Search Results (59)

An EBSL-encoding plasmid, pESBL-PH, was identified during a nosocomial outbreak of Klebsiella pneumoniae ST628 at a United Kingdom general district hospital in 2018. The plasmid from the earliest 2018 K. pneumoniae strain discovered during the outbreak was assembled using both Oxford nanopore long reads and illumina short reads, yielding a fully closed plasmid, pESBL-PH-2018. pESBL-PH-2018 was queried against the complete NCBI RefSeq Plasmid Database, comprising 93,823 plasmids, which was downloaded on 16 July 2024. To identify structurally similar plasmids, strict thresholds were applied, including a mash similarity ≥0.98. This returned 61 plasmids belonging to 13 unique sequence types (STs) hosts. The plasmids were detected from 13 unique countries, dating from 2012 to 2023. The AMR region of the plasmids varied. Interestingly IS26-mediated tandem amplification of resistance genes, including the ESBL gene bla_CTX-M-15 was identified in two independent strains, raising their copy number to three. Furthermore, the genomic background of strains carrying a pESBL-PH-2018-like plasmid were analyzed, revealing truncation of the chromosomal ompK36 porin gene and carbapenem resistance gene carriage on accessory plasmids in 17.85% and 26.78% of strains with a complete chromosome available. This analysis reveals the widespread dissemination of an ESBL-encoding plasmid in a background of resistance-encoding strains, requiring active surveillance. Full article

Functional U6 promoters are widely utilized in CRISPR gene editing systems for crops. The identification of endogenous U6 promoter activity and the establishment of CRISPR/Cas9 gene editing systems in various crops can enhance the efficiency and accuracy of gene editing in molecular breeding. In this study, four U6 snRNAs were identified in the genome of the oil flax (Linum usitatissimum L.) cultivar Longya 10, which exhibit high homology with the promoter regions of Arabidopsis thaliana U6 snRNA. We cloned and constructed fusion expression vectors with U6 promoter-driven dual-luciferase reporter genes. Transient transformation of flax and Nicotiana benthamiana was performed to measure the relative activity of dual luciferase. The U6-4 on chromosome 14 showed the highest transcriptional activity. Truncations of varying lengths from the 5′ end of this promoter were tested, revealing that a 342 bp U6 promoter fragment possesses high transcriptional activity and an optimal length. Subsequently, we constructed a CRISPR/Cas9 gene editing vector with LuU6-5P/AtU6-P driving LusPDS sgRNA. Agrobacterium-mediated infection of flax hypocotyls yielded transgenic albino flax shoots. DNA from these shoots was used as a template to amplify LusPDS fragments, which were then sequenced. Sequencing analysis revealed that CRISPR/Cas9 vectors using Lu14U6-4-5P achieved higher editing frequencies at LusPDS compared to AtU6-P-driven systems. Full article

The use of CRISPR to knockdown or knockout genes is a powerful tool for understanding the specific role of a gene in disease development. However, it can cause many unanticipated changes to the transcriptome that are not detected by DNA amplification and Sanger sequencing of the target site. Various RNA-sequencing techniques can be used to identify these changes and effectively gauge the full impact of the CRISPR knockout, thereby providing a means of selecting appropriate clones for further experimentation. Background/Objectives: RNA-seq data from 4 CRISPR knockout experiments were analyzed and techniques developed to both confirm the success of the CRISPR modifications and identify potential issues. Methods: A broad-based analysis of RNA-sequencing data identified many CRISPR-based changes not identified by PCR amplification of DNA around the CRISPR target site. These changes included an inter-chromosomal fusion event, exon skipping, chromosomal truncation, and the unintentional transcriptional modification and amplification of a neighboring gene. Conclusions: The inadvertent modifications identified by the evaluation of 4 CRISPR experiments highlight the value of using RNA-seq to identify transcriptional changes to cells altered by CRISPR, many of which cannot be recognized by evaluating DNA alone. Specific guidelines are presented for designing and analyzing CRISPR experiments using RNA-seq data. Full article

Background/Objectives: The occurrence of novel variants of staphylococcal cassette chromosome mec (SCCmec) in livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) has frequently been reported, posing significant zoonotic concern worldwide. In this study, the occurrence of novel types of SCCmec IX elements was identified in two clonal complex (CC) 398 LA-MRSA strains derived from a pig farm and a slaughterhouse in Korea. Methods: Whole-genome sequence analysis of the two CC398 MRSA-SCCmec IX strains, designated KF1A-1172 and JS1E-122, revealed that these strains are most closely related to previously characterized strains of CC398 LA-MRSA carrying SCCmec V isolated from pig farms in Korea. Results: Further structural analysis of the SCCmec IX in KF1A-1172 and JS1E-122 revealed the presence of multiple ccr gene complexes (ccrA5B3, ccrAB3, and a truncated ccrA1), including ccrA1B1 genes for SCCmec type IX. In addition, the pseudo-SCC (ΨSCC) elements, genes associated with the type 1 restriction–modification (RM) system, and zinc resistance gene czrC, were identified in the SCCmec IX. Conclusions: These findings suggest that the multiple recombination events of elements derived from various SCCmec types contributed to the emergence of the novel SCCmec IX variant. The identification of these novel SCCmec IX types in CC398 LA-MRSA also suggests that epidemiological diversification of SCCmec IX in CC398 LA-MRSA is an ongoing event, necessitating continued surveillance on the emergence of novel SCCmec variants. This study is the first to report the complete genome sequences of CC398 MRSA carrying SCCmec IX in Korea. Full article

Anderson–Fabry disease is a hereditary, progressive, multisystemic lysosomal storage disorder caused by a functional deficiency of the enzyme α-galactosidase A (α-GalA). This defect is due to mutations in the GLA gene, located in the long arm of the X chromosome (Xq21-22). Functional deficiency of the α-GalA enzyme leads to reduced degradation and accumulation of its substrates, predominantly globotriaosylceramide (Gb3), which accumulate in the lysosomes of numerous cell types, giving rise to the symptomatology. Clinical diagnosis can still be difficult today due to the peculiarities of the disease, which presents with clinical manifestations that overlap with those of other pathologies and a wide possibility of differential diagnoses, which lead to missed diagnoses, misdiagnosis, or a diagnostic delay. Patients with clinical suspicion of Fabry disease undergo a diagnostic workup that includes an evaluation of α-GALA enzyme activity, genetic analysis of the GLA gene, and the measurement of blood Lyso-Gb3, a soluble derivative of Gb3. In this paper, we describe four novel mutations identified in the GLA gene which are associated with absent or reduced α-GalA activity, pathological accumulation of the specific substrate, and characteristic clinical manifestations of Fabry disease. We identified two mutations (c.583insGAATA and p.Y207X) that result in the formation of a premature translation stop codon, resulting in a truncated protein and thus a completely non-functional enzyme. The other two identified gene alterations (p.G261C and c.786G>T, which determine p.W262C) are missense mutations that cause reduced α-GALA activity, the accumulation of blood Lyso-Gb3, and symptoms consistent with Fabry disease, and therefore may be associated with this disorder. The identification of these new mutations in patients with symptoms attributable to Fabry disease increases the molecular knowledge of the GLA gene and provides important support to the clinician, for a more accurate and timely diagnosis of the pathology. Full article

Shaker potassium channels play a crucial role in potassium (K+) nutrition and stress resistance in plants. However, systematic research on Shaker K+ channels in Chinese cabbage [Brassica rapa var. chinensis (L.) Kitamura] remains scarce. This study identified 13 Shaker K+ channel members within the cabbage genome, which are unevenly distributed across eight chromosomes. Notably, the number of Shaker K+ channel members in Chinese cabbage exceeds that found in the model plants Arabidopsis (9) and rice (10). This discrepancy is attributed to a higher number of homologous proteins in Groups II and V of Chinese cabbage, with gene segmental duplication in these two subgroups being a significant factor contributing to the expansion of the Shaker K+ channel gene family. Interspecies collinearity analysis revealed that the whole genome and the Shaker K+ channel family of Chinese cabbage show greater similarity to those of Arabidopsis than to those of rice, indicating that Shaker K+ channels from the Brassicaceae family have a closer relationship than that from the Poaceae family. Given that gene expansion occurs in Group II, we investigated whether a functional difference exists between BrKAT1.1 and BrKAT1.2 using yeast assays and promoter analysis. The expression of two BrKAT1 genes in the potassium uptake-deficient yeast mutant R5421 can restore growth under low potassium conditions, indicating their role in potassium absorption. Truncation of the N-terminal 63 amino acids of BrKAT1.2 resulted in the loss of potassium absorption capability, suggesting that the N-terminus is essential for maintaining the potassium absorption function of BrKAT1.2. Furthermore, the expression of the two BrKAT1 genes in the salt-sensitive yeast G19 enhances yeast tolerance to salt stress. These results demonstrate that BrKAT1.1 and BrKAT1.2 exhibit similar abilities in potassium uptake and salt tolerance. The difference between BrKAT1.1 and BrKAT1.2 lay in their promoter regulatory elements, suggesting that differences in transcriptional regulation contributed to the functional differentiation of BrKAT1.1 and BrKAT1.2. These findings provide a foundation for understanding the evolution and functional mechanisms of the Shaker K+ channel family in Chinese cabbage and for improving potassium nutrition and salt tolerance in this species through the manipulation of BrKAT1. Full article

Phosphatase and tensin homologue deleted on chromosome 10 (PTEN) is a crucial tumor suppressor protein with frequent mutations and alterations. Although protein therapeutics are already integral to numerous medical fields, their potential remains nascent. This study aimed to investigate the impact of stable, unphosphorylated recombinant human full-length PTEN and its truncated variants, regarding their tumor suppression activity with multiwalled-carbon nanotubes (MW-CNTs) as vehicles for their delivery in breast cancer cells (T-47D, ZR-75-1, and MCF-7). The cloning, overexpression, and purification of PTEN variants were achieved from E. coli, followed by successful binding to CNTs. Cell incubation with protein-functionalized CNTs revealed that the full-length PTEN-CNTs significantly inhibited cancer cell growth and stimulated apoptosis in ZR-75-1 and MCF-7 cells, while truncated PTEN fragments on CNTs had a lesser effect. The N-terminal fragment, despite possessing the active site, did not have the same effect as the full length PTEN, emphasizing the necessity of interaction with the C2 domain in the C-terminal tail. Our findings highlight the efficacy of full-length PTEN in inhibiting cancer growth and inducing apoptosis through the alteration of the expression levels of key apoptotic markers. In addition, the utilization of carbon nanotubes as a potent PTEN protein delivery system provides valuable insights for future applications in in vivo models and clinical studies. Full article

There are about 14,000 pseudogenes that are mutated or truncated sequences resembling functional parent genes. About two-thirds of pseudogenes are processed, while others are duplicated. Although initially thought dead, emerging studies indicate they have functional and regulatory roles. We study 14-3-3ζ, an adaptor protein that regulates cytokine signaling and inflammatory diseases, including rheumatoid arthritis, cancer, and neurological disorders. To understand how 14-3-3ζ (gene symbol YWHAZ) performs diverse functions, we examined the human genome and identified nine YWHAZ pseudogenes spread across many chromosomes. Unlike the 32 kb exon-to-exon sequence in YWHAZ, all pseudogenes are much shorter and lack introns. Out of six, four YWHAZ exons are highly conserved, but the untranslated region (UTR) shows significant diversity. The putative amino acid sequence of pseudogenes is 78–97% homologous, resulting in striking structural similarities with the parent protein. The OMIM and Decipher database searches revealed chromosomal loci containing pseudogenes are associated with human diseases that overlap with the parent gene. To the best of our knowledge, this is the first report on pseudogenes of the 14-3-3 family protein and their implications for human health. This bioinformatics-based study introduces a new insight into the complexity of 14-3-3ζ’s functions in biology. Full article

While Botrytis cinerea causes gray mold on many plants, its close relative, Botrytis fabae, is host-specifically infecting predominantly faba bean plants. To explore the basis for its narrow host range, a gapless genome sequence of B. fabae strain G12 (BfabG12) was generated. The BfabG12 genome encompasses 45.0 Mb, with 16 chromosomal telomere-to-telomere contigs that show high synteny and sequence similarity to the corresponding B. cinerea B05.10 (BcB0510) chromosomes. Compared to BcB0510, it is 6% larger, due to many AT-rich regions containing remnants of transposable elements, but encodes fewer genes (11,420 vs. 11,707), due to losses of chromosomal segments with up to 20 genes. The coding capacity of BfabG12 is further reduced by nearly 400 genes that had been inactivated by mutations leading to truncations compared to their BcB0510 orthologues. Several species-specific gene clusters for secondary metabolite biosynthesis with stage-specific expression were identified. Comparison of the proteins secreted during infection revealed high similarities, including 17 phytotoxic proteins that were detected in both species. Our data indicate that evolution of the host-specific B. fabae occurred from an ancestral pathogen with wide host range similar to B. cinerea and was accompanied by losses and degeneration of genes, thereby reducing its pathogenic flexibility. Full article

Complex structural chromosome abnormalities such as chromoanagenesis have been reported in acute myeloid leukemia (AML). They are usually not well characterized by conventional genetic methods, and the characterization of chromoanagenesis structural abnormalities from short-read sequencing still presents challenges. Here, we characterized complex structural abnormalities involving chromosomes 2, 3, and 7 in an AML patient using an integrated approach including CRISPR/Cas9-mediated nanopore sequencing, mate pair sequencing (MPseq), and SNP microarray analysis along with cytogenetic methods. SNP microarray analysis revealed chromoanagenesis involving chromosomes 3 and 7, and a pseudotricentric chromosome 7 was revealed by cytogenetic methods. MPseq revealed 138 structural variants (SVs) as putative junctions of complex rearrangements involving chromosomes 2, 3, and 7, which led to 16 novel gene fusions and 33 truncated genes. Thirty CRISPR RNA (crRNA) sequences were designed to map 29 SVs, of which 27 (93.1%) were on-target based on CRISPR/Cas9 crRNA nanopore sequencing. In addition to simple SVs, complex SVs involving over two breakpoints were also revealed. Twenty-one SVs (77.8% of the on-target SVs) were also revealed by MPseq with shared SV breakpoints. Approximately three-quarters of breakpoints were located within genes, especially intronic regions, and one-quarter of breakpoints were intergenic. Alu and LINE repeat elements were frequent among breakpoints. Amplification of the chromosome 7 centromere was also detected by nanopore sequencing. Given the high amplification of the chromosome 7 centromere, extra chromosome 7 centromere sequences (tricentric), and more gains than losses of genomic material, chromoanasynthesis and chromothripsis may be responsible for forming this highly complex structural abnormality. We showed this combination approach’s value in characterizing complex structural abnormalities for clinical and research applications. Characterization of these complex structural chromosome abnormalities not only will help understand the molecular mechanisms responsible for the process of chromoanagenesis, but also may identify specific molecular targets and their impact on therapy and overall survival. Full article

Count data consists of both observed and unobserved events. The analysis of count data often encounters overdispersion, where traditional Poisson models may not be adequate. In this paper, we introduce a tractable one-parameter mixed Poisson distribution, which combines the Poisson distribution with the improved second-degree Lindley distribution. This distribution, called the Poisson-improved second-degree Lindley distribution, is capable of effectively modeling standard count data with overdispersion. However, if the frequency of the unobserved events is unknown, the proposed distribution cannot be directly used to describe the events. To address this limitation, we propose a modification by truncating the distribution to zero. This results in a tractable zero-truncated distribution that encompasses all types of dispersions. Due to the unknown frequency of unobserved events, the population size as a whole becomes unknown and requires estimation. To estimate the population size, we develop a Horvitz–Thompson-like estimator utilizing truncated distribution. Both the untruncated and truncated distributions exhibit desirable statistical properties. The estimators for both distributions, as well as the population size, are asymptotically unbiased and consistent. The current study demonstrates that both the truncated and untruncated distributions adequately explain the considered medical datasets, which are the number of dicentric chromosomes after being exposed to different doses of radiation and the number of positive Salmonella. Moreover, the proposed population size estimator yields reliable estimates. Full article

Gamma-aminobutyric acid (GABA)-producing lactic acid bacteria (LAB) can be used as starters in the development of GABA-enriched functional fermented foods. In this work, four GABA-producing strains each of Lactococcus lactis and Streptococcus thermophilus species were isolated from cow’s milk, and their phenotypic, technological, and safety profiles determined. Genome analysis provided genetic support for the majority of the analyzed traits, namely, GABA production, growth in milk, and the absence of genes of concern. The operon harboring the glutamate decarboxylase gene (gadB) was chromosomally encoded in all strains and showed the same gene content and gene order as those reported, respectively, for L. lactis and S. thermophilus. In the latter species, the operon was flanked (as in most strains of this species) by complete or truncated copies of insertion sequences (IS), suggesting recent acquisition through horizontal gene transfer. The genomes of three L. lactis and two S. thermophilus strains showed a gene encoding a caseinolytic proteinase (PrtP in L. lactis and PrtS in S. thermophilus). Of these, all but one grew in milk, forming a coagulum of good appearance and an appealing acidic flavor and taste. They also produced GABA in milk supplemented with monosodium glutamate. Two L. lactis strains were identified as belonging to the biovar. diacetylactis, utilized citrate from milk, and produced significant amounts of acetoin. None of the strains showed any noticeable antibiotic resistance, nor did their genomes harbor transferable antibiotic resistance genes or genes involved in toxicity, virulence, or pathogenicity. Altogether these results suggest that all eight strains may be considered candidates for use as starters or components of mixed LAB cultures for the manufacture of GABA-enriched fermented dairy products. Full article

(1) Introduction: Dilated cardiomyopathy (DCM) mainly affects young individuals and is the main indication of heart transplantation. The variant c.77T>C (p.Val26Ala) of the gene coding for emerin (EMD) in chromosome Xq28 has been catalogued as a pathogenic variant for the development of DCM, exhibiting an X-linked inheritance pattern. (2) Methods: A retrospective study was conducted covering the period 2015–2023 in patients with DCM of genetic origin. The primary endpoint was patient age at onset of the first composite major cardiac event, in the form of a first episode of heart failure, malignant ventricular arrhythmia, or end-stage heart failure, according to the presence of truncating variant in titin gene (TTNtv) versus the p.Val26Ala mutation in the EMD protein. (3) Results: A total of 31 and 22 patients were included in the EMD group and TTNtv group, respectively. The primary endpoint was significantly higher in the EMD group, with a hazard ratio of 4.16 (95% confidence interval: 1.83–9.46; p = 0.001). At 55 years of age, all the patients in the EMD group had already presented heart failure, nine presented malignant ventricular arrhythmia (29%), and 13 required heart transplantation (42%). (4) Conclusions: DCM secondary to the c.77T>C (p.Val26Ala) mutation in the EMD gene is associated to an increased risk of major cardiac events compared to patients with DCM due to TTNtv, with a large proportion of transplanted patients in the fifth decade of life. Full article

A deficiency of FMRP, a canonical RNA-binding protein, causes the development of Fragile X Syndrome (FXS), which is characterised by multiple phenotypes, including neurodevelopmental disorders, intellectual disability, and autism. Due to the alternative splicing of the encoding FMR1 gene, multiple FMRP isoforms are produced consisting of full-length predominantly cytoplasmic (i.e., iso1) isoforms involved in translation and truncated nuclear (i.e., iso6) isoforms with orphan functions. However, we recently implicated nuclear FMRP isoforms in DNA damage response, showing that they negatively regulate the accumulation of anaphase DNA genomic instability bridges. This finding provided evidence that the cytoplasmic and nuclear functions of FMRP are uncoupled played by respective cytoplasmic and nuclear isoforms, potentially involving specific interactions. While interaction partners of cytoplasmic FMRP have been reported, the identity of nuclear FMRP isoform partners remains to be established. Using affinity purification coupled with mass spectrometry, we mapped the nuclear interactome of the FMRP isoform iso6 in U2OS. In doing so, we found FMRP nuclear interaction partners to be involved in RNA processing, pre-mRNA splicing, ribosome biogenesis, DNA replication and damage response, chromatin remodeling and chromosome segregation. By comparing interactions between nuclear iso6 and cytoplasmic iso1, we report a set of partners that bind specifically to the nuclear isoforms, mainly proteins involved in DNA-associated processes and proteasomal proteins, which is consistent with our finding that proteasome targets the nuclear FMRP iso6. The specific interactions with the nuclear isoform 6 are regulated by replication stress, while those with the cytoplasmic isoform 1 are largely insensitive to such stress, further supporting a specific role of nuclear isoforms in DNA damage response induced by replicative stress, potentially regulated by the proteasome. Full article

The intensively pigmented legumes belonging to Phaseolus and Vigna spp. are valued as an essential component of healthy nutrition due to their high content of flavonoids. In this context, we used the accessions of Vigna unguiculata with different colors of seed coats from the N.I. Vavilov All-Russian Institute of Plant Genetic Resources collection as the main object of this research. We applied confocal laser scanning microscopy, biochemical analysis, and wide in silico and molecular genetic analyses to study the main candidate genes for anthocyanin pigmentation within the MYB cluster on chromosome 5. We performed statistical data processing. The anthocyanin content ranged from 2.96 mg/100 g DW in reddish-brown-seeded cowpea accessions to 175.16 mg/100 g DW in black-seeded ones. Laser microscopy showed that the autofluorescence in cowpea seeds was mainly caused by phenolic compounds. The maximum fluorescence was observed in the seed coat, while its dark color, due to the highest level of red fluorescence, pointed to the presence of anthocyanins and anthocyanidins. Genes of the MYB cluster on chromosome 5 demonstrated a high homology and were segregated into a separate clade. However, amplification products were not obtained for all genes because of the truncation of some genes. Statistical analysis showed a clear correlation between the high content of anthocyanins in cowpea seeds and the presence of PCR products with primers Vigun05g0393-300-1. Full article