Patterns of Change in Nucleotide Diversity Over Gene Length.

Nucleotide diversity at a site is influenced by the relative strengths of neutral and selective population genetic processes. Therefore, attempts to estimate Effective population size based on the diversity of synonymous sites demand a better understanding of their selective constraints. The nucleotide diversity of a gene was previously found to correlate with its length. In this work, I measure nucleotide diversity at synonymous sites and uncover a pattern of low diversity towards the translation initiation site of a gene. The degree of reduction in diversity at the translation initiation site and the length of this region of reduced diversity can be quantified as "Effect Size" and "Effect Length" respectively, using parameters of an asymptotic regression model. Estimates of Effect Length across bacteria covaried with recombination rates as well as with a multitude of translation-associated traits such as the avoidance of mRNA secondary structure around translation initiation site, the number of rRNAs, and relative codon usage of ribosomal genes. Evolutionary simulations under purifying selection reproduce the observed patterns and diversity-length correlation and highlight that selective constraints on the 5'-region of a gene may be more extensive than previously believed. These results have implications for the estimation of effective population size, and relative mutation rates, and for genome scans of genes under positive selection based on "silent-site" diversity.

Detecting m6A at single-molecular resolution via direct RNA sequencing and realistic training data.

Direct RNA sequencing offers the possibility to simultaneously identify canonical bases and epi-transcriptomic modifications in each single RNA molecule. Thus far, the development of computational methods has been hampered by the lack of biologically realistic training data that carries modification labels at molecular resolution. Here, we report on the synthesis of such samples and the development of a bespoke algorithm, mAFiA (m6A Finding Algorithm), that accurately detects single m6A nucleotides in both synthetic RNAs and natural mRNA on single read level. Our approach uncovers distinct modification patterns in single molecules that would appear identical at the ensemble level. Compared to existing methods, mAFiA also demonstrates improved accuracy in measuring site-level m6A stoichiometry in biological samples.

Long extrachromosomal circular DNA identification by fusing sequence-derived features of physicochemical properties and nucleotide distribution patterns.

Long extrachromosomal circular DNA (leccDNA) regulates several biological processes such as genomic instability, gene amplification, and oncogenesis. The identification of leccDNA holds significant importance to investigate its potential associations with cancer, autoimmune, cardiovascular, and neurological diseases. In addition, understanding these associations can provide valuable insights about disease mechanisms and potential therapeutic approaches. Conventionally, wet lab-based methods are utilized to identify leccDNA, which are hindered by the need for prior knowledge, and resource-intensive processes, potentially limiting their broader applicability. To empower the process of leccDNA identification across multiple species, the paper in hand presents the very first computational predictor. The proposed iLEC-DNA predictor makes use of SVM classifier along with sequence-derived nucleotide distribution patterns and physicochemical properties-based features. In addition, the study introduces a set of 12 benchmark leccDNA datasets related to three species, namely Homo sapiens (HM), Arabidopsis Thaliana (AT), and Saccharomyces cerevisiae (SC/YS). It performs large-scale experimentation across 12 benchmark datasets under different experimental settings using the proposed predictor, more than 140 baseline predictors, and 858 encoder ensembles. The proposed predictor outperforms baseline predictors and encoder ensembles across diverse leccDNA datasets by producing average performance values of 81.09%, 62.2% and 81.08% in terms of ACC, MCC and AUC-ROC across all the datasets. The source code of the proposed and baseline predictors is available at https://github.com/FAhtisham/Extrachrosmosomal-DNA-Prediction . To facilitate the scientific community, a web application for leccDNA identification is available at https://sds_genetic_analysis.opendfki.de/iLEC_DNA/.

PPanG: a precision pangenome browser enabling nucleotide-level analysis of genomic variations in individual genomes and their graph-based pangenome.

Graph-based pangenome is gaining more popularity than linear pangenome because it stores more comprehensive information of variations. However, traditional linear genome browser has its own advantages, especially the tremendous resources accumulated historically. With the fast-growing number of individual genomes and their annotations available, the demand for a genome browser to visualize genome annotation for many individuals together with a graph-based pangenome is getting higher and higher. Here we report a new pangenome browser PPanG, a precise pangenome browser enabling nucleotide-level comparison of individual genome annotations together with a graph-based pangenome. Nine rice genomes with annotations were provided by default as potential references, and any individual genome can be selected as the reference. Our pangenome browser provides unprecedented insights on genome variations at different levels from base to gene, and reveals how the structures of a gene could differ for individuals. PPanG can be applied to any species with multiple individual genomes available and it is available at https://cgm.sjtu.edu.cn/PPanG .

Replication of [AT/TA]25 Microsatellite Sequences by Human DNA Polymerase δ Holoenzymes Is Dependent on dNTP and RPA Levels.

Fragile sites are unstable genomic regions that are prone to breakage during stressed DNA replication. Several common fragile sites (CFS) contain A+T-rich regions including perfect [AT/TA] microsatellite repeats that may collapse into hairpins when in single-stranded DNA (ssDNA) form and coincide with chromosomal hotspots for breakage and rearrangements. While many factors contribute to CFS instability, evidence exists for replication stalling within [AT/TA] microsatellite repeats. Currently, it is unknown how stress causes replication stalling within [AT/TA] microsatellite repeats. To investigate this, we utilized FRET to characterize the structures of [AT/TA]25 sequences and also reconstituted lagging strand replication to characterize the progression of pol δ holoenzymes through A+T-rich sequences. The results indicate that [AT/TA]25 sequences adopt hairpins that are unwound by the major ssDNA-binding complex, RPA, and the progression of pol δ holoenzymes through A+T-rich sequences saturated with RPA is dependent on the template sequence and dNTP concentration. Importantly, the effects of RPA on the replication of [AT/TA]25 sequences are dependent on dNTP concentration, whereas the effects of RPA on the replication of A+T-rich, nonstructure-forming sequences are independent of dNTP concentration. Collectively, these results reveal complexities in lagging strand replication and provide novel insights into how [AT/TA] microsatellite repeats contribute to genome instability.

[Non-targeted metabolomics of spleen and liver metabolism in mice treated with Pruni Semen processed with different methods].

Ultra performance liquid chromatography-quadrupole time-of-flight mass spectrometry(UPLC-Q-TOF-MS) was employed to investigate the impacts of Pruni Semen processed with different methods(raw and fried) on the liver and spleen metabolism in mice. A total of 24 male mice were randomly assigned to three groups: raw Pruni Semen group, fried Pruni Semen group, and control(deionized water) group. Mice in the three groups were orally administrated with 0.01 g·mL~(-1) Pruni Semen decoction or deionized water for one week. After that, the liver and spleen tissues were collected, and liquid chromatography-mass spectrometry(LC-MS)-based metabolomic analysis was carried out to investigate the impact of Pruni Semen on the liver and spleen metabolism in mice. Compared with thte control group, the raw Pruni Semen group showed up-regulation of 11 metabolites and down-regulation of 57 metabolites in the spleen(P<0.05), as well as up-regulation of 15 metabolites and down-regulation of 58 metabolites in the liver(P<0.05). The fried Pruni Semen group showed up-regulation of 31 metabolites and down-regulation of 10 metabolites in the spleen(P<0.05), along with up-regulation of 26 metabolites and down-regulation of 61 metabolites in the liver(P<0.05). The differential metabolites identified in the raw Pruni Semen group were primarily associated with alanine, aspartate, and glutamate metabolism, purine metabolism, amino sugar and nucleotide sugar metabolism, and D-glutamine and D-glutamate metabolism. The differential metabolites identified in the fried Pruni Semen group predominantly involved riboflavin metabolism, amino sugar and nucleotide sugar metabolism, purine metabolism, alanine, aspartate, and glutamate metabolism, D-glutamine and D-glutamate metabolism, and glutathione metabolism. The findings suggest that both raw and fried Pruni Semen have the potential to modulate the metabolism of the liver and spleen in mice by influencing the glutamine and glutamate metabolism.

Molecular detection and genotyping of Dientamoeba fragilis and Blastocystis sp. in housefly Musca domestica (Diptera: Muscidae): first report for Dientamoeba fragilis.

Dientamoeba fragilis and Blastocystis sp. are single-celled protozoan parasites of humans and animals. Although they are found in the intestines of healthy hosts, the pathogenicity of them is still unclear. To date, there is no report on D. fragilis and only two studies (without subtyping) on the occurrence of Blastocystis sp. in Musca domestica. In this study, fly samples were collected from livestock farms and their surroundings in the Kirsehir province (Central Anatolia Region) of Türkiye from May to August 2023. A total of 150 microscopically identified M. domestica samples were analyzed for the detection of D. fragilis and Blastocystis sp. molecularly. The overall prevalence of Blastocystis sp. and D. fragilis in M. domestica was determined to be 3.3% (5/150) and 8.0% (12/150), respectively. The SSU rRNA gene sequences of the isolates indicated genotype 1 of D. fragilis. Eleven isolates were identical and represented a single isolate (KAU-Dfrag1). BLAST analysis of KAU-Dfrag1 indicated identity with the isolates reported from humans, cattle, sheep, and budgerigars. The other isolate (KAU-Dfrag2) was polymorphic at two nucleotides from KAU-Dfrag1 and three nucleotides from known genotypes from GenBank and represented a variant of genotype 1. The Blastocystis sp. isolates were found to be identical and represent a single genotype (KAU-Blast1). BLAST analysis revealed that the KAU-Blast1 genotype belonged to the potentially zoonotic subtype 5 (ST5) and exhibited the highest genetic identity (ranging from 99.4 to 99.6%) with pigs, cattle, and sheep from different countries. Our study provides the first data on the molecular prevalence, epidemiology, and genotypic characterization of D. fragilis and Blastocystis sp. in M. domestica.

Identification of the novel allele, HLA-A*30:01:22, in a Saudi individual.

A single nucleotide substitution in exon 5 of HLA-A*30:01:01:01 results in the novel HLA-A*30:01:22 allele.

Microbaculum marinisediminis sp. nov., isolated from marine sediment.

A novel Gram-stain-negative and facultatively anaerobic bacterium, designated A6E488T, was isolated from intertidal sediment collected from Xiaoshi Island, Weihai, PR China (122° 1' E 37° 31' N). Cells of strain A6E488T were rod-shaped with widths of 0.3-0.4 µm and lengths of 1.1-1.8 µm. The optimal growth conditions were determined to be in 1 % (w/v) NaCl, at 37 °C, and at pH 7.0. The predominant fatty acids (≥10 %) were C19 : 0 cyclo ω8c (59.7 %) and summed feature 8 (13.8 %, C18 : 1 ω7c and/or C18 : 1 ω6c). The sole isoprenoid quinone was Q-10. Oxidase activity was negative but catalase activity was positive. The polar lipids consisted of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, one unidentified phospholipid, one unidentified aminolipid, one unidentified glycolipid, and one unidentified lipid. Based on phylogenetic analysis of 16S rRNA gene sequences, strain A6E488T showed the highest sequence similarity to Microbaculum marinum MCCC 1K03192T (97.6 %). The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values between strain A6E488T and M. marinum MCCC 1K03192T did not exceed 78 and 22 %, respectively. These values are below the recommended thresholds of 95 % (ANI) and 70 % (dDDH) for prokaryotic species delineation. On the basis of gene annotation, it was observed that strain A6E488T possesses the capability for thiosulphate oxidation, suggesting that this strain might be important in the sulphur cycle. Based on the results of phenotypic, genotypic, and chemical characterization, strain A6E488T is considered to represent a novel species of the genus Microbaculum, for which the name Microbaculum marinisediminis sp. nov. is proposed. The type strain is A6E488T (=KCTC 92197T=MCCC 1H00516T).

Benzoxazinoids secreted by wheat root weaken the pathogenicity of Fusarium oxysporum f. sp. fabae by inhibiting linoleic acid and nucleotide metabolisms.

KEY MESSAGE: The regulatory action of BXs secreted by wheat on the pathogenicity of FOF causing Fusarium wilt in faba bean were analyzed. DIMBOA and MBOA weakened the pathogenicity of FOF. A large number of pathogenic bacteria in continuous cropping soil infect faba bean plants, leading to the occurrence of wilt disease, which restricts their production. Faba bean-wheat intercropping is often used to alleviate this disease. This study investigates the effect of benzoxazinoids (BXs) secreted by wheat root on the pathogenicity of Fusarium oxysporum f. sp. Fabae (FOF) and underlying molecular mechanisms. The effects of DIMBOA(2,4-dihydroxy-7-methoxy-1,4-benzoxazine-4-one) and MBOA(6-methoxybenzoxazolin-2-one) on the activity of cell-wall-degrading enzymes in FOF(cellulase, pectinase, amylase, and protease), FOF Toxin (fusaric acid, FA) content were investigated through indoor culture experiments. The effect of BXs on the metabolic level of FOF was analyzed by metabonomics to explore the ecological function of benzoxazines intercropping control of Fusarium wilt in faba bean. The results show that the Exogenous addition of DIMBOA and MBOA decreased the activity of plant-cell-wall-degrading enzymes and fusaric acid content and significantly weakened the pathogenicity of FOF. DIMBOA and MBOA significantly inhibited the pathogenicity of FOF, and metabolome analysis showed that DIMBOA and MBOA reduced the pathogenicity of FOF by down-regulating related pathways such as nucleotide metabolism and linoleic acid metabolism, thus effectively controlling the occurrence of Fusarium wilt in faba bean.

Human transmission and outbreaks of feline-like G6 rotavirus revealed with whole-genome analysis of G6P[9] feline rotavirus.

Group A rotaviruses (RVAs) are generally highly species-specific; however, some strains infect across species. Feline RVAs sporadically infect humans, causing gastroenteritis. In 2012 and 2013, rectal swab samples were collected from 61 asymptomatic shelter cats at a public health center in Mie Prefecture, Japan, to investigate the presence of RVA and any association with human infections. The analysis identified G6P[9] strains in three cats and G3P[9] strains in two cats, although no feline RVA sequence data were available for the former. A whole-genome analysis of these G6P[9] strains identified the genotype constellation G6-P[9]-I2-R2-C2-M2-A3-N2-T3-E3-H3. The nucleotide identity among these G6P[9] strains exceeded 99.5% across all 11 gene segments, indicating the circulation of this G6P[9] strain among cats. Notably, strain RVA/Human-wt/JPN/KF17/2010/G6P[9], previously detected in a 3-year-old child with gastroenteritis, shares high nucleotide identity (>98%) with Mie20120017f, the representative G6P[9] strain in this study, across all 11 gene segments, confirming feline RVA infection and symptomatic presentation in this child. The VP7 gene of strain Mie20120017f also shares high nucleotide identity with other sporadically reported G6 RVA strains in humans. This suggests that feline-origin G6 strains as the probable source of these sporadic G6 RVA strains causing gastroenteritis in humans globally. Moreover, a feline-like human G6P[8] strain circulating in Brazil in 2022 was identified, emphasizing the importance of ongoing surveillance to monitor potential global human outbreaks of RVA.

The novel HLA-B*44:48:02 allele characterised by two different sequencing-based typing techniques.

The novel allele HLA-B*44:48:02 differs from HLA-B*44:48:01 by one synonymous nucleotide substitution in exon 3.

The novel HLA-C*07:02:147 allele characterised by two different sequencing-based typing techniques.

The novel allele HLA-C*07:02:147 differs from HLA-C*07:02:01:01 by one synonymous nucleotide substitution in exon 2.

The novel HLA-A*36:14 allele characterised by two different sequencing-based typing techniques.

The novel allele HLA-A*36:14 differs from HLA-A*36:01:01:01 by one non-synonymous nucleotide substitution in exon 4.

Identification of two novel HLA-DRB1 alleles, HLA-DRB1*08:03:13 and HLA-DRB1*08:119.

Compared with HLA-DRB1*08:03:02:01, the alleles HLA-DRB1*08:03:13 and HLA-DRB1*08:119 each show one nucleotide substitution, respectively.

The novel HLA-DRB1*03:210 allele characterised by two different sequencing-based typing techniques.

The novel allele HLA-DRB1*03:210 differs from HLA-DRB1*03:01:01:01 by one non-synonymous nucleotide substitution in exon 3.

Biophysical Characterization of RAS-SOS Complexes by Native Mass Spectrometry.

RAS is regulated by specific guanine nucleotide exchange factors, such as Son of Sevenless (SOS), that activates RAS by facilitating the exchange of inactive, GDP-bound RAS with GTP. The catalytic activity of SOS is known to be allosterically modulated by an active, GTP-bound RAS. However, it remains poorly understood how oncogenic RAS mutants interact with SOS and modulate its activity. In this chapter, we describe the application of native mass spectrometry (MS) to monitor the assembly of the catalytic domain of SOS (SOScat) with RAS and cancer-associated mutants. Results from this approach have led to the discovery of different molecular assemblies and distinct conformers of SOScat engaging KRAS. It was also found that KRASG13D exhibits high affinity for SOScat and is a potent allosteric modulator of its SOScat activity. KRASG13D-GTP can allosterically increase the nucleotide exchange rate of KRAS at the active site by more than twofold compared to the wild-type protein. Furthermore, small-molecule RAS•SOS disruptors fail to dissociate KRASG13D•SOScat complexes, underscoring the need for more potent disruptors targeting oncogenic RAS mutants. Taken together, native MS will be instrumental in better understanding the interaction between oncogenic RAS mutants and SOS, which is of crucial importance for development of improved therapeutics.

Isoptericola luteus sp. nov., a novel yellow bacterium isolated from soil.

An aerobic, non-motile, Gram-stain-positive bacterium, designated strain NEAU-Y5T, was isolated from a soil sample collected from Northeast Agricultural University, Heilongjiang province. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain NEAU-Y5T belonged to the genus and showed high 16S rRNA sequence similarity to Isoptericola variabilis (98.9 %), Isoptericola nanjingensis (98.9 %), Isoptericola cucumis (98.5 %), Isoptericola hypogeus (98.5 %), Isoptericola dokdonensis (98.5 %), Isoptericola jiangsuensis (98.3 %), and Isoptericola halalbus (98.1 %), followed by other members of the genus Isoptericola (<98 %), and phylogenetically clustered with I. dokdonensis and I. jiangsuensis. Strain NEAU-Y5T was found to grow at 4-40 °C (optimum, 28 °C), pH 6.0-12.0 (optimum, pH 7.0), and tolerated 0-6 % NaCl (w/v). The cell-wall peptidoglycan type was l-Lys-d-Asp. The whole-cell hydrolysates contained glucose, galactose, and ribose. The major polar lipids were diphosphatidylglycerol, phosphatidylethanolamine, hydroxyphosphatidylethanolamine, phosphatidylinositol, phosphatidylinositol mannoside, and glucosamine unknown phospholipid. Major fatty acids were anteiso-C15 : 0 and anteiso-C17 : 0. The predominant menaquinone was MK-9(H4). The DNA G+C content was 73.4 mol%. The digital DNA-DNA hybridization and average nucleotide identity values between strain NEAU-Y5T and the type strains of the genus Isoptericola ranged from 18.6 to 23.5 % and from 77.3 to 81.6 %, respectively. Based on morphological, physiological, chemotaxonomic, and phylogenetic data, as well as digital DNA-DNA hybridization and average nucleotide identity values, the novel strain NEAU-Y5T could be differentiated from its closest relatives. Therefore, the strain represents a novel species of the genus Isoptericola, for which the name Isoptericola luteus sp. nov. is proposed. The type strain is NEAU-Y5T (=CCTCC AA 2019087T=DSM 110637T).

Full-spectral genome analysis of natural killer/T cell lymphoma highlights impacts of genome instability in driving its progression.

BACKGROUND: Natural killer/T cell lymphoma (NKTCL) is a clinically and genetically heterogeneous disease with poor prognosis. Genome sequencing and mutation characterization provides a powerful approach for patient stratification, treatment target discovery, and etiology identification. However, previous studies mostly concentrated on base-level mutations in primary NKTCL, whereas the large-scale genomic alterations in NKTCL and the mutational landscapes in relapsed/refractory NKTCL remain largely unexplored. METHODS: Here, we assembled whole-genome sequencing and whole-exome sequencing data from 163 patients with primary or relapsed/refractory NKTCL and compared their somatic mutational landscapes at both nucleotide and structure levels. RESULTS: Our study not only confirmed previously reported common NKTCL mutational targets like STAT3, TP53, and DDX3X but also unveiled several novel high-frequency mutational targets such as PRDM9, DST, and RBMX. In terms of the overall mutational landscape, we observed striking differences between primary and relapsed/refractory NKTCL patient groups, with the latter exhibits higher levels of tumor mutation burden, copy number variants (CNVs), and structural variants (SVs), indicating a strong signal of genomic instability. Complex structural rearrangements such as chromothripsis and focal amplification are also significantly enriched in relapsed/refractory NKTCL patients, exerting a substantial impact on prognosis. Accordingly, we devised a novel molecular subtyping system (i.e., C0-C4) with distinct prognosis by integrating potential driver mutations at both nucleotide and structural levels, which further provides an informative guidance for novel treatments that target these specific driver mutations and genome instability as a whole. CONCLUSIONS: The striking differences underlying the mutational landscapes between the primary and relapsed/refractory NKTCL patients highlight the importance of genomic instability in driving the progression of NKTCL. Our newly proposed molecular subtyping system is valuable in assisting patient stratification and novel treatment design towards a better prognosis in the age of precision medicine.

Global and single-nucleotide resolution detection of 7-methylguanosine in RNA.

RNA modifications, including N-7-methylguanosine (m7G), are pivotal in governing RNA stability and gene expression regulation. The accurate detection of internal m7G modifications is of paramount significance, given recent associations between altered m7G deposition and elevated expression of the methyltransferase METTL1 in various human cancers. The development of robust m7G detection techniques has posed a significant challenge in the field of epitranscriptomics. In this study, we introduce two methodologies for the global and accurate identification of m7G modifications in human RNA. We introduce borohydride reduction sequencing (Bo-Seq), which provides base resolution mapping of m7G modifications. Bo-Seq achieves exceptional performance through the optimization of RNA depurination and scission, involving the strategic use of high concentrations of NaBH4, neutral pH and the addition of 7-methylguanosine monophosphate (m7GMP) during the reducing reaction. Notably, compared to NaBH4-based methods, Bo-Seq enhances the m7G detection performance, and simplifies the detection process, eliminating the necessity for intricate chemical steps and reducing the protocol duration. In addition, we present an antibody-based approach, which enables the assessment of m7G relative levels across RNA molecules and biological samples, however it should be used with caution due to limitations associated with variations in antibody quality between batches. In summary, our novel approaches address the pressing need for reliable and accessible methods to detect RNA m7G methylation in human cells. These advancements hold the potential to catalyse future investigations in the critical field of epitranscriptomics, shedding light on the complex regulatory roles of m7G in gene expression and its implications in cancer biology.