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PubMed2025

Viral viability markers of SARS-CoV-2: a comparison of cell culture, genomic RNA RT-PCR, and subgenomic RNA RT-PCR.

UNLABELLED: Accurate methods to assess viral viability are crucial for determining isolation duration and antiviral therapy in immunocompromised patients. Although cell culture (CC) is the gold standard, it has limitations. Cycle threshold (Ct) values from genomic RNA (gRNA) RT-PCR and subgenomic RNA (sgRNA) RT-PCR have been proposed as markers of active viral replication. This study evaluated the correlation between CC, gRNA Ct values, and sgRNA to identify the best viral viability marker. This study aimed to evaluate the correlation between CC, gRNA Ct values, and sgRNA to identify the best viral viability marker. We conducted a prospective study on immunocompromised patients with suspected SARS-CoV-2 infection at a tertiary hospital (May 2021 to May 2023). Nasopharyngeal swabs were inoculated into Vero E6 cells and tested for gRNA using RT-PCR (Cobas 6800, Roche) and for sgRNA (non-commercial RT-PCR). The sensitivity (S), specificity (SP), positive (PPV) and negative predictive value (NPV), and accuracy were calculated using CC as the gold standard. Among 285 samples from 108 patients, gRNA RT-PCR had high S and NPV (1.0) but low SP (0.24) and moderate PPV (0.63). Ct analysis improved performance in extreme but not intermediate values. A Ct ≤ 30 maximized S but had low SP; Ct ≤ 25 yielded S (0.88), SP (0.89), PPV (0.92), NPV (0.84), and accuracy (0.88); sgRNA showed the highest S (0.99), SP (0.96), PPV (0.97), NPV (0.99), and accuracy (0.98). sgRNA detection is the best marker for identifying viable SARS-CoV-2, aiding decisions on isolation, antiviral treatment, or delaying chemotherapy in immunocompromised patients. IMPORTANCE: Identifying whether a patient still has contagious SARS-CoV-2 is essential for managing isolation, antiviral treatment, and other clinical decisions-especially in immunocompromised individuals. While viral culture is the gold standard for confirming viral viability, it is slow, expensive, and not widely available. Many hospitals rely on RT-PCR tests, but these detect viral genetic material whether or not the virus is still active. This study shows that detecting subgenomic RNA (sgRNA), a molecule only present when the virus is actively replicating, is a highly accurate way, a molecule only present when the virus is actively replicating, is a highly accurate way to determine whether the virus is still viable. Compared to standard PCR or viral culture, sgRNA testing better predicts who is truly infectious. These findings support sgRNA as a useful tool to guide clinical management and infection control in vulnerable patients.

Humans↗

PubMed2004

Unusual microbial xylanases from insect guts.

Recombinant DNA technologies enable the direct isolation and expression of novel genes from biotopes containing complex consortia of uncultured microorganisms. In this study, genomic libraries were constructed from microbial DNA isolated from insect intestinal tracts from the orders Isoptera (termites) and Lepidoptera (moths). Using a targeted functional assay, these environmental DNA libraries were screened for genes that encode proteins with xylanase activity. Several novel xylanase enzymes with unusual primary sequences and novel domains of unknown function were discovered. Phylogenetic analysis demonstrated remarkable distance between the sequences of these enzymes and other known xylanases. Biochemical analysis confirmed that these enzymes are true xylanases, which catalyze the hydrolysis of a variety of substituted beta-1,4-linked xylose oligomeric and polymeric substrates and produce unique hydrolysis products. From detailed polyacrylamide carbohydrate electrophoresis analysis of substrate cleavage patterns, the xylan polymer binding sites of these enzymes are proposed.

Amino Acid Sequence↗

PubMed2004

A central role for tissue-resident dendritic cells in innate responses.

In the past 20 years, dendritic cells (DCs) have been investigated mainly with regard to antigen-specific immune responses and acquired immunity. More recently, new data have indicated a novel and fundamental role for DCs in innate immunity, which is shown by the ability of DCs to induce natural killer (NK) cell activation. In mice, the molecular mechanism by which NK cells are activated by DCs has been revealed, and it consists of the induction of interleukin-2 (IL-2) expression during the early phases of an immune response. Moreover, as indicated by genome-wide transcriptional analysis, microbially activated myeloid DCs produce other chemokines and inflammatory cytokines, such as type I interferons, which in addition to IL-2 are also involved in NK cell activation. This indicates that DCs have a key role in innate immunity. The expression of an innate receptor repertoire on DCs mediates microbial recognition and uptake, and the antigen processing and specific sorting mechanisms lead to very efficient antigen presentation by DCs. Thus, we believe that DCs participate fully in innate responses, forming a bridge between innate and acquired immunity.

Animals↗

PubMed2005

Lethality and synthetic lethality in the genome-wide metabolic network of Escherichia coli.

Recent genomic analyses on the cellular metabolic network show that reaction flux across enzymes are diverse and exhibit power-law behavior in its distribution. While intuition might suggest that the reactions with larger fluxes are more likely to be lethal under the blockade of its catalysing gene products or gene knockouts, we find, by in silico flux analysis, that the lethality rarely has correlations with the flux level owing to the widespread backup pathways innate in the genome-wide metabolism of Escherichia coli. Lethal reactions, of which the deletion generates cascading failure of following reactions up to the biomass reaction, are identified in terms of the Boolean network scheme as well as the flux balance analysis. The avalanche size of a reaction, defined as the number of subsequently blocked reactions after its removal, turns out to be a useful measure of lethality. As a means to elucidate phenotypic robustness to a single deletion, we investigate synthetic lethality in reaction level, where simultaneous deletion of a pair of nonlethal reactions leads to the failure of the biomass reaction. Synthetic lethals identified via flux balance and Boolean scheme are consistently shown to act in parallel pathways, working in such a way that the backup machinery is compromised.

Escherichia coli↗

PubMed2004

Comparative genomics identified two conserved DNA modules in a corynebacterial plasmid family present in clinical isolates of the opportunistic human pathogen Corynebacterium jeikeium.

Investigation of 62 clinical isolates of the opportunistic human pathogen Corynebacterium jeikeium revealed that 17 possessed plasmids ranging in size from 7.6 to 14.9 kb. The plasmids formed four groups on DNA restriction analysis. The complete nucleotide sequence of a representative from each group (pK43, pK64, pCJ84, and pB85766) was subsequently determined. Additionally, two plasmids (pCo455 and pCo420) were shown to be derivatives of pK43 and pK64 carrying insertion sequences of the IS3 family. Comparative genomics identified a conserved plasmid backbone consisting of two distinct DNA modules. Conserved motifs in the parAB-repA module indicated that the sequenced plasmids from C. jeikeium are new members of the pNG2 family. Recombinant derivatives of pK43 were shown to replicate in the soil bacterium Corynebacterium glutamicum and in the human pathogen Corynebacterium diphtheriae. The second plasmid module most likely encodes a novel type of DNA invertase. The respective gene is flanked by highly conserved 112-bp inverted repeats. All plasmids are 'loaded' with a characteristic set of genes encoding products of unknown function. Plasmids indistinguishable from pK43 by DNA restriction analysis were identified in different C. jeikeium strains, which revealed 16S-23S rDNA spacer length polymorphisms and specific antibiotic susceptibility profiles, implying a wide dissemination of the plasmid in clinical isolates of C. jeikeium.

Amino Acid Sequence↗

PubMed2005

Epidemiological study on Mycoplasma synoviae infection in layers.

Mycoplasma synoviae infection occurs worldwide in commercial poultry flocks and may result in severe economic losses. The prevalence of this mycoplasma in standard layers older than 60 weeks was studied in a French department and the characteristics of infected or free flocks were compared. The genomic profiles of isolates from 36 infected flocks were studied by pulsed-field gel electrophoresis and random amplified polymorphic DNA methods in order to investigate possible routes of transmission. The minimum inhibitory concentrations of antibiotics were determined. Results showed that infection was more frequent in multi-age farms. Egg production and mortality of infected flocks were respectively lower and higher than in non-infected flocks but the differences were not statistically significant. The genomic profiles of isolates were quite homogeneous, a feature which does not facilitate the understanding of routes of transmission. All isolates were susceptible to tetracyclines, macrolides (except erythromycin), spectinomycin and fluoroquinolones.

Animals↗

PubMed2006

Derivation of a novel SARS-coronavirus replicon cell line and its application for anti-SARS drug screening.

The severe acute respiratory syndrome (SARS) outbreak in 2002, which had a high morbidity rate and caused worldwide alarm, remains untreated today even though SARS was eventually isolated and controlled. Development and high-throughput screening of efficacious drugs is therefore critical. However, currently there remains a lack of such a safe system. Here, the generation and characterization of the first selectable, SARS-coronavirus (SARS-CoV)-based replicon cell line which can be used for screening is described. Partial SARS-CoV cDNAs and antibiotic resistance/reporter gene DNA were generated and assembled in vitro to produce the replicon transcription template, which was then transcribed in vitro to generate the replicon RNA. The latter was introduced into a mammalian cell line and the transfected cells were selected for by antibiotic application. For the antibiotic-resistant cell lines thus generated, the expression of reporter gene was ensured by continued monitoring using fluorescent microscopy and flow cytometry. The suitability of this replicon cell line in drug screening was demonstrated by testing the inhibitory effect of several existing drugs and the results demonstrate that the SARS-CoV replicon cell lines provide a safe tool for the identification of SARS-CoV replicase inhibitors. The replicon cell lines thus developed can be applied to high-throughput screening for anti-SARS drugs without the need to grow infectious SARS-CoV.

Animals↗

PubMed2001

An antisense-based functional genomics approach for identification of genes critical for growth of Candida albicans.

Converting the complete genome sequence of Candida albicans into meaningful biological information will require comprehensive screens for identifying functional classes of genes. Most systems described so far are not applicable to C. albicans because of its difficulty with mating, its diploid nature, and the lack of functional random insertional mutagenesis methods. We examined artificial gene suppression as a means to identify gene products critical for growth of this pathogen; these represent new antifungal drug targets. To achieve gene suppression we combined antisense RNA inhibition and promoter interference. After cloning antisense complementary DNA (cDNA) fragments under control of an inducible GAL1 promoter, we transferred the resulting libraries to C. albicans. Over 2,000 transformant colonies were screened for a promoter-induced diminished-growth phenotype. After recovery of the plasmids, sequence determination of their inserts revealed the messenger RNA (mRNA) they inhibited or the gene they disrupted. Eighty-six genes critical for growth were identified, 45 with unknown function. When used in high-throughput screening for antifungals, the crippled C. albicans strains generated in this study showed enhanced sensitivity to specific drugs.

Antifungal Agents↗

PubMed2026

Enhanced invasiveness promotes the dominance of a widely-distributed carbapenem-resistant virulence-plasmid-carrying Klebsiella pneumoniae sublineage.

Carbapenem-resistant Klebsiella pneumoniae (CRKP), a WHO's critical priority pathogen, continuously evolves to generate health-threatening high-risk (sub)lineages. Here, we conducted a 10-year surveillance of nosocomial CRKP infections, collecting and whole-genome sequencing 1513 clinical isolates, accompanied by clinical data. We applied fine-scale genome analysis for 60,724 non-local public-available K. pneumoniae genomes. We identified a predominant ST11-KL64 sublineage widely-disseminated across China and internationally-distributed. Isolates of this sublineage were more frequently recovered during seasonal influenza peaks and harbored plasmids encoding 'hypervirulence' factors but caused no increase in patient mortality. Instead, they exhibited enhanced invasiveness and translocation capacity. Mechanistically, this suggested highly invasive CRKP (hiCRKP) sublineage showed elevated resistance to macrophage-mediated phagocytosis, partly due to the virulence-encoding plasmid and the loss of two chromosomal fimD gene copies. Additionally, hiCRKP isolates carried more antimicrobial resistance genes, resulting in enhanced resistance to clinically important quinolones and tetracyclines. To address the hiCRKP-imposed challenge, we constituted a phage cocktail, which significantly improved survival in a murine infection model. Our findings unveil a clinically-relevant high-risk sublineage resulted from the ongoing evolutionary diversification of CRKP. Importantly, the 'hypervirulent' CRKP is more potent to cause diseases rather than the thought-to-be more deaths, explaining its rapid emergence in healthcare settings.

Klebsiella pneumoniae↗

PubMed2003

LexA-independent DNA damage-mediated induction of gene expression in Myxococcus xanthus.

Myxococcus xanthus, a member of the Proteobacteria delta-class, has two independent recA genes, recA1 and recA2, but only recA2 is DNA damage-inducible. The lexA gene has been isolated from M. xanthus by PCR amplification with oligonucleotides designed after sequence identification by tblastn analysis of its genome at the Cereon Microbial Sequence Database. The M. xanthus purified LexA protein is shown to bind specifically to the consensus sequence CTRHAMRYBYGTTCAGS present upstream of lexA and recA2. A degenerate copy of this motif but with important differences can be identified in the region upstream of the recA1 gene. A knock-out lexA(Def) mutant that has been generated does not differ significantly from wild type in morphology, growth rate, light-induced carotenogenesis or development. Using transcriptional lacZ fusions and quantitative RT-PCR analysis, it has been demonstrated that expression of both lexA and recA2 genes is constitutive in the lexA(Def) mutant, whereas the transcription of the DNA damage non-inducible recA1 gene is not affected in this strain. recN and ssb, whose expression in Escherichia coli are LexA-regulated, are induced by DNA damage in the M. xanthus lexA(Def) mutant. These data reveal the existence of different regulatory mechanisms for DNA damage-inducible genes in bacteria belonging to different phyla.

Bacterial Proteins↗

PubMed2005

[Expanding the known diversity and environmental distribution of cultured and uncultured bacteria].

Microorganisms represent the largest component of biodiversity in our biosphere. Traditional methods of bacterial identification depend on their culture on laboratory media and the comparison of their phenotypic characteristics. They include cellular morphology, motility, staining reactions of cell walls, ability to grow on different media and biochemical tests. These methods have many limitations and only a very small fraction of microorganisms have been cultivated. To date, molecular methods based on 16S rRNA sequences and their phylogenetic analysis are widely used for reliable identification, particularly for hard-to-culture microbial pathogens. These so-called << molecular methods >> do not require laboratory culture of isolated organisms, and many novel non-described phyla have been detected, improving our view of bacterial diversity. Novel strategies for culturing the << uncultivated >> are now under development, which are leading to the complete characterization of these new bacteria. More recently, meta- or ecogenomics, based on the complete sequencing of clones containing cosmids or bacterial artificial chromosomes with inserts, addresses the genetic potential of a sample irrespective of whether the microorganisms can be cultured or not. This has considerably extended our view of microbial diversity at the genomic level and the probability of finding new genes and their products suitable for the biotechnological and pharmaceutical industry.

Bacteria↗

PubMed2004

Yeast genome-wide drug-induced haploinsufficiency screen to determine drug mode of action.

Methods to systematically test drugs against all possible proteins in a cell are needed to identify the targets underlying their therapeutic action and unwanted effects. Here, we show that a genome-wide drug-induced haploinsufficiency screen by using yeast can reveal drug mode of action in yeast and can be used to predict drug mode of action in human cells. We demonstrate that dihydromotuporamine C, a compound in preclinical development that inhibits angiogenesis and metastasis by an unknown mechanism, targets sphingolipid metabolism. The systematic, unbiased and genome-wide nature of this technique makes it attractive as a general approach to identify cellular pathways affected by drugs.

Animals↗

PubMed2003

Novel functions of the phosphatidylinositol metabolic pathway discovered by a chemical genomics screen with wortmannin.

We report a novel connection between the phosphatidylinositol (PI) metabolic pathway and the DNA replication and damage checkpoint pathway discovered from an unbiased chemical genomics screen. Substrates and products of PI kinases are important signaling molecules that affect a wide range of biological processes. The full collection of yeast deletion strains was screened to identify genes that confer altered sensitivity to the natural product wortmannin, a PI kinase inhibitor. These experiments have allowed us to explore metabolomic and proteomic implications of PI synthesis and turnover. This study also uncovers other biological processes affected by wortmannin treatment, including proteasome-mediated degradation and chromatin remodeling. Bioinformatic analyses were used to reveal the relative distances among cellular processes affected by wortmannin and protein-protein interactions in the wortmannin-sensitive proteomic subnetwork. These results illustrate the great utility of using a whole-genome approach in annotating the biological effects of small molecules and have clear implications for pharmacogenomics. Furthermore, our discovery points to a route to overcoming genome instability, a result of defective DNA damage signaling/repair and a hallmark of cancer.

Androstadienes↗

PubMed2000

Genomewide insertional mutagenesis in Streptomyces coelicolor reveals additional genes involved in morphological differentiation.

The filamentous soil bacterium Streptomyces coelicolor undergoes a complex cycle of morphological differentiation involving the formation of an aerial mycelium and the production of pigmented antibiotics. We have developed a procedure for generating insertional mutants of S. coelicolor based on in vitro transposition of a plasmid library of cloned S. coelicolor DNAs. The insertionally mutated library was introduced into S. coelicolor, and transposon insertions were recovered at widely scattered locations around the chromosome. Many of the insertions revealed previously uncharacterized genes, and several caused novel mutant phenotypes, such as altered pigment production, enhanced antibiotic sensitivity, delayed or impaired formation of aerial hyphae, and a block in spore formation. The sporulation mutant harbored an insertion in one of three adjacent genes that are apparently unique to Streptomyces but are each represented by at least 20 paralogs at dispersed locations in the chromosome. Individual members of the three families often are found grouped together in a characteristic arrangement, suggesting that they have a common function.

Amino Acid Sequence↗

PubMed2000

A chemical genomics approach toward understanding the global functions of the target of rapamycin protein (TOR).

The target of rapamycin protein (TOR) is a highly conserved ataxia telangiectasia-related protein kinase essential for cell growth. Emerging evidence indicates that TOR signaling is highly complex and is involved in a variety of cellular processes. To understand its general functions, we took a chemical genomics approach to explore the genetic interaction between TOR and other yeast genes on a genomic scale. In this study, the rapamycin sensitivity of individual deletion mutants generated by the Saccharomyces Genome Deletion Project was systematically measured. Our results provide a global view of the rapamycin-sensitive functions of TOR. In contrast to conventional genetic analysis, this approach offers a simple and thorough analysis of genetic interaction on a genomic scale and measures genetic interaction at different possible levels. It can be used to study the functions of other drug targets and to identify novel protein components of a conserved core biological process such as DNA damage checkpoint/repair that is interfered with by a cell-permeable chemical compound.

Cell Cycle Proteins↗

PubMed2003

Natural selection and algorithmic design of mRNA.

Messenger RNA (mRNA) sequences serve as templates for proteins according to the triplet code, in which each of the 4(3) = 64 different codons (sequences of three consecutive nucleotide bases) in RNA either terminate transcription or map to one of the 20 different amino acids (or residues) which build up proteins. Because there are more codons than residues, there is inherent redundancy in the coding. Certain residues (e.g., tryptophan) have only a single corresponding codon, while other residues (e.g., arginine) have as many as six corresponding codons. This freedom implies that the number of possible RNA sequences coding for a given protein grows exponentially in the length of the protein. Thus nature has wide latitude to select among mRNA sequences which are informationally equivalent, but structurally and energetically divergent. In this paper, we explore how nature takes advantage of this freedom and how to algorithmically design structures more energetically favorable than have been built through natural selection. In particular: (1) Natural Selection--we perform the first large-scale computational experiment comparing the stability of mRNA sequences from a variety of organisms to random synonymous sequences which respect the codon preferences of the organism. This experiment was conducted on over 27,000 sequences from 34 microbial species with 36 genomic structures. We provide evidence that in all genomic structures highly stable sequences are disproportionately abundant, and in 19 of 36 cases highly unstable sequences are disproportionately abundant. This suggests that the stability of mRNA sequences is subject to natural selection. (2) Artificial Selection--motivated by these biological results, we examine the algorithmic problem of designing the most stable and unstable mRNA sequences which code for a target protein. We give a polynomial-time dynamic programming solution to the most stable sequence problem (MSSP), which is asymptotically no more complex than secondary structure prediction. We show that the corresponding least stable sequence problem (LSSP) is NP-complete, and develop two heuristics for the construction of such sequences. We have implemented these algorithms, and present experimental results placing the high/low stability sequences in context with both wildtype and random encodings. Our implementation has already been applied to the design of RNA "code-words" creating little or no secondary structure in RNA computing (Brenneman and Condon, 2001; Marathe et al., 2001), and we anticipate a variety of other applications of this work to sequence design problems (Skiena, 2001).

Algorithms↗

PubMed2006

The role of several multidrug resistance systems in Erwinia chrysanthemi pathogenesis.

The role of several multidrug resistance (MDR) systems in the pathogenicity of Erwinia chrysanthemi 3937 was analyzed. Using the blast algorithm, we have identified several MDR systems in the E. chrysanthemi genome and selected two acridine resistance (Acr)-like systems, two Emr-like systems, and one member of the major facilitator super-family family to characterize. We generated mutants in genes encoding for these systems and analyzed the virulence of the mutant strains in different hosts and their susceptibility to antibiotics, detergents, dyes, and plant compounds. We have observed that the mutant strains are differentially affected in their virulence in different hosts and that the susceptibility to toxic substances is also differential. Both Acr systems seem to be implicated in the resistance to the plant antimicrobial peptide thionin. Similarly, the emr1AB mutant is unable to grow in the presence of the potato protein tuber extract and shows a decreased virulence in this tissue. These results indicate that the function of these systems in plants could be related to the specificity to extrude a toxic compound that is present in a given host.

Bacterial Proteins↗

PubMed2005

Generation of an erythromycin-sensitive derivative of Clostridium difficile strain 630 (630Deltaerm) and demonstration that the conjugative transposon Tn916DeltaE enters the genome of this strain at multiple sites.

Erythromycin resistance in Clostridium difficile strain 630 is conferred by a genetic element termed Tn5398 which contains two erm(B) genes: erm1(B) and erm2(B). An erythromycin-sensitive derivative of strain 630 (designated 630Deltaerm) was generated by spontaneous mutation after continuous subculture for 30 days. This strain had lost the erm2(B) gene from within Tn5398 but retained erm1(B). However, the strain could revert to erythromycin resistance at a frequency of 2.79 x 10(-8), although it still contained the deletion of erm2(B). The availability of C. difficile 630Deltaerm allowed the behaviour of Tn916DeltaE to be investigated in this strain. This element entered the genome at multiple sites indicating that it could be useful as an insertional mutagen.

Blotting, Southern↗