DNA Regions Research Articles

DNA Hypomethylation Is One of the Epigenetic Mechanisms Involved in Salt-Stress Priming in Soybean Seedlings.

Salt-stress priming enhances the tolerance of plants against subsequent exposure to a similar stress. Priming-induced transcriptomic reprogramming is mediated by multiple epigenetic mechanisms, the best known of which is histone modifications. However, not much is known about other epigenetic responses. In this study, salt-stress priming resulted in global DNA hypomethylation in the leaves of soybean seedlings. The DNA methyltransferase activities in primed seedlings were reduced, contributing to the overall DNA hypomethylation. Genes associated with the hypomethylated DNA regions in primed seedlings also showed a higher mean level of the active histone mark, histone 3 lysine 4 trimethylation (H3K4me3), and a lower mean level of the repressive histone mark, H3K4me2. Transcriptomic analyses supported that DNA hypomethylation played a role in fine-tuning the chromatin status in primed seedlings to potentiate gene expressions. Motif and transcriptional network analyses revealed that DNA hypomethylation may facilitate the responses mediated by key transcription factors in the abscisic acid (ABA)-dependent pathway. A pre-treatment using a DNA methyltransferase inhibitor, 5-azacytidine, could enhance salt tolerance in non-primed soybean seedlings, similar to the priming effect, suggesting the role of DNA hypomethylation in salt-stress priming. Overall, this research furthers our understanding of the epigenetic mechanisms involved in salt-stress priming in soybean.

Circular oligomeric particles formed by Ros/MucR family members mediate DNA organization in α-proteobacteria.

The transcriptional regulator MucR from Brucella species controls the expression of many genes, including those involved in virulence, by binding AT-rich DNA regions. MucR and its homologs belong to the Ros/MucR family, whose members occur in α-proteobacteria. MucR is a recent addition to the family of histone-like nucleoid structuring (H-NS) proteins. Indeed, despite the lack of sequence homology, MucR bears many functional similarities with H-NS and H-NS-like proteins, structuring the bacterial genome and acting as global regulators of transcription. Here we present an integrated cryogenic electron microscopy(cryo-EM), nuclear magnetic resonance, modeling and biochemical study shedding light on the functional architecture of MucR from Brucella abortus and its homolog Ml5 from Mesorhizobium loti. We show that MucR and Ml5 fold in a circular quaternary assembly, which allows it to bridge and condense DNA by binding AT-rich sequences. Our results show that Ros/MucR family members are a novel type of H-NS-like proteins and, based on previous studies, provide a model connecting nucleoid structure and transcription regulation in α-proteobacteria.

Incorporation of regulatory DNA elements within a viral vector improves recombinant protein expression in plants

Plants have significant potential as recombinant protein expression chassis, as they can produce complex post-translationally modified proteins that are unobtainable using prokaryotic production systems, with almost limitless scalability and substantially reduced costs relative to eukaryotic cell cultures. Transient protein expression reduces the time taken between transformation and recombinant protein extraction and purification, however low protein yields relative to conventional stable expression systems remain a major obstacle. Here, we have assessed the effectiveness of combining several established genetic components, including a promoter, 5’ UTR, 3’ UTR, double terminator, and matrix attachment region, to modify the TMV-based pJL-TRBO expression vector for improved recombinant protein expression in plants. Using enhanced green fluorescent protein (eGFP) as a reporter, we quantified expression using fluorescence imaging in planta together with SDS-PAGE and western blotting and showed that our optimum construct resulted in a significant increase relative to pJL-TRBO-eGFP. This increase was exclusively due to the presence of the additional 5’ UTR. We anticipate that our expression constructs will be a useful tool for high-yield plant recombinant protein production and may serve as a template for further improvements.

Testing the informativeness of Y-STR and mitochondrial DNA control region markers in an attempt to predict ancestry of World War II victims from Slovenian mass grave.

Identification of human remains is a challenge in forensic genetics without relatives or personal items available. In Slovenia, a Konfin II mass grave from the Second World War (WWII) was found, containing skeletal remains of 65 victims. The archival documents detailing victims' information describe 45 persons of which 33 could be considered Germanic and 12 Slavic. This study aims to check for concordance between the victim list and actual victims found by using uniparental markers to differentiate between Slavic and non-Slavic origin by attempting to infer ancestry by analyzing the control region (CR) of mitochondrial DNA (mtDNA) and Y-chromosomal STRs. Diaphyses of femurs were used as a DNA source. Next Generation Sequencing (NGS) technology was used for mtDNA- namely HID Ion Chef™ Instrument, Precision ID mtDNA Control Region Panel, and Ion GeneStudio™ S5 System. For the Y-chromosome, PowerPlex® Y23 System (Promega) kit and SeqStudio™ for human identification (HID) were used. European DNA Profiling mtDNA Population Database (EMPOP) and Y-Chromosome STR Haplotype Reference Database (YHRD) were searched for haplotype matches. Closest haplogroups were predicted using EMPOP, Y-DNA Haplogroup Predictor- NevGen, and Whit Athey's Haplogroup Predictor. Despite mitotypes being more diverse than Y-haplotypes, the Y-haplotypes had more database matches and more unequivocal differentiation between populations. 16 victims could be considered Slavic, 15 non-Slavic, and the remaining 34 had a rather scarce informativeness- either unclear or not providing any match. To address ancestry inference more comprehensively, analysis of autosomal ancestry informative markers as well as expansion on haploid markers will be conducted in future research.

FAN1 Deletion Variant in Basenji Dogs with Fanconi Syndrome.

Background: Fanconi syndrome is a disorder of renal proximal tubule transport characterized by metabolic acidosis, amino aciduria, glucosuria, and phosphaturia. There are acquired and hereditary forms of this disorder. A late-onset form of Fanconi syndrome in Basenjis was first described in 1976 and is now recognized as an inherited disease in these dogs. In part because of the late onset of disease signs, the disorder has not been eradicated from the breed by selective mating. A study was therefore undertaken to identify the molecular genetic basis of the disease so that dogs could be screened prior to breeding in order to avoid generating affected offspring. Methods: Linkage analysis within a large family of Basenjis that included both affected and unaffected individuals was performed to localize the causative variant within the genome. Significant linkage was identified between chromosome 3 (CFA3) makers and the disease phenotype. Fine mapping restricted the region to a 2.7 Mb section of CFA3. A whole genome sequence of a Basenji affected with Fanconi syndrome was generated, and the sequence data were examined for the presence of potentially deleterious homozygous variants within the mapped region. Results: A homozygous 317 bp deletion was identified in the last exon of FAN1 of the proband. 78 Basenjis of known disease status were genotyped for the deletion variant. Among these dogs, there was almost complete concordance between genotype and phenotype. The only exception was one dog that was homozygous for the deletion variant but did not exhibit signs of Fanconi syndrome. Conclusions: These data indicate that the disorder is very likely the result of FAN1 deficiency. The mechanism by which this deficiency causes the disease signs remains to be elucidated. FAN1 has endonuclease and exonuclease activity that catalyzes incisions in regions of double-stranded DNA containing interstrand crosslinks. FAN1 inactivation may cause Fanconi syndrome in Basenjis by sensitization of kidney proximal tubule cells to toxin-mediated DNA crosslinking, resulting in the accumulation of genomic and mitochondrial DNA damage in the kidney. Differential exposure to environmental toxins that promote DNA crosslink formation may explain the wide age-at-onset variability for the disorder in Basenjis.

Abstract I003: Inhibitors of DHX9 RNA helicase as synthetic lethal cancer therapeutics

Abstract Over 100 modifications to RNA are known to occur in human cells, where they play critical roles in many aspects of normal cellular physiology, such as cell fate decisions and terminal differentiation, through effects on RNA biology such as protein and nucleic acid interactions. Our survey of human RNA-modifying enzymes suggests many are cancer essential enzymes with striking synthetic lethal profiles, including the DHX9 helicase. DHX9 is a multifunctional DExH-box RNA helicase which can unwind regions of double-stranded DNA and RNA helices but has a greater propensity for secondary structures such as DNA/RNA hybrids (R-loops) and DNA/RNA G-quadruplexes. DHX9 interacts with and regulates many proteins, including key members of DNA damage repair pathways. We have found that DHX9 knockdown is synthetic lethal in microsatellite high (MSI-H) or defective mismatch repair (dMMR) tumor models. A suite of assays was developed to identify and optimize potent and selective inhibitors of the DHX9 helicase, which recapitulate our findings with genetic tools. Profiling of DHX9 inhibitors across a broad panel of cancer cell lines reveals that tumor cells with mutations in the DNA damage repair genes BRCA1 and/or BRCA2 are also responsive to DHX9 inhibitor treatment in vitro and in vivo. DHX9 inhibition leads to increased RNA/DNA secondary structures such as R-loops and G-quadruplexes, resulting in subsequent DNA damage and increased replication stress, leading to cell cycle arrest and apoptosis. These results suggest that DHX9 inhibitors are a novel treatment modality for patients with defective DNA damage repair pathways such as dMMR and/or BRCA mutations. Citation Format: Serena J Silver. Inhibitors of DHX9 RNA helicase as synthetic lethal cancer therapeutics [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: RNAs as Drivers, Targets, and Therapeutics in Cancer; 2024 Nov 14-17; Bellevue, Washington. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(11_Suppl):Abstract nr I003.

Molecular and morphoanatomical characterization of Urocystis heteropogonis sp. nov.: a novel smut fungus infecting Heteropogon contortus

BackgroundA new species of smut fungus, Urocystis heteropogonis, was discovered infecting Heteropogon contortus in Shawar Valley, Swat district, Khyber Pakhtunkhwa, Pakistan. The study aimed to characterize this fungus based on its morpho-anatomical and molecular features and clarify its phylogenetic position within the genus Urocystis.ResultsUrocystis heteropogonis was identified as a novel species, distinct from other Urocystis species. Morphologically, it is characterized by larger spore balls (14–69 × 11–45 μm) and central spores that are 14–28 × 11–20 μm in size, with each spore containing1–8 central spores. The spore walls measure 0.9–2.5 μm in thickness and the species differs in infection patterns compared to other Urocystis species. Phylogenetic analysis based on the ITS and LSU regions of nuclear ribosomal DNA (nrDNA) further confirmed the novelty of the species, placing it within a distinct clade alongside U. agropyri, U. occulta, U. piptatheri, and U. tritici.ConclusionsThe discovery of Urocystis heteropogonis adds to the diversity of smut fungi infecting grasses and highlights the need for further research into its ecological and agricultural implications. Future studies should focus on the disease’s spread, management, and potential impact on host populations.

AcsS inhibits the hemolytic activity and thermostable direct hemolysin (TDH) gene expression in Vibrio parahaemolyticus.

Vibrio parahaemolyticus produces a key virulent factor known as thermostable direct hemolysin (TDH). TDH exhibits diverse biological activities, including hemolytic activity. The β-type hemolysis observed on Wagatsuma agar due to TDH is recognized as the Kanagawa phenomenon (KP). The tdh2 gene is primarily responsible for TDH production and the associated KP. AcsS was originally identified as an activator of swimming and swarming motility in V. parahaemolyticus. However, the extent of its potential to regulate other cellular pathways remains unclear. In this study, we explored the regulatory effects of AcsS on the hemolytic activity and tdh2 expression in V. parahaemolyticus. The data showed that V. parahaemolyticus hemolytic activity and tdh2 transcription were under the negative control of AcsS. Additionally, in vitro binding assays revealed that His-AcsS could not bind to the regulatory DNA region of tdh2. However, overexpression of AcsS in an Escherichia coli strain suppressed the expression of tdh2. Collectively, these results suggested that AcsS suppresses the hemolytic activity of V. parahaemolyticus through the downregulation of tdh2 transcription. The data enhanced our understanding of the regulatory networks governing tdh2 expression and the roles of AcsS in this bacterium.

Abstract A012: Analytical validation of a cfDNA-based fragmentomic profiling assay for the detection and quantification of 14 HPV types in cervical cancer

Abstract Purpose: Human papillomavirus (HPV) is the primary cause of cervical cancer, one of the most common cancers affecting women globally. Despite advances in prevention and screening, there remains a critical need for reliable, non-invasive assays to detect and monitor HPV-driven cervical cancers. Liquid biopsy offers a promising solution to meet this need. Here, we report the analytical validation of a plasma-based fragmentomic profiling assay designed to detect and characterize circulating tumor tissue modified viral (TTMV)-HPV DNA from 14 HPV types associated with cervical cancer. Methods: A high-complexity, laboratory-developed test was developed that employs 68 primers and 34 probes to target 14 HPV types via droplet digital PCR (ddPCR). A quantitative algorithm was applied to integrate fragment counts to generate a TTMV-HPV DNA Score, reflective of the quantity of circulating tumor-derived HPV DNA. Analytical validation was performed by determining the limit of blank (LOB), limit of quantitation (LOQ), and limit of detection (LOD) for each HPV type. To measure the limits of assay performance, engineered samples were used for reference interval studies. Synthesized target-specific DNA regions of HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59, HPV66, and HPV68 were diluted to varying concentrations to perform assay validation. Results: The assay demonstrated excellent specificity, with LOBs ranging from 0 to 0.07 copies/µL across the 14 HPV types. LODs ranged from 0.20 to 1.02 copies/µL, indicating a high level of sensitivity. The assay also showed high accuracy and precision, with coefficients of variation (CV) below 20% for all HPV types across both intra- and inter-assay precision studies. Regression analysis confirmed strong linearity (R2 > 0.99) across a wide range of analyte concentrations. Conclusion: These results demonstrate that TTMV-HPV DNA assay accurately and reproducibly detects circulating HPV DNA for 14 HPV types commonly associated with cervical cancer, offering a valuable tool for both diagnosis and ongoing surveillance in clinical settings. Future studies will present the clinical validation in order to establish the prognostic utility of the assay in predicting the likelihood of HPV-driven cervical cancer in patients. Citation Format: Sunil Kumar, Joshua Hutcheson, David Conway, Michael Horan, Syandan Chakraborty, Chloe Wiseman, Alicia Gunning, Evgeny Skyrypkin, Cassin Williams, Catherine Del Vecchio Fitz, Piyush B Gupta. Analytical validation of a cfDNA-based fragmentomic profiling assay for the detection and quantification of 14 HPV types in cervical cancer [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr A012.

Targeted Forward Genetics: Saturating Mutational Analyses of Specific Target Loci Within the Genome.

Precise allele replacement by homologous recombination (also known as "gene targeting" or "genome editing") allows scientists to engineer altered DNA sequences, insertions, or deletions at specific locations in the genome. Such reverse genetics provides powerful tools to elucidate the structure and function of regulatory DNA elements, genes, RNAs, and proteins within their natural, endogenous context. Here, we describe in detail the methodology for Targeted Forward Genetics (TFG), which supports population-scale, saturating screens of allele replacements spanning thousands of base pairs at a specific target locus in the genome. The overall approach and detailed protocols, developed for the fission yeast Schizosaccharomyces pombe, are extensible to other organisms in which gene targeting is feasible.

Evolution of 3D Chromatin Folding.

Studies examining the evolution of genomes have focused mainly on sequence conservation. However, the inner working of a cell implies tightly regulated crosstalk between complex gene networks controlled by small dispersed regulatory elements of physically contacting DNA regions. How these different levels of chromatin organization crosstalk in different species underpins the potential for genome evolutionary plasticity. We review the evolution of chromatin organization across the Animal Tree of Life. We introduce general aspects of the mode and tempo of genome evolution to later explore the multiple layers of genome organization. We argue that both genome and chromosome size modulate patterns of chromatin folding and that chromatin interactions facilitate the formation of lineage-specific chromosomal reorganizations, especially in germ cells. Overall, analyzing the mechanistic forces involved in the maintenance of chromatin structure and function of the germ line is critical for understanding genome evolution, maintenance, and inheritance.

Detection of DNA of Leishmania infantum in the brains of dogs without neurological signs in an endemic region for leishmaniasis in the state of Rio Grande do Sul, Brazil.

Canine visceral leishmaniasis (CVL) is a parasitic disease caused by the protozoan Leishmania infantum. Neurological infection occurs due to the parasite's ability to cross the blood-brain barrier. It is known that dogs can remain infected with a subclinical infection for life, potentially acting as reservoirs for L. infantum when bitten by sandflies. In this context, the objective of this study was to evaluate the occurrence of Leishmania spp. in the brains of dogs from the metropolitan region of Rio Grande do Sul, Brazil, without a history of neurological disease but residing in an endemic area for L. infantum. A total of 200 samples, from 2022 to 2023, were evaluated using conventional Polymerase Chain Reaction (PCR) with the primers Leishmini-F GGKAGGGGCGTTCTGC and Leishmini-R STATWTTACACCAACCCC, aiming to amplify a product of 120 base pairs for Leishmania spp. To identify the species, a multiplex PCR was used, differentiating L. braziliensis (127bp), L. amazonensis (100bp), and L. infantum (60bp), with the molecular target being the conserved region of the kinetoplast DNA (kDNA) minicircle, specific to Leishmania spp. Of the 200 samples evaluated, 26.5% (53/200) tested positive in the conventional PCR reaction for Leishmania spp., with the PCR multiplex the only species detected was Leishmania infantum. The average age of the positive animals was 5.08years, with 47.2% being females and 52.8% being males; among these, mixed-breed dogs were the most predominant, representing 43.4% of the total. Clinical signs varied: hepatomegaly in two dogs, pronounced neutrophilic hepatitis in one, splenomegaly in one with lymphoid hyperplasia, and glomerulonephritis and nephritis in two animals. Mild anemia and thrombocytopenia were found in eight, with pale mucous membranes in three, and diffuse alveolar edema in one case. Notable pathological findings included suspected distemper in one animal and lymphoplasmacytic meningitis in another. Histopathological findings revealed alveolar edema and acute renal failure. A third dog exhibited bilateral hydrocephalus and diffuse edema in the brain. Additional changes, such as mild inflammatory infiltrate and slight vacuolar degeneration, were observed in 11.3% of the analyzed brains. There was no clinical suspicion of leishmaniasis in any of the studied cases. Therefore, the detection of L. infantum DNA in the brains of dogs suggests that animals with subclinical infection may play a crucial role in the spread of leishmaniasis, and infection by Leishmania spp. should be considered as a differential diagnosis for neurological disease in endemic areas for the protozoan.

RNA-First Approach Identifies Deep Intronic PHEX Variants in X-Linked Hypophosphatemic Rickets.

Up to 20% of patients with X-linked hypophosphatemic rickets (XLH) have no causative variant identified on routine molecular diagnostic testing. To identify intronic variants causing PHEX mis-splicing in patients with XLH. The metabolic bone clinic of a paediatric orthopedic hospital. Four patients (age 6 to 12 years; 3 girls) with clinically diagnosed XLH and no PHEX variant on routine testing. RNA and DNA sequence analysis of PHEX. Urine-derived cells were cultured, and mRNA was extracted and transcribed to cDNA. PHEX cDNA was amplified by PCR, followed by sequencing of PCR products. Sequencing of PHEX intronic DNA regions was performed to identify variants causing mis-splicing observed on RNA analysis. PHEX mis-splicing was identified in 3 of the 4 participants, and an intronic variant was identified in all 3 cases. In a 12-year-old boy, transcript analysis showed skipping of PHEX exon 13, while sequencing of PHEX intronic regions revealed a de novo 18 bp deletion in intron 13. In a 7-year-old girl, a pseudoexon in PHEX intron 17 was found, associated with a de novo deep intronic variant (c.1768+173A>G) that activated a cryptic splice donor site. Finally, an 84 bp pseudoexon in PHEX intron 21 caused by a recurrent de novo deep intronic variant (c.2147+1197A>G) was identified in an 11-year-old girl. Analysis of RNA from urine-derived cells combined with sequencing of PHEX introns can identify deep intronic variants in individuals with XLH without a detectable PHEX variant in routine exon-centric molecular diagnosis.

SpecGMM: Integrating Spectral Analysis and Gaussian Mixture Models for taxonomic classification and identification of discriminative DNA regions

Abstract Motivation Genomic Signal Processing, which transforms biomolecular sequences into discrete signals for spectral analysis, has provided valuable insights into DNA sequence, structure, and evolution. However, challenges persist with spectral representations of variable-length sequences for tasks like species classification and in interpreting these spectra to identify discriminative DNA regions. Results We introduce SpecGMM, a novel framework that integrates sliding window-based Spectral analysis with a Gaussian Mixture Model to transform variable-length DNA sequences into fixed-dimensional spectral representations for taxonomic classification. SpecGMM’s hyperparameters were selected using a dataset of plant sequences, and applied unchanged across diverse datasets, including mitochondrial DNA, viral and bacterial genome, and 16S rRNA sequences. Across these datasets, SpecGMM outperformed a baseline method, with 9.45% average and 35.55% maximum improvement in test accuracies for a Linear Discriminant classifier. Regarding interpretability, SpecGMM revealed discriminative hypervariable regions in 16S rRNA sequences–particularly V3/V4 for discriminating higher taxa and V2/V3 for lower taxa–corroborating their known classification relevance. SpecGMM’s spectrogram video analysis helped visualize species–specific DNA signatures. SpecGMM thus provides a robust and interpretable method for spectral DNA analysis, opening new avenues in genomic signal processing research. Availability and implementation SpecGMM’s source code is available at https://github.com/BIRDSgroup/SpecGMM.

High level of aneuploidy and recurrent loss of chromosome 11 as relevant features of somatotroph pituitary tumors

BackgroundSomatotroph neuroendocrine pituitary tumors (sPitNET) are a subtype of pituitary tumors that commonly cause acromegaly. Our study aimed to determine the spectrum of DNA copy number abnormalities (CNAs) in sPitNETs and their relevance.MethodsA landscape of CNAs in sPitNETs was determined using combined whole-genome approaches involving low-pass whole genome sequencing and SNP microarrays. Fluorescent in situ hybridization (FISH) was used for microscopic validation of CNAs. The tumors were also subjected to transcriptome and DNA methylation analyses with RNAseq and microarrays, respectively.ResultsWe observed a wide spectrum of cytogenetic changes ranging from multiple deletions, recurrent chromosome 11 loss, stable genomes, to duplication of the majority of the chromosomes. The identified CNAs were confirmed with FISH. sPitNETs with multiple duplications were characterized by intratumoral heterogeneity in chromosome number variation in individual tumor cells, as determined with FISH. These tumors were separate CNA-related sPitNET subtype in clustering analyses with CNA signature specific for whole genome doubling-related etiology. This subtype encompassed GNAS-wild type, mostly densely granulated tumors with favorable expression level of known prognosis-related genes, notably enriched with POUF1/NR5A1-double positive PitNETs. Chromosomal deletions in sPitNETs are functionally relevant. They occurred in gene-dense DNA regions and were related to genes downregulation and increased DNA methylation in the CpG island and promoter regions in the affected regions. Recurrent loss of chromosome 11 was reflected by lowered MEN1 and AIP. No such unequivocal relevance was found for chromosomal gains. Comparisons of transcriptomes of selected most cytogenetically stable sPitNETs with tumors with recurrent loss of chromosome 11 showed upregulation of processes related to gene dosage compensation mechanism in tumors with deletion. Comparison of stable tumors with those with multiple duplications showed upregulation of processes related to mitotic spindle, DNA repair, and chromatin organization. Both comparisons showed upregulation of the processes related to immune infiltration in cytogenetically stable tumors and deconvolution of DNA methylation data indicated a higher content of specified immune cells and lower tumor purity in these tumors.ConclusionssPitNETs fall into three relevant cytogenetic groups: highly aneuploid tumors characterized by known prognostically favorable features and low aneuploidy tumors including specific subtype with chromosome 11 loss.

Genomic Insights into Drug Abuse: The Role of DNA and Protein-Coding Regions in Addiction and Genetic Mutation

Abstract This paper explores the genetic and epigenetic impacts of drug abuse, focusing on how chronic drug use can induce mutations in DNA, particularly within protein-coding regions, and alter gene expression through mechanisms such as DNA methylation. The study highlights the significant role of these genetic changes in the brain's reward pathways, which contribute to the development and persistence of addiction. Through a comprehensive literature review, the paper identifies key genetic factors associated with addiction susceptibility and the long-term effects of drug-induced epigenetic modifications. A proposed research framework is presented, emphasizing the need for longitudinal studies to track epigenetic changes over time, targeted genomic analysis to identify critical mutations, and the integration of multi-omics approaches to provide a holistic understanding of drug abuse's impact on the genome. The paper also addresses ethical considerations, including the importance of protecting genetic privacy and preventing stigmatization based on genetic findings. The study concludes that advancing our understanding of the genetic and epigenetic mechanisms underlying addiction can lead to more effective, personalized treatment strategies and inform public health policies aimed at mitigating the effects of drug abuse.

Application of DNA Barcoding to Identify Medicinal Plants.

Medicinal plants are valuable resources globally and are used worldwide to maintain health and treat disease; however, the presence of adulteration obstructs their development. DNA barcoding, a technique for species identification by standard DNA regions, facilitates prompt and accurate identification of traditional medicinal plants. The process of DNA barcoding entails six basic steps: 1) processing the medicinal plants, 2) extracting high-quality total DNA from the medicinal plants using centrifugal column method, 3) amplifying target DNA region internal transcribed spacer 2 (ITS2) with universal primers of plants and performing Sanger sequencing, 4) splicing and aligning sequence to obtain the target sequence, 5) matching the barcode sequence against the barcode library for identification, 6) aligning sequence, comparing intraspecific and interspecific variation, constructing phylogenetic neighbor-joining tree. As shown in the results, the universal primer can amplify the target region. Basic Local Alignment Search Tool (BLAST) demonstrates the percentage identified was 100%, and the neighbor-joining tree demonstrates that the splicing sequences were clustered with the A. sinensis OR879715.1 clade, and the clade support value is 100. This protocol provides a reference for applying DNA barcoding technology as an effective method to identify medicinal plants and adulterants.

Non-destructive and rapid identification of yeasts by nearinfrared spectroscopy and machine learning

This study aimed to apply near-infrared (NIR) spectroscopy combined with machine learning techniques to identify yeast strains rapidly and practically, comparing the results with traditional molecular identification methods. Yeasts were isolated from the digestive tracts of aquatic mining insects collected in the extreme north of the Western Amazon (Roraima), Brazil, and preserved through cryopreservation and mineral oil methods. Molecular identification involved PCR amplification and sequencing of ribosomal DNA regions. NIR spectroscopy, coupled with multivariate analysis and machine learning algorithms such as principal component analysis (PCA), hierarchical cluster analysis (HCA), k-nearest neighbor (KNN), and soft independent modeling by class analogy (SIMCA), was used to analyze and classify the yeast samples, accurately identified yeast strains at the genus and species levels, achieving 100% accuracy in both the calibration and validation sets. The results indicate that this method provides a rapid, non-destructive, and environmentally friendly alternative to traditional molecular techniques, making it suitable for real-time, in situ analysis with minimal sample manipulation.

Catalogue of fungi in China 2. Ramaria from northern China

ABSTRACT The species of Ramaria are coralloid-fungi with intricately branched and vividly coloured basidiomata. There are many studies on the genus Ramaria in China, but molecular phylogenetic analysis is rarely employed. In this study, we performed a multigene phylogenetic analysis to identify Ramaria species in Shanxi Province of northern China. Phylogenetic analyses based on four loci, the internal transcribed spacer region of nuclear ribosomal DNA (ITS), the nuclear large subunit ribosomal DNA (nrLSU), ATPase subunit 6 (atp6), and mitochondrial small subunit ribosomal DNA (mtSSU), revealed 13 Ramaria species from our collections. Combined with morphological examinations, 12 of them were identified as new species, plus a new report to China. The thirteen species were described and illustrated in this paper.

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.