediated GT. In this study, we implemented an approach to shift the repair pathway preference toward HR by using a dominant-negative ku80 mutant protein (KUDN) to disrupt the initiation of cNHEJ. The employment of KUDN conferred a 1.71- to 3.55-fold improvement in GT efficiency at the callus stage. When we screened transformants, there was a more remarkable increase in GT efficiency, ranging from 1.62- to 9.84-fold, at two specific tomato loci, SlHKT1;2 and SlEPSPS1. With practical levels of efficiency, this enhanced KUDN-based GT tool successfully facilitated a 9-bp addition at an additional locus, SlCAB13. These findings provide another promising method for more efficient and precise plant breeding.

Antioxidant effect and inhibitory effect on nitric oxide production of sea mustard fractions from Busan, Korea

We investigated the chemical characteristics and nitric oxide (NO) production and antioxidant effects of sea mustard (Undaria pinnatifida) fractions obtained from Taejongdae in Busan, Korea. Dried sea mustard was extracted using different solvents, and antioxidant effects was evaluated based on the radical scavenging and DNA oxidation assays and the effect on reactive oxygen species (ROS) production. Total phenolic content was significantly higher in the 85% aq. MeOH fraction than that in the other factions. The 85% aq. MeOH fraction was more effective in scavenging free radicals than other fractions, as assessed by 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2.2'-azino-bis (3-ethylbenothiazoline-6-sulfonic acid) diammonium salt radical cation (ABTS) assays (P<0.05). All sea mustard fractions decreased H2O2-induced cellular ROS production compared to the control (P<0.05), however, the 85% aq. MeOH fraction exhibited the greatest inhibitory effect on ROS production among all fractions. Moreover, the 85% aq. MeOH fraction significantly inhibited genomic DNA oxidation. In addition, we measured NO levels to determine the potential anti-inflammatory effects of fractions from sea mustard and observed the highest level of activity in case of the 85% aq. MeOH fraction. These results indicated that the antioxidant effects of sea mustard are associated with its higher phenol content. This study proposes that processed food products supplemented with sea mustard can be developed into functional foods to promote the health of the local populations.

Outbreak investigation of food poisoning attributed to Omphalotus japonicus using polymerase chain reaction‐based sequencing

AbstractObjectiveThis study aims to report and analyse an outbreak of Omphalotus japonicus poisoning following mushroom ingestion in Taiwan, and to employ PCR methods for accurate identification of the toxic mushrooms involved.MethodsA descriptive observational study was conducted involving a family of four who ingested the mushrooms in the mountainous rural area of Yilan County, Taiwan. Data were collected through interviews with the affected individuals and a review of their medical records. Due to the destruction of the mushroom samples, Polymerase Chain Reaction (PCR) methods were utilized for identification. Genomic DNA was extracted from the raw fruiting bodies, and specific primers targeting the internal transcribed spacer region of the nuclear ribosomal DNA were designed for PCR detection.ResultsThree family members (mother, daughter, and son) experienced gastrointestinal symptoms, including nausea, vomiting, abdominal cramps, and diarrhea, approximately 30 min after consuming the mushrooms. Upon admission to the emergency room, they were hydrated and monitored, with laboratory results showing slightly elevated alanine transaminase levels. The affected individuals recovered within 12 h and were discharged in good condition on day 2. PCR analysis of the mushroom sample confirmed the identification of Omphalotus japonicus, known to produce Illudin S and Illudin M, which cause acute gastrointestinal symptoms. This case represents the first documentation of using PCR‐based sequencing to diagnose Omphalotus japonicus poisoning in the English literature.

Development and Evaluation of Real-Time Quantitative PCR Assays for Detection of Phellinus noxius Causing Brown Root Rot Disease.

Brown root rot disease (BRRD) is a highly destructive tree disease. Early diagnosis of BRRD has been challenging because the first symptoms and signs are often observed after extensive tissue colonization. Existing molecular detection methods, all based on the internal transcribed spacer (ITS) region, were developed without testing against global Phellinus noxius isolates, other wood-decay fungi, or host plant tissues. This study aimed to develop SYBR Green real-time quantitative PCR (qPCR) assays for P. noxius. The primer pair Pn_ITS_F/Pn_ITS_R targets the ITS, and the primer pair Pn_NLR_F/Pn_NLR_R targets a P. noxius-unique group of homologous genes identified through a comparative genomics analysis. The homologous genes belong to the nucleotide-binding-oligomerization-domain-like receptor (NLR) superfamily. The new primer pairs and a previous primer pair G1F/G1R were optimized for qPCR conditions and tested for specificity using 61 global P. noxius isolates, 5 other Phellinus species, and 22 non-Phellinus wood-decay fungal species. Although all three primer pairs could detect as little as 100 fg (approximately 2.99 copies) of P. noxius genomic DNA, G1F/G1R had the highest specificity and Pn_NLR_F/Pn_NLR_R had the highest efficiency. To avoid false positives, the cutoff quantification cycle values were determined as 34 for G1F/G1R, 29 for Pn_ITS_F/Pn_ITS_R, and 32 for Pn_NLR_F/Pn_NLR_R. We further validated these qPCR assays using Ficus benjamina seedlings artificially inoculated with P. noxius, six tree species naturally infected by P. noxius, rhizosphere soil, and bulk soil. The newly developed qPCR assays provide sensitive detection and quantification of P. noxius, which is useful for long-term monitoring of BRRD status.

Evaluating different samples & techniques for hr-HPV DNA genotyping to improve the efficiency of risk profiling for oral & cervical cancers in Sikkim, India

Background & objectives Oral and genital HPV infection in men may be a source of cervical diseases in their women partners as well as disease in themselves. This study aimed to evaluate and compare the performance of Hybrid Capture 2 (HC2) in physician-collected cervical samples and qPCR in self-collected urine and oral gargle samples of women and men, respectively, for hr-HPV infection status and genotyping. Methods One thousand and two hundred biological samples were collected from 200 women (urine, oral gargle, and cervical smear) and 200 men (urine and oral gargle) visiting a referral hospital in the remote Himalayan State of Sikkim. The extracted genomic DNA from urine and gargle samples were profiled for hr-HPV genotypes using quantitative polymerase chain reaction (qPCR) and HC2 for cervical samples. Results In women, hr-HPV was detected in 17.5 per cent of cervical samples by HC2, 25.5 per cent of urine, and 7 per cent of gargle samples by qPCR. For men, hr-HPV was detected in 8 per cent urine and 5 per cent gargle samples by qPCR. Among the HPV-positive women, 56 per cent of urine samples and 20 per cent of oral samples showed single-genotype infection, while the remaining had multiple genotypes. Amongst the HPV-positive men, 62.7 per cent of urine samples and 85.7 per cent of oral samples showed single-genotype infection while the remaining had multiple genotypes. Compared to Pap, the area under ROC was good for HC2 (AUC=0.89) and for qPCR (AUC= 0.852). Interpretation & conclusions HC2 for cervical and qPCR-based HPV DNA assay for urine and gargle sample is suitable for risk profiling for cervical cancer (CC) and oral cancer (OC) screening programmes.

Azacytidine treatment affects the methylation pattern of genomic and cell-free DNA in uveal melanoma cell lines

BackgroundUveal melanoma (UM) is the most common primary intraocular tumour in adults, and approximately 50% of patients will develop metastasis. Epigenetic changes are a major factor in cancer progression. We aimed to determine whether methylation profiles could be altered using a DNA methyltransferase (DNMT) inhibitor in UM cell lines.MethodsFour primary and metastatic UM cell lines were treated with azacytidine and analysed for cell proliferation, colony formation, and BAP1 protein expression. Genomic and cell-free (cf)DNA methylation were compared.ResultsIn all cell lines, azacytidine treatment resulted in dose-dependent effects on proliferation, colony formation, and radiosensitivity. Methylation profiling revealed differences in methylation between cell lines according to BAP1 expression. Matched primary and metastatic cell lines showed very similar patterns. Alterations were seen in pathways known to be important in UM progression, such as PI3K/Akt and MAPK signaling, and in pathways involved in cancer progression, such as regulation of stemlike potential, cell motility, and invasion. These changes were maintained in genomic and cell-free DNA.ConclusionsThis data suggests that DNMT inhibitors cause changes in UM cells that are maintained in cfDNA. The results suggest that targeting methylation in UM treatment and monitoring response to treatment using cfDNA methylation could be a valuable tool.

Association of WNT Gene Polymorphism with Frequency of Cytogenetic Disorders under the Action of Ionizing Radiation

The paper presents the results of a study of the association of single nucleotide polymorphisms of the WNT genes with an increased frequency of cytogenetic disorders in the blood lymphocytes of workers at an ionizing radiation facility exposed to long-term radiation exposure at doses of 100–500 mGy.The object of the study was the blood of 95 apparently healthy workers who were subjected to long-term technogenic external exposure to γ-radiation in doses from 100 to 500 mGy in the course of their professional activities. For all examined individuals, a standard cytogenetic analysis of blood lymphocytes was performed. Genomic DNA was isolated from workers’ blood lymphocytes using a “QIAamp DNA Blood mini Kit” (Qiagen, Germany). DNA was genotyped for 116 single nucleotide polymorphisms of the WNT genes using high-density “CytoScan™ HD Array” (Affymetrix, USA) chips (DNA chips). Taking into account the Bonferroni correction, an association of single nucleotide polymorphisms of the WNT genes with a high frequency of circular chromosomes in blood lymphocytes was established, all other types of cytogenetic disorders did not show statistical significance. As a result of the study, a single nucleotide polymorphism of the WNT9B gene rs1530364 was identified, which can be considered as a potential marker of individual radiosensitivity.

Detection of PhoP-mediated colistin resistance in Gram-negative bacteria without mcr genes in human population in the Ho Municipality, Ghana

IntroductionAntimicrobial resistance (AMR) has become a global public health threat, with colistin emerging as a last-resort treatment option for multidrug-resistant Gram-negative infections. However, the emergence of colistin resistance, mediated by mechanisms like mutations in the PhoP gene, raises concerns about the future utility of this antibiotic. This study aimed to determine the prevalence of PhoP-mediated colistin resistance in Gram-negative bacteria isolated from the stool of residents in the Ho Municipality, Ghana. MethodsIn this cross-sectional study, 110 stool samples were collected from June 2021 to December 2022. Gram-negative bacteria were isolated, and colistin susceptibility was determined by broth microdilution. Genomic DNA from resistant isolates was extracted and sequenced using the Nanopore platform to detect the presence of the PhoP gene. ResultsOf the 107 Gram-negative isolates, 57 % were resistant to colistin. The PhoP gene was detected in 61.4 % of the colistin-resistant isolates, with the highest prevalence observed in Proteus mirabilis, Escherichia coli, Pseudomonas aeruginosa, and Klebsiella pneumoniae. ConclusionThe study reveals a high prevalence of PhoP-mediated colistin resistance among Gram-negative bacteria colonizing residents in the Ho Municipality, highlighting the role of the gut microbiota as a reservoir for antibiotic resistance. Continued surveillance and a collaborative One Health approach are crucial to address this growing threat.