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Biomedical subjects

David B Levin

Publications and source records attributed to David B Levin.

11 recordsLinked to original sources
PubMed2026

Optimizing a culture-enriched hybrid metagenomics pipeline to assess the AMR footprint of livestock manure in anaerobic digestate.

The role of environmental samples from livestock production systems, including manure and anaerobic digestate, as reservoirs of antimicrobial resistance genes (ARGs) is likely underestimated because conventional metagenomic approaches can overlook low-abundance ARGs and often lack the resolution to associate these genes with their microbial hosts and co-localized mobile genetic elements (MGEs). We evaluated whether culture-enriched metagenomics (CEMG), with and without antibiotic selection, enhances ARG detection in anaerobic digestate and improves the resolution of ARG-MGE-host associations using hybrid short- and long-read metagenomic assembly. CEMG increased ARG recovery; mean ARG abundance rose from 15.4 counts per million (CPM) in metagenomic fresh digestate (FD) to 124 CPM in CEMG without antibiotics and 160 CPM in antibiotic-selective CEMG. In FD, only 9 unique ARGs were detected, whereas CEMG recovered 112, including ARGs of clinical importance, such as glycopeptide resistance, beta-lactamase genes, and the cfr 23S rRNA methyltransferase conferring cross-resistance to multiple antibiotic classes. Antibiotic selection induced targeted, class-specific shifts in ARG profiles, with ARGs associated with tetracycline resistance consistently enriched across treatments. Hybrid metagenomic assembly resolved the genomic context of 784 ARGs, of which 59.3% were co-localized with at least one class of MGEs, predominantly plasmids and integrative conjugative elements/integrative mobilizable elements. Biocide and metal resistance genes frequently co-occurred with ARGs on the same contigs. Together, these findings demonstrate that antibiotic-selective culture enrichment enhances resistome surveillance by improving detection of low-abundance ARGs, while hybrid assembly provides critical genomic context for assessing their mobility and host associations.IMPORTANCELivestock manure and its byproducts, such as anaerobic digestate, are recognized as important environmental reservoirs of antimicrobial resistance genes (ARGs) and resistant bacteria, yet current metagenomic approaches may underestimate this risk by failing to detect low-abundance but clinically relevant ARGs. Here, we show that integrating culture enrichment with hybrid metagenomics improves ARG recovery and reveals ARG co-localization with mobile genetic elements and putative bacterial hosts. This approach captures a cultivable and condition-responsive fraction of the resistome that is not readily accessible through direct metagenomic sequencing alone, providing a more informative framework for environmental AMR surveillance.

anaerobic digestion↗
PubMed2006

Development and evaluation of methods to detect nucleopolyhedroviruses in larvae of the Douglas-fir tussock moth, Orgyia pseudotsugata (McDunnough).

Various molecular methods are used to detect pathogenic microorganisms and viruses within their hosts, but these methods are rarely validated by direct comparison. Southern hybridization, enzyme-linked immunosorbent assay (ELISA), and a novel DNA extraction/PCR assay were used to detect Orgyia pseudotsugata multiple nucleopolyhedrovirus (OpMNPV) in Douglas-fir tussock moth larvae. PCR was more sensitive than Southern hybridization and ELISA at detecting semipurified virus. ELISA, however, was the most accurate method for detecting virus within larvae, given that Southern hybridization and PCR produced false-negative results (31% and 2.5%, respectively). ELISA may be preferable in some applications because virus infections can be quantified (r(2) = 0.995). These results may be applicable to both applied and academic research that seeks to accurately identify the incidence of viruses and microorganisms that regulate insect populations.

Animals↗
PubMed2006

Potential for hydrogen and methane production from biomass residues in Canada.

Canada generates approximately 1.45 x 10(8)t of residual biomass per year, containing an estimated energy value of 2.28 x 10(9)GJ, which is equivalent to about 22% of Canada's current annual energy use. Anaerobic digestion of these biomass residues using conventional technologies could generate 1.14 x 10(10)m(3)/year of CH(4) with a heating value of 4.56 x 10(8)GJ. Conversion of these residues using emerging technologies that favor the synthesis of H(2) and represses the synthesis of CH(4) could generate 1.47 x 10(10)m(3)/year renewable H(2), with a heating value of 1.89 x 10(8)GJ. While CH(4)-production results in a larger amount of energy recovery, generating H(2) from waste biomass is a renewable alternative that could fuel the hydrogen economy. Additional research to further both the technical and commercial development of microbial bio-energy from biomass is warranted.

Biomass↗
PubMed2006

Sequence analysis and organization of the Neodiprion abietis nucleopolyhedrovirus genome.

Of 30 baculovirus genomes that have been sequenced to date, the only nonlepidopteran baculoviruses include the dipteran Culex nigripalpus nucleopolyhedrovirus and two hymenopteran nucleopolyhedroviruses that infect the sawflies Neodiprion lecontei (NeleNPV) and Neodiprion sertifer (NeseNPV). This study provides a complete sequence and genome analysis of the nucleopolyhedrovirus that infects the balsam fir sawfly Neodiprion abietis (Hymenoptera, Symphyta, Diprionidae). The N. abietis nucleopolyhedrovirus (NeabNPV) is 84,264 bp in size, with a G+C content of 33.5%, and contains 93 predicted open reading frames (ORFs). Eleven predicted ORFs are unique to this baculovirus, 10 ORFs have a putative sequence homologue in the NeleNPV genome but not the NeseNPV genome, and 1 ORF (neab53) has a putative sequence homologue in the NeseNPV genome but not the NeleNPV genome. Specific repeat sequences are coincident with major genome rearrangements that distinguish NeabNPV and NeleNPV. Genes associated with these repeat regions encode a common amino acid motif, suggesting that they are a family of repeated contiguous gene clusters. Lepidopteran baculoviruses, similarly, have a family of repeated genes called the bro gene family. However, there is no significant sequence similarity between the NeabNPV and bro genes. Homologues of early-expressed genes such as ie-1 and lef-3 were absent in NeabNPV, as they are in the previously sequenced hymenopteran baculoviruses. Analyses of ORF upstream sequences identified potential temporally distinct genes on the basis of putative promoter elements.

Amino Acid Motifs↗
PubMed2006

Formate synthesis by Clostridium thermocellum during anaerobic fermentation.

We have detected formate synthesis by Clostridium thermocellum 27405 cultured in both cellobiose and alpha-cellulose. While formate synthesis has been reported for one strain of C. thermocellum (strain I-1-B), numerous studies of C. thermocellum 27405 fermentation, conducted under different growth conditions, failed to detect the presence of formate. Thus, the status of formate synthesis as a fermentation end product by C. thermocellum has been uncertain. Formate synthesis competes with the synthesis of hydrogen (H2) as a fermentation end product, and thus would negatively impact H2 yields in processes designed to generate H2 from biomass. Understanding the mechanism of formate synthesis is the first step in devising means of mitigating its production. Transcription of putative pfl, fnr, and adhE genes, encoding pyruvate formate-lyase (PFL), PFL-activating enzyme (PFL-AE), and alcohol dehydrogenase E (ADH-E) enzymes, respectively, were detected by reverse transcriptase polymerase chain reactions using total RNA extracted from stationary phase C. thermocellum cultured on cellobiose. The PCR products observed correspond to the expected amplicon sizes. Nucleotide sequence analysis of the cloned PCR products followed by BLAST analyses confirmed their identity. Formate production was detected throughout growth, and PFL enzyme activity was detected in late log and stationary phase (OD600 = 0.7 and 0.9, respectively) in extracts of C. thermocellum cultured on cellobiose. BLAST analyses revealed that C. thermocellum PFL and PFL-AE have greater amino acid sequence identity with equivalent enzymes from Bacillus and Thermocynechococcus species than with other Clostridium species, but C. thermocellum ADH-E has greater amino acid sequence identity with Clostridium species.

Acetyltransferases↗
PubMed2004

Salmonid opsin sequences undergo positive selection and indicate an alternate evolutionary relationship in oncorhynchus.

Positive selection can be demonstrated by statistical analysis when non-synonymous nucleotide substitutions occur more frequently than synonymous substitutions (dN>dS). This pattern of sequence evolution has been observed in the rhodopsin gene of cichlids. Mutations in opsin genes resulting in amino acid (AA) replacement appear to be associated with the evolution of specific color patterns and the evolution of courtship behaviors. Within fish, AA replacements in opsin proteins have improved vision at great depths and have occurred in deep-sea species. Salmonids experience diverse photic environments during their life history. Furthermore, sexual selection has resulted in species-specific male and female coloration during spawning. To look for evidence of positive selection in salmonid opsins, we sequenced the RH1, RH2, LWS, SWS1, and SWS2 genes from six Pacific salmon species as well as the Atlantic salmon. These salmonids include landlocked and migratory species and species that vary in their coloration during spawning. In each opsin gene comparison from all species sampled, traditional dN:dS analysis did not indicate positive selection. However, the more sensitive Creevey-McInerney statistical analysis indicates that RH1 and RH2 experienced positive selection early in the evolution and speciation of salmonids.

Animals↗
PubMed2003

Identification of a unique transcript down-regulated in the retina of rainbow trout (Oncorhynchus mykiss) at smoltification.

Developmental and physiological changes in the retina of salmonid fishes occur during smoltification, a metamorphic event associated with thyroid hormone that prepares salmon for oceanic migration. These changes include loss of ultraviolet-sensitive (UVS) cone photoreceptors, switching of visual pigments, alterations in thyroid hormone regulation, and associated changes in behavior. This model provides an opportunity to study substantial neuronal development within an established retina. Little is known, however, about how higher order neurons are altered or how retinal gene expression changes during this transition. Here, we have used differential display RT-PCR and RACE-PCR to identify a previously uncharacterized gene transcript in Oncorhynchus mykiss under developmental regulation in the retina during smoltification, rtp12.5. This unique cDNA encodes a putative protein 112 amino acids long similar to a hypothetical human open reading frame located on chromosome 14.Q24.2. Differential expression was confirmed by RNA dot blot and in situ hybridization. We also present O. mykiss sep15 cDNA sequence and describe its expression in the vertebrate retina. Considering the expression pattern within retinal tissue observed by in situ hybridization, rtp12.5 may be under TH regulation and involved in neuronal remodeling of the retina during loss of UVS cones.

Amino Acid Sequence↗
PubMed2001

Expression, purification and characterization of the Spodoptera littoralis nucleopolyhedrovirus (SpliNPV) DNA polymerase and interaction with the SpliNPV non-hr origin of DNA replication.

The DNA polymerase from Spodoptera littoralis nucleopolyhedrovirus (SpliNPV) was expressed in, and purified from, prokaryotic and eukaryotic expression systems. While less protein was obtained from the E. coli expression system, SpliNPV DNAPOL purified from E. coli displayed similar biochemical characteristics to DNAPOL expressed in, and subsequently purified from, insect cells (Sf9) using a baculovirus expression system. Biochemical analyses suggested that the DNA polymerase and the 3'-5' exonuclease activities are intrinsic to the protein. Deletion of the first 80 amino acid residues from the N terminus of the DNAPOL affected neither the DNA polymerase nor the exonuclease activities of the enzyme. Replication products from single-stranded M13 DNA demonstrated that the DNA synthesis activity of SpliNPV DNAPOL is highly processive. Transient expression assays with a set of deletion clones containing the putative SpliNPV non-hr origin of DNA replication permitted functional characterization of sequence elements within the origin fragment. Purified SpliNPV DNAPOL stimulated origin-dependent DNA replication in a cell-free replication assay.

Animals↗
PubMed2000

Codon usage in nucleopolyhedroviruses.

Phylogenetic analyses based on baculovirus polyhedrin nucleotide and amino acid sequences revealed two major nucleopolyhedrovirus (NPV) clades, designated Group I and Group II. Subsequent phylogenetic analyses have revealed three Group II subclades, designated A, B and C. Variations in amino acid frequencies determine the extent of dissimilarity for divergent but structurally and functionally conserved genes and therefore significantly influence the analysis of phylogenetic relationships. Hence, it is important to consider variations in amino acid codon usage. The Genome Hypothesis postulates that genes in any given genome use the same coding pattern with respect to synonymous codons and that genes in phylogenetically related species generally show the same pattern of codon usage. We have examined codon usage in six genes from six NPVs and found that: (1) there is significant variation in codon use by genes within the same virus genome; (2) there is significant variation in the codon usage of homologous genes encoded by different NPVs; (3) there is no correlation between the level of gene expression and codon bias in NPVs; (4) there is no correlation between gene length and codon bias in NPVs; and (5) that while codon use bias appears to be conserved between viruses that are closely related phylogenetically, the patterns of codon usage also appear to be a direct function of the GC-content of the virus-encoded genes.

Animals↗
PubMed1999

Identification and functional analysis of a putative non-hr origin of DNA replication from the Spodoptera littoralis type B multinucleocapsid nucleopolyhedrovirus.

A putative non-hr origin of DNA replication was identified in the Spodoptera littoralis multinucleocapsid nucleopolyhedrovirus (SpliNPV) genome by transient replication assays. The putative SpliNPV ori was mapped to the PstI-J fragment between 75.1-77.9 map units in the SpliNPV genome. While the DNA sequence of the putative SpliNPV ori aligned with regions within the non-hr oris of Autographa californica, Orgyia pseudotsugata and Spodoptera exigua multinucleocapsid nucleopolyhedroviruses, it has limited DNA sequence identity with these elements. The sequence of the putative SpliNPV non-hr ori fragment contains a unique distribution of imperfect palindromes, multiple direct repeats and putative transcription factor-binding sites. Transient expression assays indicated that the putative SpliNPV ori fragment repressed SpliNPV lef-3 promoter-mediated luciferase reporter gene expression. However, the putative SpliNPV ori fragment itself was capable of directing luciferase expression in the absence of a recognizable baculovirus promoter element in an orientation-independent fashion, suggesting that DNA sequence motifs within its sequence can activate transcription. Gel mobility shift analyses confirmed that proteins within nuclear extracts from both uninfected and virus-infected cells bound with specificity to the putative SpliNPV ori fragment.

Animals↗