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Cell Aug 2023A system for programmable export of RNA molecules from living cells would enable both non-destructive monitoring of cell dynamics and engineering of cells capable of...
A system for programmable export of RNA molecules from living cells would enable both non-destructive monitoring of cell dynamics and engineering of cells capable of delivering executable RNA programs to other cells. We developed genetically encoded cellular RNA exporters, inspired by viruses, that efficiently package and secrete cargo RNA molecules from mammalian cells within protective nanoparticles. Exporting and sequencing RNA barcodes enabled non-destructive monitoring of cell population dynamics with clonal resolution. Further, by incorporating fusogens into the nanoparticles, we demonstrated the delivery, expression, and functional activity of exported mRNA in recipient cells. We term these systems COURIER (controlled output and uptake of RNA for interrogation, expression, and regulation). COURIER enables measurement of cell dynamics and establishes a foundation for hybrid cell and gene therapies based on cell-to-cell delivery of RNA.
Topics: Animals; Biological Transport; Mammals; RNA; RNA, Messenger; Viruses; Molecular Typing; Sequence Analysis, RNA; Cytological Techniques; Genetic Techniques
PubMed: 37437570
DOI: 10.1016/j.cell.2023.06.013 -
Ugeskrift For Laeger May 2023This review summarises the current knowledge of whole genome sequencing (WGS) which has become the standard method for genetic characterisation of bacteria in... (Review)
Review
This review summarises the current knowledge of whole genome sequencing (WGS) which has become the standard method for genetic characterisation of bacteria in surveillance and outbreak investigation. Although the method offers many advantages, its use in outbreak investigations is hampered by the relatively slow turn-around time. Using new approaches to perform WGS, typing and gene detection can now be completed within one day. This break-through allows clinical consequences to be taken almost immediately after detection of relevant bacteria and may have a huge impact on the future prevention of transmission of infectious diseases.
Topics: Humans; Whole Genome Sequencing; Bacterial Infections; Disease Outbreaks
PubMed: 37264858
DOI: No ID Found -
Clinical Microbiology and Infection :... Jul 2023Epidemiological and whole-genome sequencing analysis of an ongoing outbreak of Streptococcus pyogenes (Group A Streptococcus) in London (United Kingdom).
OBJECTIVES
Epidemiological and whole-genome sequencing analysis of an ongoing outbreak of Streptococcus pyogenes (Group A Streptococcus) in London (United Kingdom).
METHODS
Prospective identification of Group A Streptococcus cases from a diagnostic laboratory serving central and south London between 27 November and 10 December 2022. Case notes were reviewed and isolates were retrieved. Case numbers were compared with the previous 5 years. Whole-genome sequencing was performed with long-read, nanopore technology for emm typing and identification of superantigen genes. Associations of pathogen-related factors with an invasive disease were assessed by single-variable and multi-variable logistic regression.
RESULTS
Case numbers began increasing in October 2022 from a baseline of 2.0 cases per day, and in December 2022, the average daily case numbers reached 10.8 cases per day, four-fold the number usually seen in winter. A total of 113 cases were identified during the prospective study period. Three quarters (86/113, 76%) were paediatric cases, including 2 deaths. Of 113 cases, 11 (10%) were invasive. In total, 56 isolates were successfully sequenced, including 10 of 11 (91%) invasive isolates. The emm12 (33/56, 59%) and emm1 (9/56, 16%) types were predominant, with 7 of 9 (78%) emm1 isolates being from the M1uk clone. The majority of invasive isolates had superantigen genes spea (7/10, 70%) and spej (8/10, 80%), whereas, in non-invasive isolates, these superantigen genes were found less frequently (spea: 5/46, 11% and spej: 7/46, 15%). By multivariable analysis of pathogen-related factors, spea (OR 8.9, CI 1.4-57, p 0.020) and spej (OR 12, CI 1.8-78, p 0.011) were associated with invasive disease.
CONCLUSIONS
emm12 and emm1 types predominate in the ongoing outbreak, which mainly affects children. In this outbreak, the spea and spej superantigen genes are associated with the severity of presentation.
Topics: Child; Humans; Streptococcus pyogenes; Prospective Studies; Molecular Epidemiology; London; Antigens, Bacterial; United Kingdom; Superantigens; Disease Outbreaks; Streptococcal Infections; Bacterial Outer Membrane Proteins
PubMed: 36925107
DOI: 10.1016/j.cmi.2023.03.001 -
Cancer Medicine Aug 2023Rectal neuroendocrine neoplasms (NENs) are rare neoplasms with limited understanding of its genomic alterations and molecular typing.
BACKGROUND
Rectal neuroendocrine neoplasms (NENs) are rare neoplasms with limited understanding of its genomic alterations and molecular typing.
METHODS
The paraffin-embedded tissue specimens of 38 patients with rectal NENs after surgery were subjected to whole gene sequencing (WGS), and mutation profilings were drawn to identify high-frequency mutation genes, copy-number variations (CNVs), tumor mutation burden (TMB), signal pathways, mutation signatures, DNA damage repair (DDR) genes, and molecular types. The differences of mutated genes and signaling pathways in different pathological grades and metastatic/non-metastatic groups were compared. It helped to search for potential targets.
RESULTS
C > T and T > C transitions are the most common base substitutions in rectal NENs. DNA mismatch repair deficiency, DNA base modifications, smoking and exposure to ultraviolet light might play a role in the occurrence of rectal NENs. DAXX, KMT2C, BCL2L1, LTK, MERTK, SPEN, PKN1, FAT3, and LRP2 mutations were found in only low-grade rectal NETs, whereas APC, TP53, NF1, SOX9, and BRCA1 mutations were common in high-grade rectal NECs/MiNENs. These genes helped in distinguishing poorly-differentiated or well-differentiated rectal NENs. Alterations in P53, Wnt and TGFβ signaling pathways were more pronounced in rectal NECs and MiNENs. Alterations in Wnt, MAPK and PI3K/AKT signaling pathways promoted metastases. Rectal NENs were classified into two molecular subtypes by cluster analysis based on the mutant genes and signaling pathways combined with clinicopathological features. Patients with mutations in the LRP2, DAXX, and PKN1 gene showed a trend of well-differentiated and early-stage tumors with less metastasis (p = 0.000).
CONCLUSIONS
This study evaluated risk factors for regional lymphatic and/or distant metastases, identified high-frequency mutated genes, mutation signatures, altered signaling pathways through NGS. Rectal NENs were divided into two molecular types. This helps to evaluate the likelihood of metastasis, formulate follow-up strategies for patients and provide a target for future research on precision treatment of rectal NENs. PARP inhibitors, MEK inhibitors, mTOR/AKT/PI3K and Wnt signaling pathway inhibitors may be effective drugs for the treatment of metastatic rectal NENs.
Topics: Neuroendocrine Tumors; Rectal Neoplasms; Paraffin Embedding; Mutation; Molecular Typing; DNA Mutational Analysis; Neoplasm Staging; Humans; Male; Female; Adult; Middle Aged; Aged; Aged, 80 and over
PubMed: 37387515
DOI: 10.1002/cam4.6281 -
VBCG: 20 validated bacterial core genes for phylogenomic analysis with high fidelity and resolution.Microbiome Nov 2023Phylogenomic analysis has become an inseparable part of studies of bacterial diversity and evolution, and many different bacterial core genes have been collated and used...
BACKGROUND
Phylogenomic analysis has become an inseparable part of studies of bacterial diversity and evolution, and many different bacterial core genes have been collated and used for phylogenomic tree reconstruction. However, these genes have been selected based on their presence and single-copy ratio in all bacterial genomes, leaving out the gene's 'phylogenetic fidelity' unexamined.
RESULTS
From 30,522 complete genomes covering 11,262 species, we examined 148 bacterial core genes that have been previously used for phylogenomic analysis. In addition to the gene presence and single-copy rations, we evaluated the gene's phylogenetic fidelity by comparing each gene's phylogeny with its corresponding 16S rRNA gene tree. Out of the 148 bacterial genes, 20 validated bacterial core genes (VBCG) were selected as the core gene set with the highest bacterial phylogenetic fidelity. Compared to the larger gene set, the 20-gene core set resulted in more species having all genes present and fewer species with missing data, thereby enhancing the accuracy of phylogenomic analysis. Using Escherichia coli strains as examples of prominent bacterial foodborne pathogens, we demonstrated that the 20 VBCG produced phylogenies with higher fidelity and resolution at species and strain levels while 16S rRNA gene tree alone could not.
CONCLUSION
The 20 validated core gene set improves the fidelity and speed of phylogenomic analysis. Among other uses, this tool improves our ability to explore the evolution, typing and tracking of bacterial strains, such as human pathogens. We have developed a Python pipeline and a desktop graphic app (available on GitHub) for users to perform phylogenomic analysis with high fidelity and resolution. Video Abstract.
Topics: Humans; Phylogeny; Genes, Bacterial; RNA, Ribosomal, 16S; Genome, Bacterial; Bacteria
PubMed: 37936197
DOI: 10.1186/s40168-023-01705-9 -
Frontiers in Bioscience (Landmark... Aug 2023A broad variety of infections, ranging from skin infections to infective endocarditis can be caused by Bacterial virulence is often related to virulence genes, so we...
BACKGROUND
A broad variety of infections, ranging from skin infections to infective endocarditis can be caused by Bacterial virulence is often related to virulence genes, so we sought to investigate the relationship between virulence genes and the pathogenicity of and to explore an appropriate typing method to distinguish different pathogenic phenotypes of .
METHODS
We describe the distribution of several virulence genes in different infection types in an attempt to find the relationship between virulence genes and pathogenicity. Subsequently, we make the Matrix-Assisted Laser Desorption/Ionization-Time-of-Flight Mass Spectrometry (MALDI-TOF MS) dendrogram and -typing were performed using BioNumerics software, tried to compare the correlation between different methods and the different infectious diseases, and antimicrobial resistance of the strains, in order to obtain the epidemic characteristics and antimicrobial resistance information of based on a molecular approach.
RESULTS
The results of virulence genes showed that the seven virulence genes we have described existed in most strains, and there was no significant correlation between virulence gene distribution and infection type. Compared with the MALDI-TOF MS dendrogram, we found that -typing could better correspond to the pathogenic phenotype, with better recognition and reproducibility. In the phylogenetic tree constructed in the R-region, we found a tendency for some infection types to be distributed in clusters, new type 3 was the most dominant -type, followed by fbl47b. Bone and joint infection isolates and ear infection isolates were significantly clustered together, in addition, all the oxacillin-resistant isolates were concentrated in -type fbl45j and fbl47b.
CONCLUSIONS
In this study, we found no significant correlation between virulence genes from isolates and the site of infection. The -typing has the characteristics of convenient operation, low cost, high repeatability, and is preferable to indicate the pathogenic phenotype. Based on -typing, we described the epidemiological characteristics of in a hospital and supplemented the data for -typing. We recommend that -typing method be extended and supplemented.
Topics: Staphylococcus lugdunensis; Anti-Bacterial Agents; Drug Resistance, Bacterial; Phylogeny; Reproducibility of Results
PubMed: 37664924
DOI: 10.31083/j.fbl2808165 -
International Journal of Molecular... Oct 2023(Group B , GBS) is an important pathogen of bacterial meningitis in neonates. We aimed to investigate the clinical and genetic characteristics of neonatal GBS...
(Group B , GBS) is an important pathogen of bacterial meningitis in neonates. We aimed to investigate the clinical and genetic characteristics of neonatal GBS meningitis. All neonates with GBS meningitis at a tertiary level medical center in Taiwan between 2003 and 2020 were analyzed. Capsule serotyping, multilocus sequence typing, antimicrobial resistance, and whole-genome sequencing (WGS) were performed on the GBS isolates. We identified 48 neonates with GBS meningitis and 140 neonates with GBS sepsis. Neonates with GBS meningitis had significantly more severe clinical symptoms; thirty-seven neonates (77.8%) had neurological complications; seven (14.6%) neonates died; and 17 (41.5%) survivors had neurological sequelae at discharge. The most common serotypes that caused meningitis in neonates were type III (68.8%), Ia (20.8%), and Ib (8.3%). Sequence type (ST) is highly correlated with serotypes, and ST17/III GBS accounted for more than half of GBS meningitis cases (56.3%, = 27), followed by ST19/Ia, ST23/Ia, and ST12/Ib. All GBS isolates were sensitive to ampicillin, but a high resistance rates of 72.3% and 70.7% to erythromycin and clindamycin, respectively, were noted in the cohort. The virulence and pilus genes varied greatly between different GBS serotypes. WGS analyses showed that the presence of PezT; BspC; and ICE was likely associated with the occurrence of meningitis and was documented in 60.4%, 77.1%, and 52.1% of the GBS isolates that caused neonatal meningitis. We concluded that GBS meningitis can cause serious morbidity in neonates. Further experimental models are warranted to investigate the clinical and genetic relevance of GBS meningitis. Specific GBS strains that likely cause meningitis requires further investigation and clinical attention.
Topics: Infant, Newborn; Humans; Streptococcus agalactiae; Anti-Bacterial Agents; Streptococcal Infections; Serogroup; Serotyping; Meningitis, Bacterial; Multilocus Sequence Typing; Microbial Sensitivity Tests; Drug Resistance, Bacterial
PubMed: 37895067
DOI: 10.3390/ijms242015387 -
Frontiers in Microbiology 2023Lipid A is the hydrophobic component of bacterial lipopolysaccharide and an activator of the host immune system. Bacteria modify their lipid A structure to adapt to the...
Lipid A is the hydrophobic component of bacterial lipopolysaccharide and an activator of the host immune system. Bacteria modify their lipid A structure to adapt to the surrounding environment and, in some cases, to evade recognition by host immune cells. In this study, lipid A structural diversity within the genus was explored. The individual species have dramatically different pathogenic potential that ranges from non-infectious to life-threatening disease (leptospirosis). Ten distinct lipid A profiles, denoted L1-L10, were discovered across 31 reference species, laying a foundation for lipid A-based molecular typing. Tandem MS analysis revealed structural features of membrane lipids that might alter recognition of its lipid A by the host innate immune receptors. Results of this study will aid development of strategies to improve diagnosis and surveillance of leptospirosis, as well as guide functional studies on lipid A activity.
PubMed: 37303810
DOI: 10.3389/fmicb.2023.1181034 -
NAR Genomics and Bioinformatics Sep 2023Extrachromosomal elements of bacterial cells such as plasmids are notorious for their importance in evolution and adaptation to changing ecology. However,...
Extrachromosomal elements of bacterial cells such as plasmids are notorious for their importance in evolution and adaptation to changing ecology. However, high-resolution population-wide analysis of plasmids has only become accessible recently with the advent of scalable long-read sequencing technology. Current typing methods for the classification of plasmids remain limited in their scope which motivated us to develop a computationally efficient approach to simultaneously recognize novel types and classify plasmids into previously identified groups. Here, we introduce that can easily handle thousands of input sequences which are compressed using a unitig representation in a de Bruijn graph. Our approach offers a faster runtime than existing algorithms, with moderate memory usage, and enables an intuitive visualization, classification and clustering scheme that users can explore interactively within a single framework. platform for plasmid analysis can be easily distributed and replicated, enabling a consistent labelling of plasmids across past, present, and future sequence collections. We underscore the advantages of our approach by analysing a population-wide plasmid data set obtained from the opportunistic pathogen , studying the prevalence of the colistin resistance gene within the plasmid population, and describing an instance of resistance plasmid transmission within a hospital environment.
PubMed: 37435357
DOI: 10.1093/nargab/lqad066 -
Journal of Clinical Microbiology Dec 2023Whole genome sequencing (WGS)-based approaches for pneumococcal capsular typing have become an alternative to serological methods. serotyping from WGS has not yet been...
Whole genome sequencing (WGS)-based approaches for pneumococcal capsular typing have become an alternative to serological methods. serotyping from WGS has not yet been applied to long-read sequences produced by third-generation technologies. The objective of the study was to determine the capsular types of pneumococci causing invasive disease in Catalonia (Spain) using serological typing and WGS and to compare the performance of different bioinformatics pipelines using short- and long-read data from WGS. All invasive pneumococcal pediatric isolates collected in Hospital Sant Joan de Déu (Barcelona) from 2013 to 2019 were included. Isolates were assigned a capsular type by serological testing based on anticapsular antisera and by different WGS-based pipelines: Illumina sequencing followed by serotyping with PneumoCaT, SeroBA, and Pathogenwatch vs MinION-ONT sequencing coupled with serotyping by Pathogenwatch from pneumococcal assembled genomes. A total of 119 out of 121 pneumococcal isolates were available for sequencing. Twenty-nine different serotypes were identified by serological typing, with 24F ( = 17; 14.3%), 14 ( = 10; 8.4%), and 15B/C ( = 8; 6.7%) being the most common serotypes. WGS-based pipelines showed initial concordance with serological typing (>91% of accuracy). The main discrepant results were found at the serotype level within a serogroup: 6A/B, 6C/D, 9A/V, 11A/D, and 18B/C. Only one discrepancy at the serogroup level was observed: serotype 29 by serological testing and serotype 35B/D by all WGS-based pipelines. Thus, bioinformatics WGS-based pipelines, including those using third-generation sequencing, are useful for pneumococcal capsular assignment. Possible discrepancies between serological typing and WGS-based approaches should be considered in pneumococcal capsular-type surveillance studies.
Topics: Humans; Child; Streptococcus pneumoniae; Serotyping; Serogroup; Whole Genome Sequencing; Computational Biology; Pneumococcal Infections
PubMed: 38092657
DOI: 10.1128/jcm.00741-23