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Serotype-specific identification of polioviruses by PCR using primers containing mixed-base or deoxyinosine residues at positions of codon degeneracy.

We have developed a method for determining the serotypes of poliovirus isolates by PCR. Three sets of serotype-specific antisense PCR-initiating primers (primers seroPV1A, seroPV2A, and seroPV3A) were designed to pair with codons of VP1 amino acid sequences that are conserved within but that differ across serotypes. The sense polarity primers (primers seroPV1S, seroPV2S, and seroPV3S) matched codons of more conserved capsid sequences. The primers contain mixed-base and deoxyinosine residues to compensate for the high rate of degeneracy of the targeted codons. The serotypes of all polioviruses tested (48 vaccine-related isolates and 110 diverse wild isolates) were correctly identified by PCR with the serotype-specific primers. None of the genomic sequences of 49 nonpolio enterovirus reference strains were amplified under equivalent reaction conditions with any of the three primer sets. These primers are useful for the rapid screening of poliovirus isolates and for determining the compositions of cultures containing mixtures of poliovirus serotypes.

Amino Acid Sequence↗

Marburgvirus genomics and association with a large hemorrhagic fever outbreak in Angola.

In March 2005, the Centers for Disease Control and Prevention (CDC) investigated a large hemorrhagic fever (HF) outbreak in Uige Province in northern Angola, West Africa. In total, 15 initial specimens were sent to CDC, Atlanta, Ga., for testing for viruses associated with viral HFs known to be present in West Africa, including ebolavirus. Marburgvirus was also included despite the fact that the origins of all earlier outbreaks were linked directly to East Africa. Surprisingly, marburgvirus was confirmed (12 of 15 specimens) as the cause of the outbreak. The outbreak likely began in October 2004 and ended in July 2005, and it included 252 cases and 227 (90%) fatalities (report from the Ministry of Health, Republic of Angola, 2005), making it the largest Marburg HF outbreak on record. A real-time quantitative reverse transcription-PCR assay utilized and adapted during the outbreak proved to be highly sensitive and sufficiently robust for field use. Partial marburgvirus RNA sequence analysis revealed up to 21% nucleotide divergence among the previously characterized East African strains, with the most distinct being Ravn from Kenya (1987). The Angolan strain was less different ( approximately 7%) from the main group of East African marburgviruses than one might expect given the large geographic separation. To more precisely analyze the virus genetic differences between outbreaks and among viruses within the Angola outbreak itself, a total of 16 complete virus genomes were determined, including those of the virus isolates Ravn (Kenya, 1987) and 05DRC, 07DRC, and 09DRC (Democratic Republic of Congo, 1998) and the reference Angolan virus isolate (Ang1379v). In addition, complete genome sequences were obtained from RNAs extracted from 10 clinical specimens reflecting various stages of the disease and locations within the Angolan outbreak. While the marburgviruses exhibit high overall genetic diversity (up to 22%), only 6.8% nucleotide difference was found between the West African Angolan viruses and the majority of East African viruses, suggesting that the virus reservoir species in these regions are not substantially distinct. Remarkably few nucleotide differences were found among the Angolan clinical specimens (0 to 0.07%), consistent with an outbreak scenario in which a single (or rare) introduction of virus from the reservoir species into the human population was followed by person-to-person transmission with little accumulation of mutations. This is in contrast to the 1998 to 2000 marburgvirus outbreak, where evidence of several virus genetic lineages (with up to 21% divergence) and multiple virus introductions into the human population was found.

Angola↗

Rapid and accurate sepsis diagnostics via a novel probe-based multiplex real-time PCR system.

Sepsis is a critical clinical emergency that requires prompt diagnosis and intervention. Its prevalence has increased due to the aging population and increased antibiotic resistance. Early identification and the use of innovative technologies are crucial for improving patient outcomes. Modern methodologies are needed to minimize the turnaround time for diagnosis and improve outcomes. Rapid diagnostic tests and multiplex PCR are effective but have limitations in identifying a range of pathogens and target genes. Our study evaluated two novel probe-based multiplex real-time PCR systems: the SEPSI ID and SEPSI DR panels. These systems can quickly identify bacterial and fungal pathogens, alongside antibiotic resistance genes. The assays cover 29 microorganisms (gram-negative bacteria, gram-positive bacteria, yeast, and mold species), alongside 23 resistance genes and four virulence factors. A streamlined workflow uses 2 µL of broth from positive blood cultures (BCs) without nucleic acid extraction and provides results in approximately 1 h. We present the results from an evaluation of 228 BCs and 22 isolates previously characterized by whole-genome sequencing. In comparison to the reference methods, the SEPSI ID panel demonstrated a sensitivity of 96.88%, a specificity of 100%, and a PPV of 100%, whereas the SEPSI DR panel showed a sensitivity of 97.8%, a PPV of 89.7%, and a specificity of 96.7%. Both panels also identified additional pathogens and resistance-related targets not detected by conventional methods. This assay shows promise for rapidly and accurately diagnosing sepsis. Future studies should validate its performance in various clinical settings to enhance sepsis management and improve patient outcomes.IMPORTANCEWe present a new diagnostic method that enables the quick and precise identification of pathogens and resistance genes from positive blood cultures, eliminating the need for nucleic acid extraction. This technique can also be used on fresh pathogen cultures. It has the potential to greatly improve treatment protocols, leading to better patient outcomes, more responsible antibiotic use, and more efficient management of healthcare resources.

Humans↗

Complete genomes from a xenic Dolichospermum flosaquae FBCC-A233 culture reveal genome-inferred metabolic asymmetry with associated bacteria.

Cyanobacteria form phycosphere communities with associated bacteria, but genome-resolved resources are needed to formulate testable hypotheses about their metabolic interactions. Here, we reconstructed three complete circular genomes from a unialgal xenic culture, including Dolichospermum flosaquae FBCC-A233 and two associated alphaproteobacterial genomes assigned to Sphingorhabdus sp. and Brevundimonas sp. Genome-wide read mapping and genome-quality assessment supported the three recovered genomes as high-quality circular reconstructions. Comparative genome analysis placed the cyanobacterial genome within the Dolichospermum flosaquae species cluster under the GTDB framework, while the associated bacterial genomes represented Sphingorhabdus sp. and a putative undescribed Brevundimonas species-level lineage. Genome architecture analysis indicated reduced genome size and gene content in Brevundimonas relative to genus-level references although additional metrics did not support a strong conclusion of classical genome streamlining. Selected KEGG module and KO-level reconstructions indicated genome-inferred metabolic asymmetries across the consortium. FBCC-A233 encoded photosynthesis- and nitrogen-related modules and a BioU-mediated de novo biotin biosynthesis route, whereas the associated bacteria lacked complete de novo biotin biosynthesis but retained biotin-dependent carboxylase genes. FBCC-A233 also encoded extensive anaerobic corrinoid biosynthesis potential; however, canonical DMB-containing cobalamin completion, cobamide identity, and complete transporter systems were not resolved. Together, these complete genomes provide a genome-resolved resource for investigating genome-inferred metabolic differentiation and ecological interactions in cyanobacteria-associated bacterial consortia.IMPORTANCEPhycosphere interactions between cyanobacteria and associated bacteria can shape aquatic microbial communities, but many proposed interactions remain difficult to evaluate without genome-resolved resources. This study provides three complete circular genomes from a unialgal xenic Dolichospermum flosaquae culture, capturing the cyanobacterium and two co-maintained bacterial associates. Our analysis identifies genome-inferred metabolic asymmetries, particularly in biotin- and cobamide-related pathways. D. flosaquae FBCC-A233 encoded candidate de novo biotin and corrinoid biosynthesis capacity, whereas the associated bacteria lacked complete de novo pathways but retained cofactor-dependent enzymes. These findings nominate cofactor-related dependencies as experimentally testable hypotheses while emphasizing unresolved uptake, export, cobamide identity, and growth-dependence mechanisms. The complete genomes and KO-level reconstructions generated here provide a resource for future studies of cyanobacteria-associated consortia.

Genome, Bacterial↗

Proteomic strategies and their application in cancer research.

The understanding of carcinogenesis and tumor progression on a molecular basis needs a detailed study of proteins as effector molecules and as critical components of the multiple interconnected signaling pathways that drive the neoplastic phenotype. Thus, the proteomic approach represents a powerful tool for the challenge of the post-genomic era. The term "cancer proteome" refers to the collection of proteins expressed by a given cancer cell and should be considered as a highly dynamic entity within the cell, which affects a variety of cellular activities. The emerging proteomic analysis platforms including 2D-PAGE, mass spectrometry technologies, and protein microarrays represent powerful tools to study and understand cancer. These systems aim to not only identify, catalogue, and characterize cancer proteins, but also to unveil how they interact to affect overall tumor progression. Moreover, recent studies on various cancers have reported promising results concerning the detection of novel molecular biomarkers useful in the early diagnosis of cancer and in drug discovery. Thus, a new subdiscipline named clinical proteomics, concomitant with new molecular technologies that are developed, demonstrates promise to discover new cancer biomarkers. The early diagnosis of cancer, even in a premalignant state, is crucial for the successful treatment of this disease. For these reasons, it is clear that the identification of biomarkers for the early diagnosis of cancer should represent one of the main goals of this emerging field of study.

Animals↗

Array-CGH and breast cancer.

The introduction of comparative genomic hybridization (CGH) in 1992 opened new avenues in genomic investigation; in particular, it advanced analysis of solid tumours, including breast cancer, because it obviated the need to culture cells before their chromosomes could be analyzed. The current generation of CGH analysis uses ordered arrays of genomic DNA sequences and is therefore referred to as array-CGH or matrix-CGH. It was introduced in 1998, and further increased the potential of CGH to provide insight into the fundamental processes of chromosomal instability and cancer. This review provides a critical evaluation of the data published on array-CGH and breast cancer, and discusses some of its expected future value and developments.

Breast Neoplasms↗

The phylogenetic relationships of insectivores with special reference to the lesser hedgehog tenrec as inferred from the complete sequence of their mitochondrial genome.

The complete mitochondrial genome of a lesser hedgehog tenrec Echinops telfairi was determined in this study. It is an endemic African insectivore that is found specifically in Madagascar. The tenrec's back is covered with hedgehog-like spines. Unlike other spiny mammals, such as spiny mice, spiny rats, spiny dormice and porcupines, lesser hedgehog tenrecs look amazingly like true hedgehogs (Erinaceidae). However, they are distinguished morphologically from hedgehogs by the absence of a jugal bone. We determined the complete sequence of the mitochondrial genome of a lesser hedgehog tenrec and analyzed the results phylogenetically to determine the relationships between the tenrec and other insectivores (moles, shrews and hedgehogs), as well as the relationships between the tenrec and endemic African mammals, classified as Afrotheria, that have recently been shown by molecular analysis to be close relatives of the tenrec. Our data confirmed the afrotherian status of the tenrec, and no direct relation was recovered between the tenrec and the hedgehog. Comparing our data with those of others, we found that within-species variations in the mitochondrial DNA of lesser hedgehog tenrecs appear to be the largest recognized to date among mammals, apart from orangutans, which might be interesting from the view point of evolutionary history of tenrecs on Madagascar.

Animals↗

Clinical genomics data standards for pharmacogenetics and pharmacogenomics.

This special report concerns a talk on data standards given at a workshop entitled 'An International Perspective on Pharmacogenetics: The Intersections between Innovation, Regulation and Health Delivery', which was held by the Organization for Economic Co-operation and Development (OECD) on October 17-19, 2005, in Rome, Italy. The worlds of healthcare and life sciences (HCLS) are extremely fragmented in terms of their underlying information technology, making it difficult to semantically exchange information between disparate entities. While we have reached the point where functional interoperability is ubiquitous, we are still far from achieving true semantic interoperability where a receiving system can use incoming data as though it was created internally. The critical enablers of semantic interoperability are information standards dedicated to HCLS data, spanning all the way from biological research data to clinical research and clinical trials, and finally to healthcare clinical data. The challenge lies in integrating various data standards based on predetermined goals, thereby improving the quality of care provided to patients.

Clinical Medicine↗

[Expression of the fusion protein CFP10-ESAT6 of Mycobacterium tuberculosis and the study of its immunogenicity].

OBJECTIVE: To express a recombinant fusion protein CFP10-ESAT6 of Mycobacterium tuberculosis, and obtain the polyclonal antibodies of this fusion protein by immune rabbit. METHODS: The 630 bp cfpl0-esat6 fusion gene fragments were amplified from the genomic DNA of a Mycobacterium tuberculosis reference strain H37Rv and inserted into the expression plasmid pET32a (+) to generate the recombinant plasmid pET-cfp10-esat6. The recombinat expression plasmid was transformed into E. coli BL21 (DE3). The fused protein CFP10-ESAT6 with His-tag was expressed after inducing with IPTG and purified with affinity chromatography. This protein was used to immune the rabbit to obtained the polyclonal antibodies, and been analyzed with Western-blot and ELISA. RESULTS: The recombinant plasmid pET-cfp10-esat6 was success fully constructed, the recombinant fusion protein CFP10-ESAT6 could be expressed at relatively high levels, and the polyclonal antibodies of fusion protein were obtained. CONCLUSION: The successful construction and expression of the recombinant fusion protein CFP10-ESAT6 and the obtained polyclonal antibodies will be very helpful for the development of new anti-tuberculosis vaccine and the clinical serologic diagnosis.

Animals↗

Detection and characterization of neonatal cytomegalovirus through nanopore sequencing using flongle flow cells: Pilot study in Philadelphia, Pennsylvania.

BACKGROUND: Cytomegalovirus (CMV) remains a significant infection in neonates and its early detection can aid with further treatment (antiviral, audiology). However, current diagnostics do not provide genetic information. OBJECTIVE: We explored the use of the portable and comprehensive sequencing method from Oxford Nanopore Technologies, utilizing low-cost Flongle flow cells to detect and perform sequence-level characterization of neonatal urine samples that tested positive for CMV by PCR. STUDY DESIGN: We performed a pilot study based on a retrospective cohort study of neonates who were positive for CMV by PCR, who were admitted at two birth hospitals in Philadelphia, PA. We leveraged deep and long-read sequencing results to analyze the reads in two forms: by comparing them against a reference-based strain and by reconstructing the genome through de novo assembly with phylogenetic tree analysis. RESULTS: We assayed seven clinical samples, including a positive and negative control sample, from newborns ranging from 23 weeks' gestation to term, with testing performed for microcephaly, hearing test results, small gestational age, and thrombocytopenia. Each sample showed multiple differences compared to the reference strain, and the phylogenetic tree analysis of the de novo assembly depicted the genetic diversity of the samples. CONCLUSION: This pilot study shows that nanopore sequencing with low-cost Flongle flow cells can detect and characterize CMV strains from clinical neonatal urine samples. This, coupled with current screening and diagnostic criteria, could further our genomic understanding of neonatal CMV, such as viral genome diversity, genotype-phenotype associations, and spread of strains.

Humans↗

Mosaic bacterial chromosomes: a challenge en route to a tree of genomes.

In a recent analysis J.G. Lawrence and H. Ochman [Proc Natl Acad Sci USA 1998;95:9413-9417 (Reference 1)] surmised that about 10% of the current E. coli genome consists of genes that were acquired in over 200 events of lateral gene transfer, which occurred subsequent to the divergence of E. coli and Salmonella some 100 million years ago. Overall, the data suggest that no less than 18% of E. coli's genes might be relatively recent foreign acquisitions, and that the average rate of acquisition may be close to about 16 kb per million years. These quantitative estimates of comparatively recent genome flux have profound impact on evolutionary genome comparisons. They tend to suggest that a search should be on to identify principles that might ultimately govern gene distribution patterns across prokaryotic genomes.

Biological Evolution↗

DBTBS: database of transcriptional regulation in Bacillus subtilis and its contribution to comparative genomics.

DBTBS (http://dbtbs.hgc.jp) was originally released in 1999 as a reference database of published transcriptional regulation events in Bacillus subtilis, one of the best studied bacteria. It is essentially a compilation of transcription factors with their regulated genes as well as their recognition sequences, which were experimentally characterized and reported in the literature. Here we report its major update, which contains information on 114 transcription factors, including sigma factors, and 633 promoters of 525 genes. The number of references cited in the database has increased from 291 to 378. It also supports a function to find putative transcription factor binding sites within input sequences by using our collection of weight matrices and consensus patterns. Furthermore, though preliminarily, DBTBS now aims to contribute to comparative genomics by showing the presence or absence of potentially orthologous transcription factors and their corresponding cis-elements on the promoters of their potentially orthologously regulated genes in 50 eubacterial genomes.

Bacillus subtilis↗

Occurrence of multiple genomovars of Burkholderia cepacia in cystic fibrosis patients and proposal of Burkholderia multivorans sp. nov.

We performed an integrated genotypic and phenotypic analysis of 128 strains of the genera Burkholderia, Ralstonia, and Pseudomonas in order to study the taxonomic structure of Burkholderia cepacia and its relationships with other Burkholderia species. Our data show that presumed B. cepacia strains isolated from cystic fibrosis patients belong to at least five distinct genomic species, one of which was identified as Burkholderia vietnamiensis. This group of five phenotypically similar species is referred to as the B. cepacia complex. The name Burkholderia multivorans is proposed for one of these genomic species, which was formerly referred to as B. cepacia genomovar II; the remaining B. cepacia groups are referred to as genomovars I, III, and IV, pending additional differential phenotypic tests. The role and pathogenic potential of each of these taxa, particularly in view of the potentially fatal infections in cystic fibrosis patients, need further evaluation. The data presented also demonstrate that Pseudomonas glathei and Pseudomonas pyrrocinia should be reclassified as Burkholderia species.

Bacterial Proteins↗

Identification of two processed psuedogenes of the human Tom20 gene.

The open reading frame (ORF) of the human Tom20 gene (hTom20) was amplified by PCR from a HeLa cDNA library using primers based on the sequence of HUMRSC145 and cloned into a pET15b vector. Amplification of human genomic DNA using these primers yielded a DNA fragment of the same size as that of the ORF of hTom20 cDNA. Sequencing of this fragment revealed that: (1) it has the same number of base pairs as the ORF of hTom20 cDNA (438 bp); and (2) the two sequences differ by 14 single base pair substitutions (97% similarity) causing eight changes in the amino acid sequence and two premature stop codons. Further amplification of human genomic DNA adaptor-ligated libraries using primers based on HUMRSC145 revealed three different sequence-related genomic regions; one corresponding to the fragment referred above, another corresponding to the hTom20 gene, and a third fragment of which the sequence differs from the ORF of hTom20 cDNA by only 22 base pair substitutions and a deletion of 4 bp. We conclude that, in addition to the hTom20 gene, there are two genomic DNA sequences (psi1Tom20 and psi2Tom20) that are processed pseudogenes of hTom20. Aspects concerning their evolutionary origin are discussed.

Base Sequence↗

Nonrandom location of IS1 elements in the genomes of natural isolates of Escherichia coli.

We have studied the spatial distribution of IS1 elements in the genomes of natural isolates comprising the ECOR reference collection of Escherichia coli. We find evidence for nonrandomness at three levels. Many pairs of IS1 elements are in much closer proximity (< 10 kb) than can be accounted for by chance. IS1 elements in close proximity were identified by long-range PCR amplification of the genomic sequence between them. Each amplified region was sequenced and its map location determined by database screening of DNA hybridization. Among the ECOR strains with at least two IS1 elements, 54% had one or more pairs of elements separated by < 10 kb. We propose that this type of clustering is a result of "local hopping," in which we assume that a significant proportion of tranposition events leads to the insertion of a daughter IS element in the vicinity of the parental element. A second level of nonrandomness is found in strains with a modest number of IS1 elements that are mapped through the use of inverse PCR to amplify flanking genomic sequences: in these strains, the insertion sites tend to be clustered over a smaller region of chromosome than would be expected by chance. A third level of nonrandomness is observed in the composite distribution of IS elements across strains: among 20 mapped IS1 elements, none were found in the region of 48-77 minutes, a significant gap. One region of the E. coli chromosome, at 98 min, had a cluster of IS1 elements in seven ECOR strains of diverse phylogenetic origin. We deduce from sequence analysis that this pattern of distribution is a result of initial insertion in the most recent common ancestor of these strains and therefore not a hot spot of insertion. Analysis using long-range PCR with primers for IS2 and IS3 also yielded pairs of elements in close proximity, suggesting that these elements may also occasionally transpose by local hopping.

Chromosome Mapping↗

Compositional biases of bacterial genomes and evolutionary implications.

We compare and contrast genome-wide compositional biases and distributions of short oligonucleotides across 15 diverse prokaryotes that have substantial genomic sequence collections. These include seven complete genomes (Escherichia coli, Haemophilus influenzae, Mycoplasma genitalium, Mycoplasma pneumoniae, Synechocystis sp. strain PCC6803, Methanococcus jannaschii, and Pyrobaculum aerophilum). A key observation concerns the constancy of the dinucleotide relative abundance profiles over multiple 50-kb disjoint contigs within the same genome. (The profile is rhoXY* = fXY*/fX*fY* for all XY, where fX* denotes the frequency of the nucleotide X and fY* denotes the frequency of the dinucleotide XY, both computed from the sequence concatenated with its inverted complementary sequence.) On the basis of this constancy, we refer to the collection [rhoXY*] as the genome signature. We establish that the differences between [rhoXY*] vectors of 50-kb sample contigs of different genomes virtually always exceed the differences between those of the same genomes. Various di- and tetranucleotide biases are identified. In particular, we find that the dinucleotide CpG=CG is underrepresented in many thermophiles (e.g., M. jannaschii, Sulfolobus sp., and M. thermoautotrophicum) but overrepresented in halobacteria. TA is broadly underrepresented in prokaryotes and eukaryotes, but normal counts appear in Sulfolobus and P. aerophilum sequences. More than for any other bacterial genome, palindromic tetranucleotides are underrepresented in H. influenzae. The M. jannaschii sequence is unprecedented in its extreme underrepresentation of CTAG tetranucleotides and in the anomalous distribution of CTAG sites around the genome. Comparative analysis of numbers of long tetranucleotide microsatellites distinguishes H. influenzae. Dinucleotide relative abundance differences between bacterial sequences are compared. For example, in these assessments of differences, the cyanobacteria Synechocystis, Synechococcus, and Anabaena do not form a coherent group and are as far from each other as general gram-negative sequences are from general gram-positive sequences. The difference of M. jannaschii from low-G+C gram-positive proteobacteria is one-half of the difference from gram-negative proteobacteria. Interpretations and hypotheses center on the role of the genome signature in highlighting similarities and dissimilarities across different classes of prokaryotic species, possible mechanisms underlying the genome signature, the form and level of genome compositional flux, the use of the genome signature as a chronometer of molecular phylogeny, and implications with respect to the three putative eubacterial, archaeal, and eukaryote domains of life and to the origin and early evolution of eukaryotes.

Bacteria↗

Plant genomics: the third wave.

Completing the primary genomic sequence of Arabidopsis thaliana was a major milestone, being the first plant genome and only the third high-quality finished eukaryotic genome sequence. Understanding how the genome sequence comprehensively encodes developmental programs and environmental responses is the next major challenge for all plant genome projects. This requires fully characterizing the genes, the regulatory sequences, and their functions. We discuss several functional genomics approaches to decode the linear sequence of the reference plant Arabidopsis thaliana, including full-length cDNA collections, microarrays, natural variation, knockout collections, and comparative sequence analysis. Genomics provides the essential tools to speed the work of the traditional molecular geneticist and is now a scientific discipline in its own right.

Arabidopsis↗