A Comprehensive Guide to Quality Assessment and Data Submission for Genomic Surveillance of Enteric Pathogens.

This document outlines the steps necessary to assemble and submit the standard data package required for contributing to the global genomic surveillance of enteric pathogens. Although targeted to GenomeTrakr laboratories and collaborators, these protocols are broadly applicable for enteric pathogens collected for different purposes. There are five protocols included in this chapter: (1) quality control (QC) assessment for the genome sequence data, (2) validation for the contextual data, (3) data submission for the standard pathogen package or Pathogen Data Object Model (DOM) to the public repository, (4) viewing and querying data at NCBI, and (5) data curation for maintaining relevance of public data. The data are available through one of the International Nucleotide Sequence Database Consortium (INSDC) members, with the National Center for Biotechnology Information (NCBI) being the primary focus of this document. NCBI Pathogen Detection is a custom dashboard at NCBI that provides easy access to pathogen data plus results for a standard suite of automated cluster and genotyping analyses important for informing public health and regulatory decision-making.

Protocol for culturing patient-derived organoids of cervical cancer.

Herein, we present a protocol for culturing patient-derived organoids (PDOs) of cervical cancer that includes workflows for tumor biopsy/resection tissue and cytobrush-sampled cells. We describe steps for PDO culture initiation, including rinsing, gentle dissociation, Lymphoprep separation, and cell assessment, as well as seeding cells from surgical and cytobrush tissue digestion. We then provide guidance on PDO maintenance and passage and techniques for producing conditioned medium. Overall, this protocol serves as a valuable guide for establishing and maintaining cervical cancer PDOs. For complete details on the use and execution of this protocol, please refer to Colbert et al.1.

Protocol for simultaneous detection of mRNAs and (phospho-)proteins with ARTseq-FISH in mouse embryonic stem cells.

Understanding the molecular signatures of individual cells within complex biological systems is crucial for deciphering cellular heterogeneity and uncovering regulatory mechanisms. Here, we present a protocol for simultaneous multiplexed detection of selected mRNAs and (phospho-)proteins in mouse embryonic stem cells using spatial single-cell profiling. We describe steps for employing single-stranded DNA (ssDNA)-labeled antibo'dies, padlock probes, and rolling circle amplification to achieve simultaneous visualization of mRNAs and (phospho-)proteins at subcellular resolution. This protocol has potential application in identifying cells in heterogeneous biological microenvironments. For complete details on the use and execution of this protocol, please refer to Hu et al.1.

Potential vs challenges of expanding the protein universe with genetic code expansion in eukaryotic cells.

Following decades of innovation and perfecting, genetic code expansion has become a powerful tool for in vivo protein modification. Some of the major hurdles that had to be overcome include suboptimal performance of GCE-specific translational components in host systems, competing cellular processes, unspecific modification of the host proteome and limited availability of codons for reassignment. Although strategies have been developed to overcome challenges, there is critical need for further improvement. Here we discuss the current state-of-the-art in genetic code expansion technology and the issues that still need to be addressed to unleash the full potential of this method in eukaryotic cells.

Efficient Strategy for Synthesizing Vector-Free and Oncolytic Herpes Simplex Type 1 Viruses.

Synthesizing viral genomes plays an important role in fundamental virology research and in the development of vaccines and antiviral drugs. Herpes simplex virus type 1 (HSV-1) is a large DNA virus widely used in oncolytic virotherapy. Although de novo synthesis of the HSV-1 genome has been previously reported, the synthetic procedure is still far from efficient, and the synthesized genome contains a vector sequence that may affect its replication and application. In the present study, we developed an efficient vector-free strategy for synthesis and rescue of synthetic HSV-1. In contrast to the conventional method of transfecting mammalian cells with a completely synthesized genome containing a vector, overlapping HSV-1 fragments synthesized by transformation-associated recombination (TAR) in yeast were linearized and cotransfected into mammalian cells to rescue the synthetic virus. Using this strategy, a synthetic virus, F-Syn, comprising the complete genome of the HSV-1 F strain, was generated. The growth curve and electron microscopy of F-Syn confirmed that its replication dynamics and morphogenesis are similar to those of the parental virus. In addition, by combining TAR with in vitro CRISPR/Cas9 editing, an oncolytic virus, F-Syn-O, with deleted viral genes ICP6, ICP34.5, and ICP47 was generated. The antitumor effect of F-Syn-O was tested in vitro. F-Syn-O established a successful infection and induced dose-dependent cytotoxic effects in various human tumor cell lines. These strategies will facilitate convenient and systemic manipulation of HSV-1 genomes and could be further applied to the design and construction of oncolytic herpesviruses.

Prospective Evaluation of Extubation Failure in Neonates and Infants After Cardiac Surgery.

Background: Extubation failure and its associated complications are not uncommon after pediatric cardiac surgery, especially in neonates and young infants. We aimed to identify the frequency, etiologies, and clinical characteristics associated with extubation failure after cardiac surgery in neonates and young infants. Methods: We conducted a single center prospective observational study of patients ≤180 days undergoing cardiac surgery between June 2022 and May 2023 with at least one extubation attempt. Patients who failed extubation, defined as reintubation within 72 h of first extubation attempt, were compared with patients extubated successfully using χ2, Fisher exact, or Wilcoxon rank-sum tests as appropriate. Results: We prospectively enrolled 132 patients who met inclusion criteria, of which 11 (8.3%) failed extubation. Median time to reintubation was 25.5 h (range 0.4-55.8). Extubation failures occurring within 12 h (n = 4) were attributed to upper airway obstruction or apnea, whereas extubation failures occurring between 12 and 72 h (n = 7) were more likely to be due to intrinsic lung disease or cardiac dysfunction. Underlying genetic anomalies, greater weight relative to baseline at extubation, or receiving positive end expiratory pressure (PEEP) > 5 cmH2O at extubation were significantly associated with extubation failure. Conclusions: In this study of neonates and young infants recovering from cardiac surgery, etiologies of early versus later extubation failure involved different pathophysiology. We also identified weight relative to baseline and PEEP at extubation as possible modifiable targets for future investigations of extubation failure in this patient population.

Integrating genomics as clinical biomarkers in pediatric pulmonology.

Respiratory diseases often result from complex interactions between an individual's genetic predisposition and their exposure to various environmental and other risk factors. Here we will briefly review how various types of "omics", particularly epigenomics and transcriptomics, hold promise for translation into clinical biomarkers in pediatric pulmonary medicine, using asthma and cystic fibrosis as examples.

The ROSMAP project: aging and neurodegenerative diseases through omic sciences.

The Religious Order Study and Memory and Aging Project (ROSMAP) is an initiative that integrates two longitudinal cohort studies, which have been collecting clinicopathological and molecular data since the early 1990s. This extensive dataset includes a wide array of omic data, revealing the complex interactions between molecular levels in neurodegenerative diseases (ND) and aging. Neurodegenerative diseases (ND) are frequently associated with morbidity and cognitive decline in older adults. Omics research, in conjunction with clinical variables, is crucial for advancing our understanding of the diagnosis and treatment of neurodegenerative diseases. This summary reviews the extensive omics research-encompassing genomics, transcriptomics, proteomics, metabolomics, epigenomics, and multiomics-conducted through the ROSMAP study. It highlights the significant advancements in understanding the mechanisms underlying neurodegenerative diseases, with a particular focus on Alzheimer's disease.

Impact of Hashimoto's thyroiditis on the tumor microenvironment in papillary thyroid cancer: insights from single-cell analysis.

This study investigates the impact of Hashimoto's thyroiditis (HT), an autoimmune disorder, on the papillary thyroid cancer (PTC) microenvironment using a dataset of 140,456 cells from 11 patients. By comparing PTC cases with and without HT, we identify HT-specific cell populations (HASCs) and their role in creating a TSH-suppressive environment via mTE3, nTE0, and nTE2 thyroid cells. These cells facilitate intricate immune-stromal communication through the MIF-(CD74+CXCR4) axis, emphasizing immune regulation in the TSH context. In the realm of personalized medicine, our HASC-focused analysis within the TCGA-THCA dataset validates the utility of HASC profiling for guiding tailored therapies. Moreover, we introduce a novel, objective method to determine K-means clustering coefficients in copy number variation inference from bulk RNA-seq data, mitigating the arbitrariness in conventional coefficient selection. Collectively, our research presents a detailed single-cell atlas illustrating HT-PTC interactions, deepening our understanding of HT's modulatory effects on PTC microenvironments. It contributes to our understanding of autoimmunity-carcinogenesis dynamics and charts a course for discovering new therapeutic targets in PTC, advancing cancer genomics and immunotherapy research.

Feature sequence-based genome mining uncovers the hidden diversity of bacterial siderophore pathways.

Microbial secondary metabolites are a rich source for pharmaceutical discoveries and play crucial ecological functions. While tools exist to identify secondary metabolite clusters in genomes, precise sequence-to-function mapping remains challenging because neither function nor substrate specificity of biosynthesis enzymes can accurately be predicted. Here, we developed a knowledge-guided bioinformatic pipeline to solve these issues. We analyzed 1928 genomes of Pseudomonas bacteria and focused on iron-scavenging pyoverdines as model metabolites. Our pipeline predicted 188 chemically different pyoverdines with nearly 100% structural accuracy and the presence of 94 distinct receptor groups required for the uptake of iron-loaded pyoverdines. Our pipeline unveils an enormous yet overlooked diversity of siderophores (151 new structures) and receptors (91 new groups). Our approach, combining feature sequence with phylogenetic approaches, is extendable to other metabolites and microbial genera, and thus emerges as powerful tool to reconstruct bacterial secondary metabolism pathways based on sequence data.

Comparative genomic analysis of Acanthamoeba from different sources and horizontal transfer events of antimicrobial resistance genes.

Acanthamoeba species are among the most common free-living amoeba and ubiquitous protozoa, mainly distributed in water and soil, and cause Acanthamoeba keratitis (AK) and severe visual impairment in patients. Although several studies have reported genomic characteristics of Acanthamoeba, limited sample sizes and sources have resulted in an incomplete understanding of the genetic diversity of Acanthamoeba from different sources. While endosymbionts exert a significant influence on the phenotypes of Acanthamoeba, including pathogenicity, virulence, and drug resistance, the species diversity and functional characterization remain largely unexplored. Herein, our study sequenced and analyzed the whole genomes of 19 Acanthamoeba pathogenic strains that cause AK, and by integrating publicly available genomes, we sampled 29 Acanthamoeba strains from ocular, environmental, and other sources. Combined pan-genomic and comparative functional analyses revealed genetic differences and evolutionary relationships among the different sources of Acanthamoeba, as well as classification into multiple functional groups, with ocular isolates in particular showing significant differences that may account for differences in pathogenicity. Phylogenetic and rhizome gene mosaic analyses of ocular Acanthamoeba strains suggested that genomic exchanges between Acanthamoeba and endosymbionts, particularly potential antimicrobial resistance genes trafficking including the adeF, amrA, and amrB genes exchange events, potentially contribute to Acanthamoeba drug resistance. In conclusion, this study elucidated the adaptation of Acanthamoeba to different ecological niches and the influence of gene exchange on the evolution of ocular Acanthamoeba genome, guiding the clinical diagnosis and treatment of AK and laying a theoretical groundwork for developing novel therapeutic approaches. IMPORTANCE: Acanthamoeba causes a serious blinding keratopathy, Acanthamoeba keratitis, which is currently under-recognized by clinicians. In this study, we analyzed 48 strains of Acanthamoeba using a whole-genome approach, revealing differences in pathogenicity and function between strains of different origins. Horizontal transfer events of antimicrobial resistance genes can help provide guidance as potential biomarkers for the treatment of specific Acanthamoeba keratitis cases.

Emerging trends in plant natural products biosynthesis: a chemical perspective.

Biosynthetic pathways are multistep processes transforming simple substrates into more complex structures. Over the past two decades, our understanding of these pathways, especially for specialized plant metabolites, has significantly increased. This surge is due to numerous scientific advancements such as next-generation sequencing, improved analytical platforms, and metabolite-transcript networks. The uprising of data sharing through public databases has also fostered collaboration and knowledge dissemination. Growing concerns about the supply of therapeutic natural products and their environmental impact have led to exploring sustainable alternatives like heterologous expression, which requires extensive knowledge of these pathways. Herein, we review emerging approaches in biosynthetic pathway elucidations and their prospects for their efficient integration.

Global distribution and genomic characteristics analysis of avian-derived mcr-1-positive Escherichia coli.

The prevalence of avian-derived Escherichia coli (E. coli) carrying mcr-1 poses a significant threat to the development of the poultry industry and public health safety. Despite ongoing in-depth epidemiological research worldwide, a comprehensive macroscopic study based on genomics is still lacking. In response, this study collected 1104 genomic sequences of avian-derived mcr-1-positive E. coli (MCRPEC) from the NCBI public database, covering 31 countries. The majority of sequences originated from China (48.82 %), followed by the Netherlands (10.41 %). In terms of avian hosts, chicken accounted for the largest proportion (44.11 %), followed by gallus (24.09 %). Avian-derived MCRPEC also serves as a reservoir for other antibiotic resistance genes (ARGs), with 179 ARGs coexisting with mcr-1 identified. A total of 206 virulence-associated genes were also identified, revealing the pathogenic risks of MCRPEC. Pan-genome analysis revealed that avian-derived MCRPEC from different hosts, countries of origin, and serotypes exhibit minor SNP differences, indicating a high risk of cross-regional and cross-host transmission. The ST types of MCRPRC are diverse, with ST10 being the most prevalent (n=70). Spearman analysis showed a significant correlation between the number of ARGs and the insertion sequences (ISs) as well as plasmid replicon in ST10 strains. Furthermore, ST10 strains share a similar genetic basis with human-derived MCRPEC, suggesting the possibility of clonal dissemination. Pan-genome-wide association studies (pan-GWAS) indicated that the differential genes of MCRPEC from different countries and host sources are significantly different, mainly related to genes encoding type IV secretion systems and mobile genetic elements (MGEs). Plasmid mapping of showed that the prevalent plasmid types vary by country and host, with IncI2 and IncX4 being the main mcr-1-positive plasmids. Among the 12 identified mcr-1 genetic contexts with ISs, the Tn6330 transposon was the predominant carrier of mcr-1. In summary, the potential threat of avian-derived MCRPEC cannot be ignored, and long-term and comprehensive monitoring are essential.

An integrative strategy used by the aphid Uroleucon formosanum to counter host sesquiterpene lactone defense: Insights from combined genomic and transcriptomic analysis.

Insect herbivores adapt and develop strategies to counteract plant chemical defenses. The aphid Uroleucon formosanum is a serious sap-sucking pest that infests lettuces containing toxic sesquiterpene lactones (STLs). Herein, we employed a combination of genome sequencing and RNA-seq transcriptome profiling to understand the mechanisms underlying phytotoxin tolerance in U. formosanum. We generated the first chromosome-level genome assembly for U. formosanum, with a total size of 453.26 Mb and a scaffold N50 of 33.22 Mb. Comparative genomic analyses revealed an enrichment of signals for positive selection and gene family expansion in immune-related pathways. Specifically, the expanded set of heat shock protein 70 (HSP70) genes showed upregulation after treatment with lactucin, suggesting that they may play a role in the immune response against STLs. The expression of takeout-like genes and cuticle-associated genes was also significantly increased in the lactucin-treated samples. Additionally, 53 cytochrome P450 monooxygenase, 30 carboxylesterase, 19 glutathione S-transferase, 32 uridine diphosphate glycosyltransferase and 63 ATP-binding cassette (ABC) transporter genes were identified in the U. formosanum genome. CYP4C1, CYP6A13 and 7 ABC genes were strongly upregulated in response to lactucin treatment, indicating the involvement of detoxifying enzymes in the tolerance of U. formosanum to STLs. Our findings suggest that the cuticle barrier, immune response and enzyme-mediated metabolic detoxification jointly enhance the tolerance of U. formosanum to phytotoxins and promote its adaptation to host plants. This study presents a valuable genomic resource and provides insights into insect adaptation to plant chemical challenges and future technological developments for pest management.

Chromosome-level genome assembly and population genomic analysis provide insights into the genetic diversity and adaption of Schizopygopsis younghusbandi on the Tibetan Plateau.

The Yarlung Tsangpo River on the Tibetan Plateau provides a unique natural environment for studying fish evolution and ecology. However, the genomes and genetic diversity of plateau fish species have been rarely reported. Schizopygopsis younghusbandi, a highly specialized Schizothoracine species and economically important fish inhabiting the Yarlung Tsangpo River, is threatened by overfishing and biological invasion. Herein, we generated a chromosome-level genome of S. younghusbandi and whole-genome resequencing data for 59 individuals from six locations of the river. The results showed that the divergence time between S. younghusbandi and other primitive Schizothoracine species was ∼4.2 Mya, coinciding with the major phase of the Neogene Tibetan uplift. The expanded gene families enriched in DNA integration and replication, ion binding and transport, energy storage, and metabolism likely contribute to the adaption of this species. The S. younghusbandi may have diverged from other highly specialized Schizothoracine species in the Zanda basin during the Pliocene epoch, which underwent major population reduction possibly due to the drastic climate change during the last glacial period. Population analysis indicated that the ancient population might have originated upstream before gradually adapting to evolve into the populations inhabiting the mid-stream and downstream regions of the Yarlung Tsangpo River. In conclusion, the chromosome-level genome and population diversity of S. younghusbandi provide valuable genetic resources for the evolution, ecology, and conservation studies of endemic fishes on the Tibetan Plateau.

Genomic epidemiology describes introduction and outbreaks of antifungal drug-resistant Candida auris.

Candida auris is a globally emerged fungal pathogen causing nosocomial invasive infections. Here, we use cutting-edge genomic approaches to elucidate the temporal and geographic epidemiology of drug-resistant C. auris within the UK. We analysed a representative sample of over 200 isolates from multiple UK hospitals to assess the number and timings of C. auris introductions and infer subsequent patterns of inter- and intra-hospital transmission of azole drug-resistant isolates. We identify at least one introduction from Clade I and two from Clade III into the UK, and observe temporal and geographical evidence for multiple transmission events of antifungal drug resistant isolates between hospitals and identified local within-hospital patient-to-patient transmission events. Our study confirms outbreaks of drug-resistant C. auris are linked and that transmission amongst patients occurs, explaining local hospital outbreaks, and demonstrating a need for improved epidemiological surveillance of C. auris to protect patients and healthcare services.

Genomic analysis of Enterobacteriaceae from colorectal cancer patients at a tertiary hospital in Ghana: a case-control study.

Colorectal cancer (CRC) is a severe gastrointestinal cancer and a leading cause of cancer-related deaths in Ghana. The potential role of gut Enterobacteriaceae in the increasing incidence of CRC in Ghana is yet to be thoroughly investigated. In this study, Enterobacteriaceae from CRC patients and healthy control participants were analyzed by whole genome sequencing to identify genomic features that are associated with CRC. Socio-demographic data showed a significant association between age and alcohol consumption and CRC. Escherichia coli was the most abundant Enterobacteriaceae isolated from the study participants and they were predominantly intestinal commensals. Escherichia coli isolates belonging to phylogroup D encoded the highest number of virulence genes. The agn43 and int genes were widespread in Escherichia coli isolates from the CRC patients. Multilocus sequence types of potentially pathogenic Escherichia coli from the CRC patients also encoded genes involved in aggregation, adherence and biofilm formation. The ampC2 and ampH antimicrobial resistance genes were also widespread in the genome of the Escherichia coli isolates. This study highlights the virulence tendencies of Escherichia coli from CRC patients and their ability to transfer virulence determinants to other Enterobacteriaceae residing in the gut.

Decoding the identity of rare tumors.

Solitary fibrous tumors have gene expression signatures similar to those of neuroendocrine tumors.

Genome-wide association studies for milk production traits in two autochthonous Aosta cattle breeds.

Genome-wide association studies (GWASs) are used to identify quantitative trait loci for phenotypic traits of interest. The use of multilocus mixed models allows to correct for population stratification and account for long-range linkage disequilibrium. In this study, GWASs were conducted to identify the genetic bases of milk production (milk yield, protein and fat composition, and yield) in two autochthonous dual-purpose cattle breeds from the Aosta Valley. Using either the breeding values or the deregressed proofs, common significative single nucleotide polymorphisms have been identified for milk yield, protein percentage, and fat percentage. Two major quantitative trait loci regions have been identified on the chromosomes 5 and 14 for the fat percentage, harbouring the MGST1, CYHR1, VPS28, and CPSF1 genes. For the protein percentage, a candidate region has been identified on BTA 6; in this region, the CSN1S1, CSN2, HSTN, CSN3, and RUFY3 genes are annotated. Most of the identified genes have already been associated with milk composition in other studies on cosmopolitan and local cattle. These results show that the genes involved in milk composition quantitative traits in the Aosta cattle are common also in other cattle breeds and they can be further investigated with the use of whole genome sequencing data.