Nucleotide Substitutions Research Articles

Central role of the ramAR locus in the multidrug resistance in ESBL-Enterobacterales.

The aim of this study was to evaluate the proportion of resistance to a temocillin, tigecycline, ciprofloxacin, and chloramphenicol phenotype called t2c2 that resulted from mutations within the ramAR locus among extended-spectrum β-lactamases-Enterobacterales (ESBL-E) isolated in three intensive care units for 3 years in a French university hospital. Two parallel approaches were performed on all 443 ESBL-E included: (i) the minimal inhibitory concentrations of temocillin, tigecycline, ciprofloxacin, and chloramphenicol were determined and (ii) the genomes obtained from the Illumina sequencing platform were analyzed to determine multilocus sequence types, resistomes, and diversity of several tetR-associated genes including ramAR operon. Among the 443 ESBL-E strains included, isolates of Escherichia coli (n = 194), Klebsiella pneumoniae (n = 122), and Enterobacter cloacae complex (Ecc) (n = 127) were found. Thirty-one ESBL-E strains (7%), 16 K. pneumoniae (13.1%), and 15 Ecc (11.8%) presented the t2c2 phenotype in addition to their ESBL profile, whereas no E. coli presented these resistances. The t2c2 phenotype was invariably reversible by the addition of Phe-Arg-β-naphthylamide, indicating a role of resistance-nodulation-division pumps in these observations. Mutations associated with the t2c2 phenotype were restricted to RamR, the ramAR intergenic region (IR), and AcrR. Mutations in RamR consisted of C- or N-terminal deletions and amino acid substitutions inside its DNA-binding domain or within key sites of protein-substrate interactions. The ramAR IR showed nucleotide substitutions involved in the RamR DNA-binding domain. This diversity of sequences suggested that RamR and the ramAR IR represent major genetic events for bacterial antimicrobial resistance.IMPORTANCEMorbimortality caused by infectious diseases is very high among patients hospitalized in intensive care units (ICUs). A part of these outcomes can be explained by antibiotic resistance, which delays the appropriate therapy. The transferable antibiotic resistance gene is a well-known mechanism to explain the high rate of multidrug resistance (MDR) bacteria in ICUs. This study describes the prevalence of chromosomal mutations, which led to additional antibiotic resistance among MDR bacteria. More than 12% of Klebsiella pneumoniae and Enterobacter cloacae complex strains presented mutations within the ramAR locus associated with a dysregulation of an efflux pump called AcrAB-TolC and a porin: OmpF. These dysregulations led to an increase in antibiotic output notably tigecycline, ciprofloxacin, and chloramphenicol associated with a decrease of input for beta-lactam, especially temocillin. Mutations within transcriptional regulators such as ramAR locus played a major role in antibiotic resistance dissemination and need to be further explored.

Structure, Evolution, and Mitochondrial Genome Analysis of Mussel Species (Bivalvia, Mytilidae).

Based on the nucleotide sequences of the mitochondrial genome (mitogenome) of specimens taken from two mussel species (Arcuatula senhousia and Mytilus coruscus), an investigation was performed by means of the complex approaches of the genomics, molecular phylogenetics, and evolutionary genetics. The mitogenome structure of studied mussels, like in many other invertebrates, appears to be much more variable than in vertebrates and includes changing gene order, duplications, and deletions, which were most frequent for tRNA genes; the mussel species' mitogenomes also have variable sizes. The results demonstrate some of the very important properties of protein polypeptides, such as hydrophobicity and its determination by the purine and pyrimidine nucleotide ratio. This fact might indirectly indicate the necessity of purifying natural selection for the support of polypeptide functionality. However, in accordance with the widely accepted and logical concept of natural cutoff selection for organisms living in nature, which explains its action against deleterious nucleotide substitutions in the nonsynonymous codons (mutations) and its holding of the active (effective) macromolecules of the polypeptides in a population, we were unable to get unambiguous evidence in favor of this concept in the current paper. Here, the phylogeny and systematics of mussel species from one of the largest taxons of bivalve mollusks are studied, the family known as Mytilidae. The phylogeny for Mytilidae (order Mytilida), which currently has no consensus in terms of systematics, is reconstructed using a data matrix of 26-27 mitogenomes. Initially, a set of 100 sequences from GenBank were downloaded and checked for their gender: whether they were female (F) or male (M) in origin. Our analysis of the new data confirms the known drastic differences between the F/M mitogenome lines in mussels. Phylogenetic reconstructions of the F-lines were performed using the combined set of genetic markers, reconstructing only protein-coding genes (PCGs), only rRNA + tRNA genes, and all genes. Additionally, the analysis includes the usage of nucleotide sequences composed of other data matrices, such as 20-68 mitogenome sequences. The time of divergence from MRCA, estimated via BEAST2, for Mytilidae is close to 293 Mya, suggesting that they originate in the Silurian Period. From all these data, a consensus for the phylogeny of the subfamily of Mytilinae and its systematics is suggested. In particular, the long-debated argument on mussel systematics was resolved as to whether Mytilidae, and the subfamily of Mytilinae, are monophyletic. The topology signal, which was strongly resolved in this paper and in the literature, has refuted the theory regarding the monophyly of Mytilinae.

Comparative genomic analysis of Brazilian Mesomycoplasma ovipneumoniae strains revealed genomic differences associated with the geographic origin and health status and mutations in the gyrA

Sheep respiratory disease (SRD) is a multifactorial illness commonly affecting sheep. Mesomycoplasma (Mycoplasma) ovipneumoniae is one of the most important etiological agents of SRD and should be better understood, especially in countries where it was recently detected, such as Brazil. Also, the intensive use of quinolones in mycoplasmal infections increases the selective pressure for resistance to this drug class, and no data about antimicrobial resistance in Brazil is available. Therefore, this study aimed to perform a comparative genomic analysis of newly isolated Brazilian M. ovipneumoniae strains, identify point mutations in target genes that may be associated with antibiotic resistance, and perform a phylogenomic analysis of these strains with available genome representatives of M. ovipneumoniae. Glucose-fermenting fried egg-like colonies identified as M. ovipneumoniae were obtained after a culture of tracheobronchial lavage from infected sheep. The genomes were sequenced, de novo assembled and comparatively evaluated. Important putative virulence factors were detected in all isolates: the analysis of the average nucleotide homology of all these genes with the M. ovipneumoniae ATCC 29419 revealed associations between clpB, lgt, tuf, and dnaJ genes and geographic location. In addition, nucleotide substitutions in a few positions of the Quinolone-Resistant Determinant Region of the gyrA gene, including the Ser83Ala, were detected. The phylogenomic analysis showed that the Brazilian isolates belonged to two different clades corresponding to geographic location, and the isolates from São Paulo showed high similarity, which differs from isolates from Rio de Janeiro. This first genomic analysis of the Brazilian M. ovipneumoniae genomes demonstrates strain segregation according to location and health status, reinforcing the importance of continuous surveillance and diagnostics of this bacteria causing sheep respiratory disease in the Brazilian flocks.

First report of occurrence of Babesia gibsoni in captive Indian wolves

Blood samples from fifteen captive Indian wolves (Canis lupus pallipes) maintained at Arignar Anna Zoological Park, Vandalur, Chennai were screened for the presence of Babesia spp., Ehrlichia canis and Trypnosoma evansi DNA by PCR. Out of 15 wolf samples, 3 samples were found positive for Babesia spp. The amplified 18S rRNA gene fragments from 3 wolves were sequenced and confirmed as Babesia gibsoni. A maximum likelihood tree was constructed using the three sequences along with other Babesia spp. sequences derived from GenBank adopting HKY nucleotide substitution model based on the Bayesian Information Criterion. The phylogenetic analysis confirmed that the three sequences were of Babesia gibsoni and highly divergent from Babesia canis, B. vogeli and B. vulpes. This might be a possible spill over event of B. gibsoni from community dogs through blood feeding dog ticks. This is the first report and molecular confirmation of B. gibsoni infection in captive Indian wolves.

Improved specificity and efficacy of base-editing therapies with hybrid guide RNAs.

Phenylketonuria (PKU), hereditary tyrosinemia type 1 (HT1), and mucopolysaccharidosis type 1 (MPSI) are autosomal recessive disorders linked to the phenylalanine hydroxylase (PAH) gene, fumarylacetoacetate hydrolase (FAH) gene, and alpha-L-iduronidase (IDUA) gene, respectively. Potential therapeutic strategies to ameliorate disease include corrective editing of pathogenic variants in the PAH and IDUA genes and, as a variant-agnostic approach, inactivation of the 4-hydroxyphenylpyruvate dioxygenase (HPD) gene, a modifier of HT1, via adenine base editing. Here we evaluated the off-target editing profiles of therapeutic lead guide RNAs (gRNAs) that, when combined with adenine base editors correct the recurrent PAH P281L variant, PAH R408W variant, or IDUA W402X variant or disrupt the HPD gene in human hepatocytes. To mitigate off-target mutagenesis, we systematically screened hybrid gRNAs with DNA nucleotide substitutions. Comprehensive and variant-aware specificity profiling of these hybrid gRNAs reveal dramatically reduced off-target editing and reduced bystander editing. Lastly, in a humanized PAH P281L mouse model, we showed that when formulated in lipid nanoparticles (LNPs) with adenine base editor mRNA, selected hybrid gRNAs revert the PKU phenotype, substantially enhance on-target editing, and reduce bystander editing in vivo. These studies highlight the utility of hybrid gRNAs to improve the safety and efficacy of base-editing therapies.

Individual from the kurgan burial of the XII century in the Middle Oka — experience of complex archaeological and genetic research

For the first time, this paper presents a complex study of the burial of the ancient Russian woman from the classic “Vyatich” mound from the Kremenyie burial site (Moscow region). The mounds and synchronized ground cremation burials are combined at this unique 12th-century burial site. The aim of this research is to examine the historical details of the person from the mound using conventional archaeological, anthropological, and archae-oparasitology methodologies along with modern paleogenetics methods. The burial site is characterized by a general “archaic” rite, manifested in the late preservation of the cremation rite along with the burial. According to anthropological data, an elderly woman, over forty, was buried there. She was identified as a member of the local population by radiogenic strontium isotope (87Sr/86Sr) study. Rich grave content and the fact that the body was buried in the middle of the mound indicate the woman's high social standing in the community. According to ar-chaeoparasitological data, the eating habits of the woman appear to be characterized by the consumption of un-cooked or insufficiently thermally prepared freshwater fish. The complete mtDNA sequence reconstruction indi-cates that it belongs to haplogroup V1a1, one of the Western Eurasian haplogroup V branches. To date, this represents the first case of the V1a1 mitogroup being found in the Russian Plain during ancient times. A repre-sentative of the Danish Viking clan from the Oxford burial site of the XI century was the closest of the ancient samples to the mtDNA of the studied woman, which indicates a genetic relationship on the maternal lineage with the medieval Northwestern European population. Single nucleotide substitution A7299G in the mitogenome of the woman clusters her with present-day Russians from the Belgorod and Pskov regions and shows the continuity of the modern Russian population with the Ancient Russians. Thus, the results of our study demonstrate the private details of the individual as common historical features, which include the ceremonial side of burial and belonging to the maternal genetic lineage preserved in the modern gene pool of the Russian population.

A Coiled-Coil Nucleotide-Binding Domain Leucine-Rich Repeat Receptor Gene MeRPPL1 Plays a Role in the Replication of a Geminivirus in Cassava.

Disease resistance gene (R gene)-encoded nucleotide-binding leucine-rich repeat proteins (NLRs) are critical players in plant host defence mechanisms because of their role as receptors that recognise pathogen effectors and trigger plant effector-triggered immunity (ETI). This study aimed to determine the putative role of a cassava coiled-coil (CC)-NLR (CNL) gene MeRPPL1 (Manes.12G091600) (single allele) located on chromosome 12 in the tolerance or susceptibility to South African cassava mosaic virus (SACMV), one of the causal agents of cassava mosaic disease (CMD). A transient protoplast system was used to knock down the expression of MeRPPL1 by clustered regularly interspaced short palindromic repeats-CRISPR-associated protein 9 (CRISPR-Cas9). The MeRPPL1-targeting CRISPR vectors and/or SACMV DNA A and DNA B infectious clones were used to transfect protoplasts isolated from leaf mesophyll cells from the SACMV-tolerant cassava (Manihot esculenta) cultivar TME3. The CRISPR/Cas9 silencing vector significantly reduced MeRPPL1 expression in protoplasts whether with or without SACMV co-infection. Notably, SACMV DNA A replication was higher in protoplasts with lower MeRPPL1 expression levels than in non-silenced protoplasts. Mutagenesis studies revealed that protoplast co-transfection with CRISPR-MeRPPL1 silencing vector + SACMV and transfection with only SACMV induced nucleotide substitution mutations that led to altered amino acids in the highly conserved MHD motif of the MeRPPL1-translated polypeptide. This may abolish or alter the regulatory role of the MHD motif in controlling R protein activity and could contribute to the increase in SACMV-DNA A accumulation observed in MeRPPL1-silenced protoplasts. The results herein demonstrate for the first time a role for a CNL gene in tolerance to a geminivirus in TME3.

Investigating the Genetic Cause of Ichthyosis Disease in a Family with Exome Sequencing

Background: Ichthyosis is a diverse skin disease characterized by dry skin and itching, particularly affecting the hands. Most forms of this condition are hereditary. Objectives: In this study, we investigated potential new mutations associated with this disease using the new technique of whole-exome sequencing. Methods: In this study, after collecting the patient's blood sample in an EDTA tube and extracting DNA using the salting-out method, next-generation sequencing was performed using the WES method. Following the analysis of the results and identification of the candidate gene, PCR technique and Chromas software were used to confirm the findings. Results: The results indicated the presence of a new variant, NM_001099287: Exon 6:c.C1083A: p.Y361X, in the NIPAL4 gene in a heterozygous form in the parents and a homozygous form in the patient. This variant occurred as a nucleotide substitution in the exon 6 region of the NIPAL4 mRNA sequence. Conclusions: Using the WES technique, it is possible to investigate the presence of new variants related to ichthyosis in a short time and at a low cost.

Multi-dimensional cell-free DNA-based liquid biopsy for sensitive early detection of gastric cancer

BackgroundGastric cancer is the fifth most common cancer type. Most patients are diagnosed at advanced stages with poor prognosis. A non-invasive assay for the detection of early-stage gastric cancer is highly desirable for reducing associated mortality.MethodsWe collected a prospective study cohort of 110 stage I–II gastric cancer patients and 139 non-cancer individuals. We performed whole-genome sequencing with plasma samples and profiled four types of cell-free DNA (cfDNA) characteristics, fragment size pattern, copy number variation, nucleosome coverage pattern, and single nucleotide substitution. With these differential profiles, we developed an ensemble model to detect gastric cancer signals. Further, we validated the assay in an in-house first validation cohort of 73 gastric cancer patients and 94 non-cancer individuals and an independent second validation cohort of 47 gastric cancer patients and 49 non-cancer individuals. Additionally, we evaluated the assay in a hypothetical 100,000 screening population by Monte Carlo simulation.ResultsOur cfDNA-based assay could distinguish early-stage gastric cancer from non-cancer at an AUROC of 0.962 (95% CI: 0.942–0.982) in the study cohort, 0.972 (95% CI: 0.953–0.992) in the first validation cohort and 0.937 (95% CI: 0.890–0.983) in the second validation cohort. The model reached a specificity of 92.1% (128/139) and a sensitivity of 88.2% (97/110) in the study cohort. In the first validation cohort, 91.5% (86/94) of non-cancer individuals and 91.8% (67/73) of gastric cancer patients were correctly identified. In the second validation cohort, 89.8% (44/49) of non-cancer individuals and 87.2% (41/47) of gastric cancer patients were accurately classified.ConclusionsWe introduced a liquid biopsy assay using multiple dimensions of cfDNA characteristics that could accurately identify early-stage gastric cancer from non-cancerous conditions. As a cost-effective non-invasive approach, it may provide population-wide benefits for the early detection of gastric cancer.Trial registrationThis study was registered on ClinicalTrials.gov under the identifier NCT05269056 on March 7, 2022.

Phylogenetic relationships of <i>Mesocestoides</i> vaillant, 1863 tetrathyridia from small mammals of Eastern Russia and Alaska based on <i>18S rRNA</i> gene

A fragment of 875 bp of the 18S rRNA gene was studied in 12 samples of Mesocestoides tetrathyridia from small mammals of 7 species collected in geographically distant localities. Five haplotypes were identified, differing from each other by 27 nucleotide substitutions in 24 sites. It has been found that the 18S rRNA haplotypes belong to two genetically distinct haplogroups. Molecular diversity indices were calculated for each of them. The conducted analysis allowed to suggest the following: 1) Mesocestoides sp. haplogroups A and B belong to two different species, but do not belong to any of the confirmed species of the genus; 2) the deletion of bp 729–747 and the insertion at site 761 of guanine can be regarded as a genetic marker for the species Mesocestoides litteratus.

Mitochondrial genome complexity in Stemona sessilifolia: nanopore sequencing reveals chloroplast gene transfer and DNA rearrangements.

Mitochondria are semi-autonomous organelles in eukaryotic cells with their own genome. Plant mitogenomes differ from animal mitogenomes in size, structure, and repetitive DNA sequences. Despite larger sizes, plant mitogenomes do not have significantly more genes. They exhibit diverse structures due to variations in size, repetitive DNA, recombination frequencies, low gene densities, and reduced nucleotide substitution rates. In this study, we analyzed the mitochondrial genome of Stemona sessilifolia using Nanopore and Illumina sequencing. De-novo assembly and annotation were conducted using Unicycler, Geseq, tRNAscan-SE and BLASTN, followed by codon usage, repeat sequence, RNA-editing, synteny, and phylogenetic analyses. S. sessilifolia's mitogenome consisted of one linear contig and six circular contigs totaling 724,751bp. It had 39 protein-coding genes, 27 tRNA genes, and 3 rRNA genes. Transfer of chloroplast sequences accounted for 13.14% of the mitogenome. Various analyses provided insights into genetic characteristics, evolutionary dynamics, and phylogenetic placement. Further investigations can explore transferred genes' functions and RNA-editing's role in mitochondrial gene expression in S. sessilifolia.

Strength of selection in lung tumors correlates with clinical features better than tumor mutation burden

Single nucleotide substitutions are the most common type of somatic mutations in cancer genome. The goal of this study was to use publicly available somatic mutation data to quantify negative and positive selection in individual lung tumors and test how strength of directional and absolute selection is associated with clinical features. The analysis found a significant variation in strength of selection (both negative and positive) among tumors, with median selection tending to be negative even though tumors with strong positive selection also exist. Strength of selection estimated as the density of missense mutations relative to the density of silent mutations showed only a weak correlation with tumor mutation burden. In the “all histology together” analysis we found that absolute strength of selection was strongly correlated with all clinically relevant features analyzed. In histology-stratified analysis selection was strongest in small cell lung cancer. Selection in adenocarcinoma was somewhat higher compared to squamous cell carcinoma. The study suggests that somatic mutation- based quantifying of directional and absolute selection in individual tumors can be a useful biomarker of tumor aggressiveness.

High-throughput mutational analysis of a methyltransferase ribozyme

Methyltransferase ribozyme 1 (MTR1) is a catalytic RNA that has been isolated from a random RNA pool by in vitro selection. The ribozyme catalyzes site-specific formation of 1-methyl adenosine (m1A) using 6-methyl guanine (m6G) as a methyl group donor. The ribozyme has been extensively characterized by biochemical and structural analyses. Here, we describe high-throughput screening of single point mutants in the catalytic domain of MTR1 and determine their effect on ribozyme activity. Our mutational profiling method successfully assessed the activity of the 141 MTR1 variants tested in each experiment and revealed that the ribozyme is very sensitive to nucleotide substitutions in the catalytic core domain. Our technique can be applied to methyltransferase ribozymes that catalyze formation of different modifications such as 7-methylguanosine (m7G) or 3-methylcytidine (m3C).

Detection of the HLA-C*08:66 allele in a Taiwanese individual.

Two nucleotide substitutions in codon 152 of HLA-C*08:01:01:01 result in a novel allele HLA-C*08:66.

Recognition of the HLA-DQB1*03:96 allele in a Taiwanese individual.

One nucleotide substitution in codon 130 of HLA-DQB1*03:03:02:01 results in a novel allele HLA-DQB1*03:96.

Characterisation of the novel HLA-B*38:01:01:18 allele by next-generation sequencing.

HLA-B*38:01:01:18 differs from the HLA-B*38:01:01:01 allele by one nucleotide substitution in the 5'UTR.

Multi-dimensional cell-free DNA-based liquid biopsy for sensitive detection of head and neck squamous cell carcinomas.

e18054 Background: Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer type worldwide. The mortality from HNSCC is closely linked to stage. However, the majority of HNSCC patients were diagnosed at an advanced stage. Therefore, a non-invasive assay for the early detection of HNSCCs is highly desirable for reducing the associated mortality. Methods: We collected a study cohort of 281 participants including 140 pathologically confirmed with head and neck squamous cell carcinomas (HNSCCs) and 141 with non-cancerous conditions. A plasma sample was collected from each participant along with general health screening examinations before any invasive examinations. We performed low-pass whole-genome sequencing with plasma samples and profiled three types of cell-free DNA (cfDNA) characteristics, fragment size pattern (FSP), copy number variation (CNV), and single nucleotide substitution (SNS). We developed an ensemble model with these differential profiles to identify HNSCC patients from non-cancer individuals. We further collected a validation cohort consisting of 90 HNSCC patients and 95 non-cancer individuals to validate the cfDNA-based assay. Results: The liquid biopsy assay that incorporated three types of cfDNA characteristics was able to identify HNSCC patients from non-cancer individuals at an AUROC of 0.989 in the study cohort and 0.984 in the validation cohort. At a specificity of 95.7% (135/141), it achieved a sensitivity of 95.7% (134/140) in the study cohort. With this threshold (0.580), in the validation cohort, 93.7% (89/95) of non-cancer individuals and 93.3% (84/90) of HNSCC patients were correctly identified. Specifically, the sensitivities in early stage (I&II) HNSCCs were 94.5 % (52/55) and 87.5% (28/32) in the study and validation cohort, respectively. Of note, the inferred probabilities of cancer showed consistent trends to increase with pathological stages in both cohorts (P=0.032 and 0.047, respectively). On the contrary, the differentiation grades and sub-locations did not significantly affect the performance of the assay. Conclusions: We introduced a liquid biopsy assay using multiple dimensions of cfDNA characteristics that could accurately identify HNSCCs from non-cancerous conditions. As a cost-effective non-invasive approach, it may be conveniently incorporated into annual general physical examinations to provide supplementary information about the risk of HNSCCs.

Genomic full-length sequence of the novel HLA-C*06:364 allele.

HLA-C*06:364 differs from HLA-C*06:02:01:01 by a non-synonymous nucleotide substitution in exon 3.

A single nucleotide substitution in exon 5 generated the novel KIR2DL3*00112 allele.

The novel KIR2DL3*00112 allele differs from the closest allele KIR2DL3*00101 by a single same sense mutation.

A single nucleotide substitution in BnaC02.LBD6 promoter causes blade shape variation in Brassica napus

Leaf shape is a part of ideotype. Appropriate leaf rolling aids compact plant architecture. Oilseed is a crucial crop, but the genetic basis for leaf shape development in rapeseed (Brassica napus) is unclear, and no corresponding genetic improvement germplasm resources are available. We identified a dominant mutant, INSIDE-ROLLING LEAF1 (IRL1), in rapeseed with internal rolling of leaves due to abnormal mesophyll cell development. It had drooped siliques and a semi-dwarf stature, reducing of one to two effective branch numbers. Map-based cloning and complementary validation confirmed BnaC02G0201100ZS as causal gene IRL1. BnaC02G0201100ZS encodes LATERAL ORGAN BOUNDARIES DOMAIN6 (BnaC02.LBD6). Phenotypic variation of the irl1 mutant was due to an increase in BnaC02.LBD6 expression owing to a single nucleotide substitution at the DNA binding site in the promoter. Phenotype of plants overexpressing BnaC02.LBD6 was similar to that of the irl1 mutant. Haploid analysis showed a rare variation in BnaC02.LBD6 promoter, resulting in a unique phenotype in the irl1 mutant. Transcriptome analysis showed the differential expression of genes related to leaf adaxial–abaxial polarity development, secondary metabolic processes, and hormone signalling pathways. We provide novel insights into rapeseed: the genetic basis of variation in leaf characteristics and valuable genetic resources for plant architecture improvement.