62 research outputs found
Author response
No full textDistinct microbial ecosystems have evolved to meet the challenges of indoor environments, shaping the microbial communities that interact most with modern human activities. Microbial transmission in food-processing facilities has an enormous impact on the qualities and healthfulness of foods, beneficially or detrimentally interacting with food products. To explore modes of microbial transmission and spoilage-gene frequency in a commercial food-production scenario, we profiled hop-resistance gene frequencies and bacterial and fungal communities in a brewery. We employed a Bayesian approach for predicting routes of contamination, revealing critical control points for microbial management. Physically mapping microbial populations over time illustrates patterns of dispersal and identifies potential contaminant reservoirs within this environment. Habitual exposure to beer is associated with increased abundance of spoilage genes, predicting greater contamination risk. Elucidating the genetic landscapes of indoor environments poses important practical implications for food-production systems and these concepts are translatable to other built environments
Comparative genomic and plasmid analysis of beer-spoiling and non-beer-spoiling Lactobacillus brevis isolates
No full textBeer-spoilage-related lactic acid bacteria (BSR LAB) belong to multiple genera and species; however, beer-spoilage capacity is isolate-specific and partially acquired via horizontal gene transfer within the brewing environment. Thus, the extent to which genus-, species- or environment- (i.e., brewery-) level genetic variability influences beer-spoilage phenotype is unknown. Publicly available Lactobacillus brevis genomes were analyzed via BlAst Diagnostic Gene findEr (BADGE) for BSR genes and assessed for pangenomic relationships. Also analyzed were functional coding capacities of plasmids of LAB inhabiting extreme niche environments. Considerable genetic variation was observed in L. brevis isolated from clinical samples, whereas 16 candidate genes distinguish BSR and non-BSR L. brevis genomes. These genes are related to nutrient scavenging of gluconate/pentoses, mannose, and metabolism of pectin. BSR L. brevis isolates also have higher average nucleotide identity and stronger pangenome association to one another, though isolation source (i.e., specific brewery) also appears to influence the plasmid coding capacity of BSR LAB. Finally, it is shown that niche-specific adaptation and phenotype are plasmid-encoded for both BSR and non-BSR LAB. The ultimate combination of plasmid-encoded genes dictates the ability of L. brevis to survive in the most extreme beer environment, namely, gassed/pressurized beer.The accepted manuscript in pdf format is listed with the files at the bottom of this page. The presentation of the authors' names and (or) special characters in the title of the manuscript may differ slightly between what is listed on this page and what is listed in the pdf file of the accepted manuscript; that in the pdf file of the accepted manuscript is what was submitted by the author
HIV-1 Glycoprotein 120-Specific Exosome-Targeted CD8+ T Cell Vaccine
No full textImmunosuppression is a hallmark of human immunodeficiency virus-1 (HIV-1) infection. Upon binding to cluster of differentiation (CD) 4 receptor via trimeric glycoprotein (Gp) 120, HIV-1 enters and multiplies in CD4+ T cells, leading to the death of these cells. CD4+ T helper (Th) cells are required for the generation and maintenance of CD8+ T cells, which are crucial to control HIV-1 proliferation. The stimulation of HIV-1-specific CD8+ T cell responses in CD4-deficient environment is a major scientific challenge. In addition, dendritic cells (DCs) expressing C-type lectin and dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) with high affinity for Gp120, appear to act as “Trojan horses”, facilitating the spread of HIV-1 from mucosal surfaces to T cells in lymph nodes, and these HIV-1-infected T cells have been found to be impaired. Currently, highly active antiretroviral therapy (HAART) is the only means to halt progression of acquired immunodeficiency syndrome (AIDS). Although HAART suppresses viral replication and significantly improves prognosis, toxicity and cost of the treatment have become major limitations for its use. In addition, with long-term use, HAART also decreases HIV-1-specific CD4+ Th1 and CD8+ T cell responses, causing a functional decrease in capacity of HIV-1-capturing DCs in initiating adaptive immune responses. As a result, HIV patients are unable to eliminate infected cells and proviral latent reservoirs. Therefore, how to stimulate efficient CD8+ T cell responses in AIDS patients is one of the major challenges in HIV-1 patient therapy.
Previously, it was demonstrated that novel ovalbumin (OVA)-specific exosome (EXO)-targeted CD4+ T cell vaccine (CD4+aTexo) was capable of stimulating CD4+ T cell-independent CD8+ T cell responses and antitumor immunity to a highly metastasizing tumor challenge in wild-type mice. Since CD4+ T cells are killed by HIV-1, the present study proposed to use active CD8+ T cells rather than active CD4+ T cells for vaccine development. First, OVA-specific CD8+aTexo (OVA-aTexo) vaccine was prepared by pulsing concanavalin A (Con A)-stimulated CD8+ T cells with OVA-pulsed DCs (DCOVA)-released exosomes (EXOOVA). In wild-type mice, OVA-aTexo vaccine stimulated CD4-independent OVA-specific CD8+ T cell responses via CD40L and interleukin (IL)-2 signaling, and exosomal peptide major histo-compatibility complex (pMHC)-I targeting. To further provide insight into whether CD8+aTexo vaccine induces similar cellular immune response in the context of human immune system, transgenic A2-Kb mice expressing α1 and α2 domains of human leukocyte antigen (HLA)-A2 and α3 domain of mouse H2-Kb were used. Adenovirus (AdVGp120) expressing HIV-1 envelope protein Gp120 was used to transfect bone-marrow DCs to generate DC expressing Gp120 and DCGp120-released EXO were purified (EXOGp120). EXOGp120 were also purified from the culture supernatant of DC2.4Gp120-transfected cells. Next, Gp120-specific CD8+aTexo (Gp120-aTexo) vaccine was prepared by pulsing ConA-stimulated CD8+ T cells with EXOGp120. In wild-type and A2-Kb mice, Gp120-aTexo vaccine stimulated Gp120-specific effector and memory CD8+ cytotoxic T lymphocyte (CTL) responses, and provided preventive immunity to BL610-Gp120 and BL6-10A2Kb/Gp120 tumor cells, respectively. Gp120-aTexo vaccine also provided therapeutic immunity against 3 and 6-day lung tumor metastasis in transgenic A2-Kb mice. Taken together, these results represent a novel approach to the induction of immunity for the treatment of AIDS patients with CD4+ T cell deficiency or for use in AIDS patients on HAART to clear virus-infected cells
The mutant-prevention concentration concept and its application to Staphylococcus aureus
No full textStaphylococcus aureus is a ubiquitous organism causing world-wide morbidity and mortality. This species readily develops resistance to antimicrobial agents. Current dosing strategies are based, in part, on minimum inhibitory concentrations (MICs). This susceptibility test fails to detect the presence of first-step resistant mutants often present in large heterogeneous populations of infecting bacteria. Dosing strategies based on MIC results may, in fact, allow for the selective proliferation of resistant subpopulations. The mutant-prevention concentration (MPC) is the drug concentration at which all first-step resistant mutants will be eradicated along with the susceptible cells. Determination of the mutant-selection window (MSW) is possible using MIC and MPC data. When considered together with achievable drug concentrations in human bodily sites, the MSW helps determine which antimicrobials are likely to select for resistance.
MIC and MPC testing on clinical isolates of methicillin-susceptible (MSSA) and -resistant (MRSA) S. aureus was performed. Characterization via the polymerase chain reaction, sequencing, and electron microscopy (EM) was done on selected organisms recovered from MPC studies (MPC-recovered). MIC and MPC testing was performed on organisms isolated sequentially from patients with recurring S. aureus infections. Pulsed field gel electrophoresis was performed on these sequential isolates.
Based on the MIC and the MPC values, the most potent agents for systemic MSSA and MRSA infections are gemifloxacin and vancomycin, respectively. Re-testing MPC-recovered populations by the MIC showed increased MIC results compared to the parent populations. Macrolide-resistance genes were discovered in S. aureus MPC-recovered populations; in contrast, parental isolates lacked these resistance determinants. EM revealed an increase in cell wall thickness of a vancomycin MPC-recovered population compared to its parental population. Moxifloxacin and vancomycin had the lowest and narrowest MSWs for systemic MSSA and MRSA
infections, respectively, compared to the other agents tested. Sequential isolates showed no change in MIC and MPC values.
The data presented provides evidence for the application of the MPC test to S. aureus organisms. The MPC data is significant when determining appropriate dosing strategies aimed at preventing resistance
Plasmid analysis, comparative genomics and transcriptomics of beer-spoilage lactic acid bacteria emphasizing the role of dissolved carbon dioxide and traditional beer-spoilage markers
No full textSpecific isolates of lactic acid bacteria (LAB) are capable of growing in and spoiling beer, and are the cause of product and process contamination, and financial loss for brewers the world over. To date, our understanding of how these contaminants are able to grow in beer is limited to analysis of hop-tolerance mechanisms, with a limited number of putative hop-tolerance genes having been described. In order to demonstrate that these hop-tolerance genes are incomplete descriptors of overall beer-spoilage ability, the transcriptional activity of these genes in two different beer-spoilage related (BSR) LAB isolates, and the prevalence and sequence conservation of hop-tolerance gene horC in BSR LAB with varying beer-spoilage ability is examined. This analysis is followed by work demonstrating that the total plasmid profile of a beer-spoilage LAB, and not just plasmids harboring hop-tolerance genes, contributes to the isolate’s overall beer-spoilage phenotype and highlights redundancy in potential beer-spoilage mechanisms. The next chapter provides evidence that the presence of dissolved CO2 (dCO2) in beer selects for the ability of LAB to spoil packaged beer, and that tolerance to this stress is not correlated with hop-tolerance, indicating that dCO2 stress is an important part of the total beer environment. This is followed by the presentation and analysis of the genome of the rapid beer-spoiling isolate Lactobacillus brevis BSO 464 and subsequent RNA sequencing for this isolate when grown in degassed and gassed beer so as to elucidate which genes are active when grown in beer, and when grown specifically in the presence of dCO2. Global transcriptome sequencing of this L. brevis isolate and Pediococcus claussenii ATCC BAA-344T when each were grown in growth-limiting concentrations of hops was also performed in order to clarify the hop-specific transcriptional response from that of the response when these isolates grow in the total beer environment. Lastly, comparison is made between available genomes of BSR LAB to reveal that the specific brewery environment a BSR LAB is recovered from, influences genetic variability and that comparison within a given LAB species reveals genetic differences that can be exploited as beer-spoilage genetic markers. This comparative analysis reveals that the total plasmid-coding capacity strongly influences individual BSR LAB beer-spoilage phenotype and the environment they are able to grow in. Overall, beer-spoilage ability is shown to be adaptive and acquired incrementally and not solely as a result of the presence of hop-tolerance genes
A CXCR1/CXCR2 and heterologous GPCR antagonism in melanoma development
No full textBeing the most aggressive human skin cancers, melanoma has always occurred with a poor prognosis. It is responsible for 80% of skin cancer. Treatments for melanoma include surgical removal, and radio- and chemotherapy, which are not effective toward the advanced stages of the disease. Only three chemotherapy drugs, hydroxyurea, dacarbazine and interleukin-2, are currently approved by the Food and Drug Administration for metastatic melanoma, and the therapeutic response rate is only 5%-20%. Thus, there is a need for novel therapies that can target tumours, especially when the tumour cells become refractory to chemotherapy.
ELR–CXC chemokines with a Glutamine – leucine – arginine (ELR) motif (for example, interleukin-8/CXCL8) are able to chemoattract neutrophils during inflammation responses via their receptors, CXCR1 and CXCR2, which can be expressed by human malignant tumour cells, keratinocytes, endothelial cells, and fibroblasts. CXCR1 and CXCR2 play very important roles in melanoma by promoting tumour cell proliferation, angiogenesis, and metastasis. They are also involved in the tumour’s becoming refractory to chemotherapy.
An ELR–CXC chemokine antagonist developed by our lab, CXCL8(3-72)K11R/G31P (G31P), effectively blocks CXCR1- and CXCR2- induced inflammatory responses, and further antagonizes the functions of heterologous G protein–coupled receptor’s (GPCR). The tumour–associated GPCRs, along with ELR–CXC chemokines and their receptors, have been shown to simultaneously increase in several tumour models, including melanoma. Thus, given the knowledge regarding the importance of the ELR-CXC chemokines and heterologous GPCRs’ in melanoma and G31P’s ability to block ELR-CXC chemokines and at least some heterologous GPCRs, we hypothesize that G31P is a viable therapeutic option for melanoma cancers by virtue of its success in blocking tumour progression in mouse models.
Our data indicated that ELR-CXC chemokine antagonism with G31P had no significant impact on tumour growth or tumour-induced angiogenesis, which suggested that blockade of CXCR1 and CXCR2 alone was insufficient to block tumour development in this melanoma mouse model. Evaluation of other tumour-related parameters (e.g., angiogenic patterns and stress protein level) are recommended as a means of determining what parameters beyond CXCR1 and CXCR2 signaling are important in tumour progression in our matrigel model
Creation, evaluation, and use of PSI, a program for identifying protein-phenotype relationships and comparing protein content in groups of organisms
No full textRecent advances in DNA sequencing technology have enabled entire genomes to be sequenced quickly and accurately, resulting in an exponential increase in the number of organisms whose genome sequences have been elucidated. While the genome sequence of a given organism represents an important starting point in understanding its physiology, the functions of the protein products of many genes are still unknown; as such, computational methods for studying protein function are becoming increasingly important. In addition, this wealth of genomic information has created an unprecedented opportunity to compare the protein content of different organisms; among other applications, this can enable us to improve taxonomic classifications, to develop more accurate diagnostic tests for identifying particular bacteria, and to better understand protein content relationships in both closely-related and distantly-related organisms.
This thesis describes the design, evaluation, and use of a program called Proteome Subtraction and Intersection (PSI) that uses an idea called genome subtraction for discovering protein-phenotype relationships and for characterizing differences in protein content in groups of organisms. PSI takes as input a set of proteomes, as well as a partitioning of that set into a subset of "included" proteomes and a subset of "excluded" proteomes. Using reciprocal BLAST hits, PSI finds orthologous relationships among all the proteins in the proteomes from the original set, and then finds groups of orthologous proteins containing at least one orthologue from each of the proteomes in the "included" subset, and none from any of the proteomes in the "excluded" subset.
PSI is first applied to finding protein-phenotype relationships. By identifying proteins that are present in all sequenced isolates of the genus Lactobacillus, but not in the related bacterium Pediococcus pentosaceus, proteins are discovered that are likely to be responsible for the difference in cell shape between the lactobacilli and P. pentosaceus. In addition, proteins are identified that may be responsible for resistance to the antibiotic gatifloxacin in some lactic acid bacteria.
This thesis also explores the use of PSI for comparing protein content in groups of organisms. Based on the idea of genome subtraction, a novel metric is proposed for comparing the difference in protein content between two organisms. This metric is then used to create a phylogenetic tree for a large set of bacteria, which to the author's knowledge represents the largest phylogenetic tree created to date using protein content. In addition, PSI is used to find the proteomic cohesiveness of isolates of several bacterial species in order to support or refute their current taxonomic classifications.
Overall, PSI is a versatile tool with many interesting applications, and should become more and more valuable as additional genomic information becomes available
SOLID-PHASE ENZYME IMMUNOASSAY OF IgM-CLASS RHEUMATOID FACTOR: COMPARISON OF THREE METHODS FOR PREPARATION OF THE SOLID-PHASE TARGET IgG
No full textIdentification and analysis of the flagellin gene and protein from the genus pectinatus
No full textThe use of reduced oxygen-packaging techniques has resulted in anaerobic bacteria emerging as a problem for the brewing industry over the last twenty-five years. The genus Pectinatus, consisting of the speciesP. cerevisiiphilus and P. frisingensis, is a concern for producers of unpasteurized beer. As a result, there is an ongoing need to both understand this genus and develop rapid detection methodologies to combat its presence in the brewery. The objectives of this study were to sequence and characterize the flagellin genes from both Pectinatus species and evaluate the genes and proteins from a taxonomic and detection-suitability standpoint. A combination of micro-protein sequencing, polymerase chain reaction (PCR) and Bubble-PCR was used to completely sequence one flagellin gene from each Pectinatus species. This knowledge was then utilized to sequence the flagellin gene from four additional Pectinatus isolates, two from each species. To confirm the identity of the flagellin genes, one flagellin gene from each species was cloned, expressed and detected with Pectinatus-specific antibodies. A discrepancy between of the predicted protein size and the actual protein size led to tests for glycosylation, a post-translational modification. Taxonomic analyses, based on the flagellin genes, were conducted at both the superkingdom and genus levels. Finally, genus- and species-specific PCR primer sets were designed and tested for the specific detection of Pectinatus in the brewery. Cloning and expression data confirmed the identity of the sequenced genes as Pectinatus flagellin genes. Glycosylation was positively confirmed to be a post-translational modification for five of the six strains tested. Phylogenetic analysis revealed that both of the Pectinatus species grouped with the phylum Firmicutes (low G+C, Gram-positive bacteria) and that there was more diversity at the species level within the P. frisingensis flagellin gene than the P. cerevisiiphilus flagellin gene. As a final point, the detection of most Pectinatus isolates was achieved with the preliminary PCR primer sets designed, however, some non-Pectinatus beer spoilage organisms, primarily wort spoilage organisms, were also detected. Both the basic science and the applied results generated from this study will aid the brewing industry in its ongoing battle to control Pectinatus contamination
Comparison of DNA sequence assembly algorithms using mixed data sources
No full textDNA sequence assembly is one of the fundamental areas of bioinformatics. It involves the correct formation of a genome sequence from its DNA fragments ("reads") by aligning and merging the fragments. There are different sequencing technologies -- some support long DNA reads and the others, shorter DNA reads. There are sequence assembly programs specifically designed for these different types of raw sequencing data.
This work explores and experiments with these different types of assembly software in order to compare their performance on the type of data for which they were designed, as well as their performance on data for which they were not designed, and on mixed data. Such results are useful for establishing good procedures and tools for sequence assembly in the current genomic environment where read data of different lengths are available. This work also investigates the effect of the presence or absence of quality information on the results produced by sequence assemblers.
Five strategies were used in this research for assembling mixed data sets and the testing was done using a collection of real and artificial data sets for six bacterial organisms. The results show that there is a broad range in the ability of some DNA sequence assemblers to handle data from various sequencing technologies, especially data other than the kind they were designed for. For example, the long-read assemblers PHRAP and MIRA produced good results from assembling 454 data. The results also show the importance of having an effective methodology for assembling mixed data sets. It was found that combining contiguous sequences obtained from short-read assemblers with long DNA reads, and then assembling this combination using long-read assemblers was the most appropriate approach for assembling mixed short and long reads. It was found that the results from assembling the mixed data sets were better than the results obtained from separately assembling individual data from the different sequencing technologies. DNA sequence assemblers which do not depend on the availability of quality information were used to test the effect of the presence of quality values when assembling data. The results show that regardless of the availability of quality information, good results were produced in most of the assemblies.
In more general terms, this work shows that the approach or methodology used to assemble DNA sequences from mixed data sources makes a lot of difference in the type of results obtained, and that a good choice of methodology can help reduce the amount of effort spent on a DNA sequence assembly project
- …
