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PubMed2005

Human metapneumovirus infections in Mexico: epidemiological and clinical characteristics.

The human metapneumovirus (hMPV) is a recently described respiratory RNA virus that mainly affects children. To date there has not been a report that describes the detection of this virus in Mexico. This study was performed to detect hMPV in hospitalized Mexican children with respiratory infections, and describe their epidemiological and clinical characteristics. Nasal wash samples from 558 children younger than 3 years of age with the admission diagnosis of a respiratory tract infection were evaluated. Respiratory viruses were detected in 221 children [respiratory syncytial virus (RSV), 193 (34.6 %); influenza virus, 13 (2.3 %); parainfluenza viruses, 15 (2.7 %)]. Respiratory secretions of 323 children in whom the presence of these viruses was excluded (131 admitted during the 2002-2003 respiratory season and 192 during the 2003-2004 season) were tested for hMPV infection. The hMPV genome was detected in 34 specimens by amplification using real-time RT-PCR. Sequencing of amplicons and phylogenetic analysis indicated the predominance of genotype A hMPV. The months with the highest number of hMPV detections were February and March. During the 2002-2003 season hMPV activity peaked after the RSV season. During the 2003-2004 season hMPV and RSV epidemics occurred simultaneously. The clinical presentation of an hMPV infection was indistinguishable from other respiratory infections. Except for one death, the outcomes of the hMPV infections were good. In this study, among the viruses routinely tested for, hMPV was the second most common agent, after RSV, in children younger than 3 years of age hospitalized with respiratory tract infections.

Child, Preschool↗

PubMed2004

The epidemiology of HIV-1 subtypes in infected patients from northeastern Thailand.

In total, 117 HIV-1 infected patients from several provinces in Northeastern Thailand were analysed. All blood samples collected from individuals were confirmed by EIA and Western blot and partially by HIV-1 gag-, pol- and env-PCR. By serotyping with a V3-peptide ELISA, 108 (92.3%) of the sera samples belonged to subtype E, 9 (7.7%) were serotype B. For 10 Thai HIV-1 infections, the serotype and genotype were determined. The genotype was determined by phylogenetic analysis of directly sequenced PCR amplicons, 8 were subtype E, 2 subtype B. For these patients the serotype did correlate with the genotype. Tracing back the origin of Thai patients, it seems that most were infected within early years of the epidemic and the Thai subtype B infected patients have been imported directly from foreign countries via sexual contact. The findings suggest there are two district subtypes in Thailand with the majority being subtype E. The relatively high prevalence of subtype B in Northeastern Thailand may be due to the increasing intermix of the two strains (subtypes E and B) and the migration for employment from foreign countries. This may lead to public health concerns regarding surveillance of HIV-1 subtypes and the regulation of potentially infected workers returning from abroad to the country.

Blotting, Western↗

PubMed2002

Diversity of ribosomal mutations conferring resistance to macrolides, clindamycin, streptogramin, and telithromycin in Streptococcus pneumoniae.

Mechanisms of resistance were studied in 22 macrolide-resistant mutants selected in vitro from 5 parental strains of macrolide-susceptible Streptococcus pneumoniae by serial passage in various macrolides (T. A. Davies, B. E. Dewasse, M. R. Jacobs, and P. C. Appelbaum, Antimicrob. Agents Chemother., 44:414-417, 2000). Portions of genes encoding ribosomal proteins L22 and L4 and 23S rRNA (domains II and V) were amplified by PCR and analyzed by single-strand conformational polymorphism analysis to screen for mutations. The DNA sequences of amplicons from mutants that differed from those of parental strains by their electrophoretic migration profiles were determined. In six mutants, point mutations were detected in the L22 gene (G95D, P99Q, A93E, P91S, and G83E). The only mutant selected by telithromycin (for which the MIC increased from 0.008 to 0.25 microg/ml) contained a combination of three mutations in the L22 gene (A93E, P91S, and G83E). L22 mutations were combined with an L4 mutation (G71R) in one strain and with a 23S rRNA mutation (C2611A) in another strain. Nine other strains selected by various macrolides had A2058G (n = 1), A2058U (n = 2), A2059G (n = 1), C2610U (n = 1), and C2611U (n = 4) mutations (Escherichia coli numbering) in domain V of 23S rRNA. One mutant selected by clarithromycin and resistant to all macrolides tested (MIC, >32 microg/ml) and telithromycin (MIC, 4 microg/ml) had a single base deletion (A752) in domain II. In six remaining mutants, no mutations in L22, L4, or 23S rRNA could be detected.

Amino Acid Sequence↗

PubMed2002

Identification of a novel NCF-1 (p47-phox) pseudogene not containing the signature GT deletion: significance for A47 degrees chronic granulomatous disease carrier detection.

The p47-phox gene, NCF-1, has 2 nearly identical pseudogenes (psiNCF-1) in proximity at chromosomal locus 7q11.23. A dinucleotide deletion (DeltaGT) at the beginning of exon 2 that leads to a frameshift and premature stop codon is considered the signature sequence of the pseudogenes. It is also the most prevalent mutation in p47-phox-deficient (A47 degrees ) chronic granulomatous disease (CGD) as a result of the insertion of a DeltaGT-containing fragment of pseudogene into NCF-1. Extending our study of the relationship between NCF-1 and psiNCF-1 to 53 unaffected control individuals, we found that although in most (n = 44), the ratio of pseudogene (DeltaGT) to functional gene (GTGT) sequence in amplicons spanning exon 2 was 2:1, as previously observed, surprisingly, in 7 persons the ratio was 1:1, and in 2 persons the ratio was 1:2. The lowered ratios are explained by the presence, in a heterozygous or homozygous state, respectively, of a pseudogene that contains GTGT rather than DeltaGT. It is possible that this pseudogene has not undergone deletion of GT, but more likely, based on analysis of additional NCF-1/psiNCF-1 markers, it represents the previously unidentified product of the reciprocal crossover of DNA fragments between the functional gene and one of its pseudogenes. The mutated NCF-1 resulting from this event is the predominant A47 degrees CGD allele. The existence of 2 extended haplotypes encompassing NCF-1/psiNCF-1 further complicates the detection of A47 degrees CGD carriers. Although most have a DeltaGT/GTGT ratio of 5:1, some have a ratio of 2:1 and are indistinguishable by this means from unaffected individuals.

Chromosomes, Human, Pair 7↗

PubMed2006

Effects of water regime on archaeal community composition in Arctic soils.

Effects of water regime on archaeal communities in Arctic soils from Spitsbergen were studied using denaturing gradient gel electrophoresis (DGGE) of amplified 16S rRNA genes, with subsequent sequencing of amplicons and ordination analysis of binary DGGE data. Samples with major differences in soil water regime showed significant differences in their archaeal community profiles. Methanomicrobiales, Methanobacteriaceae and Methanosaeta were detectable only in environments that were wet during most of the growth season, while a novel euryarchaeotal cluster was detected only in less reduced solifluction material. Group 1.3b of Crenarchaeota had a high relative abundance within the archaeal community in a wide range of wet soils. Along a natural soil moisture gradient, changes in archaeal community composition were observed only in upper soil layers. The results indicated that members of Methanomicrobiales were relatively tolerant to soil aeration. Differences in archaeal community composition associated with soil water regime were predominant over regional and seasonal variation, and over differences between individual wetlands. The results suggest that the observed 'on-off switch' mechanism of soil hydrology for large-scale variations in methane emissions from northern wetlands is at least partly caused by differences in the community structure of organisms involved in methane production.

Archaea↗

PubMed2026

Dual-transcriptomic analysis of human nasal transcriptome and microbiome reveals host-bacteria associations in symptomatic respiratory infection.

BACKGROUND: The human nasopharynx is colonized by a diverse community of commensal microbiota linked to many respiratory diseases, yet their associations with the host remain unclear. RESULTS: In this study, we introduced a dual-transcriptomics analysis strategy, which can characterize the host transcriptome and microbiome from nasal samples simultaneously. We applied this workflow to a local SARS-CoV-2 cohort with 76 asymptomatic infected patients, among whom 52 (68.42%) developed symptomatic infection during a 1-week follow-up period. Nasal swabs were collected from all 76 patients at enrollment and from 73 patients at one-week later follow-up. We detected a median of 8.94% reads that did not map to the human genome across all 149 samples, among which around half (median 49.68%) were successfully mapped to microbiome genome. Meta-transcriptomic analysis detected significantly higher SARS-related coronavirus loads in samples from the symptomatic group at enrollment (P = 0.004), and both groups showed decreased loads one week later (symptomatic, P = 0.001; asymptomatic, P = 0.035). Compared with benchmarking 16 S rRNA sequencing on 53 samples, our computational strategy showed high correlation of relative abundance in all top 20 genera (median Rho = 0.90, Pmax < 0.001). A total of 670 bacteria species were identified to show a relative abundance ≥ 0.01% in at least 10% samples. Differential abundance analysis identified 76 species (DASs) from six phyla with significantly decreased abundance in samples from the symptomatic group (log2(fold change or FC) < -1 and adjusted P < 0.05) compared to the asymptomatic group at enrollment. Integrating these symptom-associated DASs with host's gene expression using an expression quantitative trait bacteria (eQTB) model, we found 45 symptom-associated DASs identified at enrollment were significantly associated with one to 14 genes (adjusted P < 0.05). GSEA showed a series of symptom-associated DASs were significantly correlated with pathways related to olfactory function, keratinocyte differentiation, and DNA methylation. CONCLUSIONS: In summary, our dual-transcriptomic analysis strategy effectively characterized host-microbiome associations, offering insights into microbial contributions to respiratory diseases.

Humans↗

PubMed1993

Homologous recombination between direct repeat sequences yields P-glycoprotein containing amplicons in arsenite resistant Leishmania.

The protozoan parasite Leishmania often responds to drug pressure by amplifying part of its genome. At least two loci derived from the same 800 kb chromosome were amplified either as extrachromosomal circles or linear fragments after sodium arsenite selection. A 50 kb linear amplicon was detected in six independent arsenite mutants and revertants grown in absence of arsenite rapidly lost the amplicon and part of their resistance. The circular extrachromosomal amplicons, all derived from the H locus of Leishmania, were characterized more extensively. In all cases, direct repeated sequences appeared to be involved in the formation of circular amplicons. Most amplicons were generated after homologous recombination between two linked P-glycoprotein genes. This recombination event was, in two cases, associated with the loss of one allele of the chromosomal copy. A novel rearrangement point was found in a mutant where the amplicon was created by recombination between two 541 bp direct repeats surrounding the P-glycoprotein gene present at the H locus. It is also at one of these repeats that an H circle with large inverted duplications was formed. We propose that the presence of repeated sequences in the H locus facilitates the amplification of the drug resistance genes concentrated in this locus.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2002

Sequence-specific electrochemical detection of asymmetric PCR amplicons of traditional Chinese medicinal plant DNA.

In this study, an electrochemistry-based approach to detect nucleic acid amplification products of Chinese herbal genes is reported. Using asymmetric polymerase chain reaction and electrochemical techniques, single-stranded target amplicons are produced from trace amounts of DNA sample and sequence-specific electrochemical detection based on the direct hybridization of the crude amplicon mix and immobilized DNA probe can be achieved. Electrochemically active intercalator Hoechst 33258 is bound to the double-stranded duplex formed by the target amplicon hybridized with the 5'-thiol-derivated DNA probe (16-mer) on the gold electrode surface. The electrochemical current signal of the hybridization event is measured by linear sweep voltammetry, the response of which can be used to differentiate the sequence complementarities of the target amplicons. To improve the reproducibility and sensitivity of the current signal, issues such as electrode surface cleaning, probe immobilization, and target hybridization are addressed. Factors affecting hybridization efficiency including the length and binding region of the target amplicon are discussed. Using our approach, differentiation of Chinese herbal species Fritillaria (F. thunbergii and F. cirrhosa) based on the 16-mer unique sequences in the spacer region of the 5S-rRNA is demonstrated. The ability to detect PCR products using a nonoptical electrochemical detection technique is an important step toward the realization of portable biomicrodevices for on-spot bacterial and viral detections.

DNA Probes↗

PubMed2006

Molecular analysis of the subgingival microbiota in health and disease.

This investigation provides molecular analyses of the periodontal microbiota in health and disease. Subgingival samples from 47 volunteers with healthy gingivae or clinically diagnosed chronic periodontitis were characterized by PCR-denaturing gradient gel electrophoresis (DGGE) with primers specific for the V2-V3 region of the eubacterial 16S rRNA gene. A hierarchical dendrogram was constructed from band patterns. All unique PCR amplicons (DGGE bands) were sequenced for identity. Samples were also analyzed for the presence of Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, and Tannerella forsythensis by multiplex PCR. Associations of patient age, gender, and smoking status together with the presence of each unique band and putative periodontal pathogens with disease were assessed by logistic regression. Periodontal pockets were colonized by complex eubacterial communities (10 to 40 distinct DGGE bands) with substantial individual variation in the community profile. Species diversity in health and disease was determined by the Shannon-Weaver index of diversity and compared by the Mann-Whitney U test. Sequence analyses of DGGE amplicons indicated the occurrence of many nontypical oral species and eubacteria previously associated with this environment. With the exception of T. forsythensis, the putative pathogens were not detected by DGGE. Multiplex PCR, however, detected T. forsythensis, A. actinomycetemcomitans, and P. gingivalis in 9% 16%, and 29% of the patients with disease, respectively. The presence of A. actinomycetemcomitans was significantly associated with disease (P < 0.01). Statistical analyses indicated that the presence of Treponema socranskii and Pseudomonas sp. was a significant predictor of disease (P < 0.05) and that there was no significant difference (P > 0.05) in terms of eubacterial species diversity between health and disease.

Actinobacillus Infections↗

PubMed2002

Presence of anaerobic bacteroides in aerobically grown microbial granules.

Microbial granules were grown in a column-type sequential aerobic sludge blanket reactor inoculated with activated sludge flocs taken from a wastewater treatment plant and containing a medium with glucose as the main carbon source. The reactor selected for granules that could settle rapidly by employing a short settling time of 2 min. Matured granules with diameters between 2 and 3 micro m were examined for anaerobic bacteria as their presence can signal the onset of diffusion limitation problems that can potentially diminish granule stability due to the bacterial production of fermentation gases and organic acids under anaerobic conditions. To detect the anaerobes in the granules, clones were constructed from 16S rRNA PCR amplicons. Two sequence types associated with a strict anaerobe Bacteroides spp. were identified from these clones. Fluorescence in situ hybridization (FISH) followed by confocal laser scanning microscopy (CLSM) demonstrated that cells of Bacteroides spp. were concentrated at a depth of approximately 800 micro m below the surface of the granule. Cell enumeration using flow cytometry showed that the percentage of labeled cells of Bacteroides spp. compared to total bacterial cells in the granules was 0.56%. This is the first study to use a suite of culture-independent techniques to report the presence of a defined species of anaerobic bacteria in aerobically grown microbial granules.

Aerobiosis↗

PubMed1999

Single nucleotide polymorphism (SNP) discovery and linkage mapping of bovine cytokine genes.

Polymorphic markers at bovine gene loci facilitate the integration of cattle genetic maps with those of humans and mice. To this end, 31 single nucleotide polymorphism (SNP) markers were developed for seven bovine chemokine genes. Loci were amplified from bovine genomic DNA by the polymerase chain reaction, and candidate amplicons were sequenced to determine their identity. Amplified loci from 24 founding parents and select progeny from a beef cattle reference population were sequenced and analyzed for SNPs. SNP haplotype alleles were determined by examining segregation patterns and used to establish the locus position on the bovine linkage map. Loci for growth-related proteins (GRO3, GRO1, and GROX) were clustered with the related CXC chemokine genes, interleukin (IL) 8, and epithelial cell inflammatory protein 1, at 84 cM from the centromeric end of the bovine chromosome (BTA) 6 linkage group. Bovine loci for a cluster of IL8 receptors, a stromal cell-derived factor 1, interferon-gamma, and tumor necrosis factor-alpha were mapped at 90, 55, 59, and 34 cM, respectively, from the centromeric ends of the BTA 2, 28, 5, and 23 linkage groups. The positions of these bovine loci were compared with those of orthologous loci on the human map to refine the boundaries of conserved synteny. These seven loci provide examples of SNP development in which the efficiency was largely dependent on the availability of bovine genomic or cDNA sequence. The polymorphic nature of these SNP haplotype markers suggests that they will be useful for mapping complex traits in cattle, such as resistance to infectious disease.

Animals↗

PubMed2003

Molecular variation of Potato yellow vein virus isolates.

To evaluate the variation of Potato yellow vein virus from potato fields, 12 isolates were collected from Colombia and one was collected from Peru. Double-stranded RNA was extracted from the plants and used as a template for RT-PCR amplification of the coat protein ( CP) gene and, in separate reactions the C-terminal region of the heat shock protein 70 homologue ( Hsp70h) gene and the N-terminal region of the p60 open reading frame. The CP amplicons were subjected to single-strand conformation polymorphism (SSCP) analysis and, together with the other amplicon, nucleotide sequence analysis. These analyses suggested that there is low genetic diversity in the PYVV isolates examined and that the Peruvian isolate of PYVV may have originated in Colombia.

Capsid Proteins↗

PubMed2006

Comparison of preservation methods of Rhipicephalus appendiculatus (Acari: Ixodidae) for reliable DNA amplification by PCR.

Five differently preserved groups of adult Rhipicephalus appendiculatus specimens were compared for quality of DNA extracted. Three methods were used to extract DNA from specimens i.e. two simple mosquito validated DNA extraction methods and a tick validated method. Extraction of DNA from tick legs was attempted. The quality of DNA extracted was evaluated by the success of PCR amplification of the ITS2 gene and the mitochondrial COI gene fragment. Fresh specimens (i.e. killed just before extraction) had the highest success of DNA amplification followed by specimens killed in ethanol and subsequently stored in the refrigerator (4 degrees C). There was no significant difference in amplification success between cryopreserved and 70% ethanol preserved specimens. It was possible to amplify DNA from legs of ticks. Sequenced ITS2 amplicon of template obtained from legs of ticks was as legible as those from whole tick extract. The two mosquito validated DNA extraction methods showed a significantly lower amplification success than the tick validated protocol.

Animals↗

PubMed1994

Identification by extrachromosomal amplification and overexpression of a zeta-crystallin/NADPH-oxidoreductase homologue constitutively expressed in Leishmania spp.

A gene which overexpresses a 36-kDa protein (p36) in tunicamycin-resistant Leishmania was mapped by transfection and overexpression to the upstream region of the drug maker in the extrachromosomal amplicon. Complete sequencing of this region revealed a single open reading frame of about 1 kb. Authenticity of the cloned gene is verified by immunologic specificity of its recombinant products and sequence identity with a p36 peptide. The gene shares an overall sequence similarity of about 50% with members of the eukaryote alcohol dehydrogenase family at the amino acid level, including essentially all 13 evolutionarily conserved residues and a nucleotide-binding domain. The binding ligands for both structurally and catalytically important zinc atoms are absent, similar to the zeta-crystallin/NADPH:quinone oxidoreductase gene. Consistent with hydrophilicity of its primary sequence and the presence of a nucleotide binding site, p36 is a soluble molecule non-sedimentable at 105,000 x g and binds Blue Sepharose, elutable only with NADPH. The p36 gene is expressed constitutively in both stages of the wild-type and is conserved among all Leishmania species examined, suggestive of its functional significance different from evolutionarily related homologues.

Alcohol Dehydrogenase↗

PubMed2025

Defining APOBEC-induced mutation signatures and modifying activities in yeast.

APOBEC cytidine deaminases guard cells in a variety of organisms from invading viruses and foreign nucleic acids. Recently, several human APOBECs have been implicated in mutating evolving cancer genomes. Expression of APOBEC3A and APOBEC3B in yeast allowed experimental derivation of the substitution patterns they cause in dividing cells, which provided critical links to these enzymes in the etiology of the COSMIC single base substitution (SBS) signatures 2 and 13 in human tumors. Additionally, the ability to scale yeast experiments to high-throughput screens allows use of this system to also investigate cellular pathways impacting the frequency of APOBEC-induced mutation. Here, we present validated methods utilizing yeast to determine APOBEC mutation signatures, genetic interactors, and chromosomal substrate preferences. These methods can be employed to assess the potential of other human APOBECs and APOBEC orthologs in different species to contribute to cancer genome evolution as well as define the pathways that protect the nuclear genome from inadvertent APOBEC activity during viral restriction.

Humans↗

PubMed2004

The rapid identification of European Armillaria species from soil samples by nested PCR.

New specific primers AR1 and AR2 were successfully used for the amplification of a specific part of internal transcribed spacer (ITS) of rDNA of Armillaria isolated from soil samples. DNA was isolated from 0.5 g of forest soil and ITS region was amplified by nested PCR reaction with external primers ITS1 and ITS4 and internal primers AR1 and AR2. The individual species were distinguished by restriction fragment length polymorphisms (RFLPs) analysis with restriction endonuclease HinfI. The fragments were analysed by ion-exchange HPLC that is more sensible and more rapid than electrophoresis. The amplicons were sequenced to improve the discrimination between the species. The method enables the identification of Armillaria species within one day directly from soil samples without the need for previous isolation and cultivation of mycelium of Armillaria.

Agaricales↗

PubMed2005

Characterization of a reuterin-producing Lactobacillus coryniformis strain isolated from a goat's milk cheese.

Lactobacillus coryniformis CECT 5711, a strain isolated from a goat's milk cheese, displayed a broad-spectrum antimicrobial activity; as a consequence, its ability to produce the antagonistic compounds associated to lactic acid bacteria, including bacteriocins, hydrogen peroxide, lactic acid, acetic acid, and reuterin (3-hydroxypropionaldehyde, 3-HPA) was investigated. Production of bacteriocins or hydrogen peroxide by this strain could not be detected. However, in addition to lactic acid and acetic acid, it produced reuterin and cobalamin, a cofactor required for conversion of glycerol to 3-HPA through a glycerol dehydratase. The gene encoding a glycerol dehydratase subunit was detected by PCR and the corresponding amplicon was sequenced. This strain showed a high survival after exposition to conditions simulating those existing in the gastrointestinal tract as well as a notable ability to adhere to intestinal cells, which suggests that its reuterin-producing ability may be used for the host benefit. In addition, the strain showed a strong beta-galactosidase activity. Production of biogenic amines and degradation of mucin could not be detected.

Aldehydes↗

PubMed2006

Seasonal distribution and phylogenetic analysis of human metapneumovirus among children in Osaka City, Japan.

BACKGROUND: Human metapneumovirus (hMPV) is one of the etiological agents of respiratory tract infection (RTI). Because clinical symptoms of hMPV resemble those caused by respiratory syncytial virus (RSV), clinical diagnosis of hMPV infection is difficult. Moreover, hMPV isolation using cultured cells is generally difficult and not efficient compared with reverse transcription-PCR (RT-PCR). OBJECTIVES: To assess infection and seasonal distribution of hMPV associated with RTI among children in Osaka City, Japan. STUDY DESIGN: To detect the hMPV gene, we extracted viral RNA from clinical specimens of patients with RTI and performed RT-PCR or nested-PCR for the fusion (F) gene. hMPV-specific amplicons were sequenced and subjected to phylogenetic analysis. RESULTS: From June 2004 to May 2005, we detected 29 (20.1%) hMPV strains among 144 clinical specimens. Fifteen strains were detected by RT-PCR and the remaining 14 by nested-PCR. Prevalence was principally in winter and spring with incidence peaking in April. We also detected the hMPV RNA from a patient with encephalitis. Approximately 80% of the hMPV-positive patients were younger than 3 years. To analyze these isolates precisely, a phylogenetic analysis using F gene was performed and demonstrated that Osaka City isolates of hMPV consists of two major genetic lineages each comprising two sublineages. CONCLUSIONS: We found two major genetic lineages of hMPV in Osaka City, Japan. We also found that nested-PCR was an efficient method for detecting the hMPV gene in clinical specimens. Of the 28 patients presenting with hMPV infection, 1 patient had associated encephalitis suggesting that hMPV infection might play a role in inducing encephalitis.

Adolescent↗