Matching Items (12)

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Rapid Characterization of Microalgae and Microalgae Mixtures Using Matrix-Assisted Laser Desorption Ionization Time-Of-Flight Mass Spectrometry (MALDI-TOF MS)

Description

Current molecular methods to characterize microalgae are time-intensive and expensive. Matrix Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) may represent a rapid and economical alternative approach. The objectives of

Current molecular methods to characterize microalgae are time-intensive and expensive. Matrix Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) may represent a rapid and economical alternative approach. The objectives of this study were to determine whether MALDI-TOF MS can be used to: 1) differentiate microalgae at the species and strain levels and 2) characterize simple microalgal mixtures. A common protein extraction sample preparation method was used to facilitate rapid mass spectrometry-based analysis of 31 microalgae. Each yielded spectra containing between 6 and 56 peaks in the m/z 2,000 to 20,000 range. The taxonomic resolution of this approach appeared higher than that of 18S rDNA sequence analysis. For example, two strains of Scenedesmus acutus differed only by two 18S rDNA nucleotides, but yielded distinct MALDI-TOF mass spectra. Mixtures of two and three microalgae yielded relatively complex spectra that contained peaks associated with members of each mixture. Interestingly, though, mixture-specific peaks were observed at m/z 11,048 and 11,230. Our results suggest that MALDI-TOF MS affords rapid characterization of individual microalgae and simple microalgal mixtures.

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Date Created
  • 2015-08-13

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A Designed Experiments Approach to Optimizing MALDI-TOF MS Spectrum Processing Parameters Enhances Detection of Antibiotic Resistance in Campylobacter jejuni

Description

MALDI-TOF MS has been utilized as a reliable and rapid tool for microbial fingerprinting at the genus and species levels. Recently, there has been keen interest in using MALDI-TOF MS

MALDI-TOF MS has been utilized as a reliable and rapid tool for microbial fingerprinting at the genus and species levels. Recently, there has been keen interest in using MALDI-TOF MS beyond the genus and species levels to rapidly identify antibiotic resistant strains of bacteria. The purpose of this study was to enhance strain level resolution for Campylobacter jejuni through the optimization of spectrum processing parameters using a series of designed experiments. A collection of 172 strains of C. jejuni were collected from Luxembourg, New Zealand, North America, and South Africa, consisting of four groups of antibiotic resistant isolates. The groups included: (1) 65 strains resistant to cefoperazone (2) 26 resistant to cefoperazone and beta-lactams (3) 5 strains resistant to cefoperazone, beta-lactams, and tetracycline, and (4) 76 strains resistant to cefoperazone, teicoplanin, amphotericin, B and cephalothin. Initially, a model set of 16 strains (three biological replicates and three technical replicates per isolate, yielding a total of 144 spectra) of C. jejuni was subjected to each designed experiment to enhance detection of antibiotic resistance. The most optimal parameters were applied to the larger collection of 172 isolates (two biological replicates and three technical replicates per isolate, yielding a total of 1,031 spectra). We observed an increase in antibiotic resistance detection whenever either a curve based similarity coefficient (Pearson or ranked Pearson) was applied rather than a peak based (Dice) and/or the optimized preprocessing parameters were applied. Increases in antimicrobial resistance detection were scored using the jackknife maximum similarity technique following cluster analysis. From the first four groups of antibiotic resistant isolates, the optimized preprocessing parameters increased detection respective to the aforementioned groups by: (1) 5% (2) 9% (3) 10%, and (4) 2%. An additional second categorization was created from the collection consisting of 31 strains resistant to beta-lactams and 141 strains sensitive to beta-lactams. Applying optimal preprocessing parameters, beta-lactam resistance detection was increased by 34%. These results suggest that spectrum processing parameters, which are rarely optimized or adjusted, affect the performance of MALDI-TOF MS-based detection of antibiotic resistance and can be fine-tuned to enhance screening performance.

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Created

Date Created
  • 2016-05-31

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Biomarker- and similarity coefficient-based approaches to bacterial mixture characterization using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS)

Description

MALDI-TOF MS profiling has been shown to be a rapid and reliable method to characterize pure cultures of bacteria. Currently, there is keen interest in using this technique to identify

MALDI-TOF MS profiling has been shown to be a rapid and reliable method to characterize pure cultures of bacteria. Currently, there is keen interest in using this technique to identify bacteria in mixtures. Promising results have been reported with two- or three-isolate model systems using biomarker-based approaches. In this work, we applied MALDI-TOF MS-based methods to a more complex model mixture containing six bacteria. We employed: 1) a biomarker-based approach that has previously been shown to be useful in identification of individual bacteria in pure cultures and simple mixtures and 2) a similarity coefficient-based approach that is routinely and nearly exclusively applied to identification of individual bacteria in pure cultures. Both strategies were developed and evaluated using blind-coded mixtures. With regard to the biomarker-based approach, results showed that most peaks in mixture spectra could be assigned to those found in spectra of each component bacterium; however, peaks shared by two isolates as well as peaks that could not be assigned to any individual component isolate were observed. For two-isolate blind-coded samples, bacteria were correctly identified using both similarity coefficient- and biomarker-based strategies, while for blind-coded samples containing more than two isolates, bacteria were more effectively identified using a biomarker-based strategy.

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Date Created
  • 2015-11-05

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Identification of Transcriptomic Biomarkers for use in Diagnosis of Irritable Bowel Syndrome (IBS)

Description

Irritable bowel syndrome (IBS) is a common gastrointestinal disorder that afflicts more than 20% of the population in the United States. Symptoms include mild to severe abdominal discomfort accompanied by

Irritable bowel syndrome (IBS) is a common gastrointestinal disorder that afflicts more than 20% of the population in the United States. Symptoms include mild to severe abdominal discomfort accompanied by a change in stool character and form ranging from constipation to diarrhea. Additionally, IBS is associated with secondary effects including depression, anxiety, poor quality of life, insomnia and sexual dysfunction. Despite the known association of secondary effects, patients are often tested for potential illnesses that share similar pathological symptoms. This process can be costly and protracted and yet not deliver a completely accurate diagnosis. The aim of this research is to identify gene expression-based biological signatures and unique biomarkers for the detection of IBS. Through the use of quantitative polymerase chain reaction (qPCR), comparison of pooled samples of non-IBS patient-derived RNA were used to identify differentially expressed genes in patients with IBS. Data obtained from preliminary DNA microarray analysis demonstrated a degree of success in differentiating between IBS and asymptomatic patients. Additional comprehensive DNA microarray analyses have led to the identification of a series of 858 differentially expressed genes, including genes associated with serotonin metabolism, which may characterize the IBS pathological state. The microarray results were screened using a combination of gene ontological analysis and qPCR. Real-time PCR revealed repressed levels of tryptophan hydroxylase (TPH1), an enzyme involved in the rate- limiting step in serotonin biosynthesis, in IBS patients relative to controls. Lower concentrations of serum 25(OH)D were also observed among the IBS cohort relative to asymptomatic patients, especially among IBS-D subtype. Vitamin D was shown to modulate differentially expressed genes in IBS patients, suggesting that IBS pathophysiology may involve vitamin D insufficiency and/or an irregularity in serotonin metabolism. Additional qPCR analysis of 32 differentially expressed genes in IBS patients identified 7 putative genetic biomarkers proposed for a potential IBS diagnostic panel. Based on the quality of these results, we may be able to develop, test, and market a diagnostic kit for IBS.

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Date Created
  • 2017-12

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Understanding the Role of the Repair Response during Localized Tissue Damage in D. melanogaster

Description

Proper developmental fidelity ensures uninterrupted progression towards sexual maturity and species longevity. However, early development, the time-frame spanning infancy through adolescence, is a fragile state since organisms have limited mobility

Proper developmental fidelity ensures uninterrupted progression towards sexual maturity and species longevity. However, early development, the time-frame spanning infancy through adolescence, is a fragile state since organisms have limited mobility and responsiveness towards their environment. Previous studies have shown that damage during development leads to an onset of developmental delay which is proportional to the extent of damage accrued by the organism. In contrast, damage sustained in older organisms does not delay development in response to tissue damage. In the fruit fly, Drosophila melanogaster, damage to wing precursor tissues is associated with developmental retardation if damage is sustained in young larvae. No developmental delay is observed when damage is inflicted closer to pupariation time. Here we use microarray analysis to characterize the genomic response to injury in Drosophila melanogaster in young and old larvae. We also begin to develop tools to examine in more detail, the role that the neurotransmitter dopamine might play in mediating injury-induced developmental delays.

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Date Created
  • 2016-05

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Identification of Differentially Expressed Genes as Biomarkers for Diagnosis of Irritable Bowel Syndrome (IBS): A Pilot Gene Discovery Hypothesis Generating Study

Description

The diagnosis of irritable bowel syndrome (IBS) is currently based on symptomatic criteria that exclude other conditions affecting the gastrointestinal tract, such as celiac disease, food allergies, and infections. The

The diagnosis of irritable bowel syndrome (IBS) is currently based on symptomatic criteria that exclude other conditions affecting the gastrointestinal tract, such as celiac disease, food allergies, and infections. The absence of appropriate diagnostic and therapeutic approaches for IBS places a significant burden on the patient and the health care system due to direct and indirect costs of care. Limitations associated with the application of symptomatic criteria include inappropriate use and/or intrinsic limitations such as the population to which these criteria are applied. The lack of biomarkers specific for IBS, non-specific abdominal symptoms, and considerable variability in the disease course creates additional uncertainty during diagnosis. This project involved screening tissue samples from patients with verified IBS to identify gene expression-based biomarkers associated with IBS. Through validation of microarray gene chip data on the tissue samples using PCR, it was determined that a number of genes within the diseased IBS patient tissue samples were differentially expressed in comparison to the healthy subjects. These findings could potentially lead to the diagnosis of IBS on the basis of a genetic "fingerprint".

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Date Created
  • 2013-12

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Identification of Mycobacterium smegmatis and antibiotic resistance through the utilization of high titer mycobacteriophage concentrations and MALDI-TOF MS

Description

The diagnosis of bacterial infections based on phage multiplication has the potential for profound clinical implications, particularly for antibiotic-resistant strains and the slow-growing Mycobacterium tuberculosis. The possibility of hastening the

The diagnosis of bacterial infections based on phage multiplication has the potential for profound clinical implications, particularly for antibiotic-resistant strains and the slow-growing Mycobacterium tuberculosis. The possibility of hastening the diagnosis of antibiotic-resistant mycobacterial infections was accomplished via the study of Mycobacterium smegmatis, a generally non-pathogenic, comparatively fast growing microorganism to M. tuberculosis. These proof-of-concept studies established that after transduction of M. smegmatis cells with bacteriophages, MALDI-TOF MS could be used to detect increased amounts of phage proteins. Recording the growth of M. smegmatis over an 8-hour period, starting with very low OD600 measurements, simulated bacterial loads in clinical settings. For the purposes of MALDI-TOF MS, the procedure for the most effective lethal exposure for M. smegmatis was determined to be a 1-hour incubation in a 95°C water bath. Successful precipitation of the lytic mycobacteriophages D29 and Giles was performed using chloroform and methanol and overlaid with 1-2 μL of α-cyano-4-hydoxycinnaminic acid, which allowed for more distinct and repeatable MALDI-TOF MS spectra. Phage D29 was found to produce an m/z peak at 18.477 kDa, which may have indicated a 2+-charged ion of the 34.8 kDa minor tail protein. The Giles proteins that were identified with MALDI-TOF MS have not been directly compared to protein values reported in the scientific literature. However, the MALDI-TOF MS spectra suggested that distinct peaks existed between M. smegmatis mc2155 and mycobacteriophages, indicating that successful infection with lytic phage and replication thereafter may have occurred. The distinct peaks between M. smegmatis and the phage can be used as indicators of the presence of mycobacteria. At this point, the limits of detection of each phage must be elucidated in order for MALDI-TOF MS spectra to be successfully implemented as a mechanism to rapidly detect antibiotic-resistant mycobacteria.

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Date Created
  • 2015-05

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A Designed Experiments Approach to Optimization of Automated Data Acquisition during Characterization of Bacteria with MALDI-TOF Mass Spectrometry

Description

MALDI-TOF MS has been shown capable of rapidly and accurately characterizing bacteria. Highly reproducible spectra are required to ensure reliable characterization. Prior work has shown that spectra acquired manually can

MALDI-TOF MS has been shown capable of rapidly and accurately characterizing bacteria. Highly reproducible spectra are required to ensure reliable characterization. Prior work has shown that spectra acquired manually can have higher reproducibility than those acquired automatically. For this reason, the objective of this study was to optimize automated data acquisition to yield spectra with reproducibility comparable to those acquired manually. Fractional factorial design was used to design experiments for robust optimization of settings, in which values of five parameters (peak selection mass range, signal to noise ratio (S:N), base peak intensity, minimum resolution and number of shots summed) commonly used to facilitate automated data acquisition were varied. Pseudomonas aeruginosa was used as a model bacterium in the designed experiments, and spectra were acquired using an intact cell sample preparation method. Optimum automated data acquisition settings (i.e., those settings yielding the highest reproducibility of replicate mass spectra) were obtained based on statistical analysis of spectra of P. aeruginosa. Finally, spectrum quality and reproducibility obtained from non-optimized and optimized automated data acquisition settings were compared for P. aeruginosa, as well as for two other bacteria, Klebsiella pneumoniae and Serratia marcescens. Results indicated that reproducibility increased from 90% to 97% (p-value [~ over =] 0.002) for P. aeruginosa when more shots were summed and, interestingly, decreased from 95% to 92% (p-value [~ over =] 0.013) with increased threshold minimum resolution. With regard to spectrum quality, highly reproducible spectra were more likely to have high spectrum quality as measured by several quality metrics, except for base peak resolution. Interaction plots suggest that, in cases of low threshold minimum resolution, high reproducibility can be achieved with fewer shots. Optimization yielded more reproducible spectra than non-optimized settings for all three bacteria.

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Date Created
  • 2014-03-24

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Genetic and Biochemical Insights into the Mycobacterial PrrAB System as a Regulator of Respiration and Central Metabolism

Description

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, is the 10th leading cause of death, worldwide. The prevalence of drug-resistant clinical isolates and the paucity of newly-approved antituberculosis drugs impedes

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, is the 10th leading cause of death, worldwide. The prevalence of drug-resistant clinical isolates and the paucity of newly-approved antituberculosis drugs impedes the successful eradication of Mtb. Bacteria commonly use two-component systems (TCS) to sense their environment and genetically modulate adaptive responses. The prrAB TCS is essential in Mtb, thus representing an auspicious drug target; however, the inability to generate an Mtb ΔprrAB mutant complicates investigating how this TCS contributes to pathogenesis. Mycobacterium smegmatis, a commonly used M. tuberculosis genetic surrogate was used here. This work shows that prrAB is not essential in M. smegmatis. During ammonium stress, the ΔprrAB mutant excessively accumulates triacylglycerol lipids, a phenotype associated with M. tuberculosis dormancy and chronic infection. Additionally, triacylglycerol biosynthetic genes were induced in the ΔprrAB mutant relative to the wild-type and complementation strains during ammonium stress. Next, RNA-seq was used to define the M. smegmatis PrrAB regulon. PrrAB regulates genes participating in respiration, metabolism, redox balance, and oxidative phosphorylation. The M. smegmatis ΔprrAB mutant is compromised for growth under hypoxia, is hypersensitive to cyanide, and fails to induce high-affinity respiratory genes during hypoxia. Furthermore, PrrAB positively regulates the hypoxia-responsive dosR TCS response regulator, potentially explaining the hypoxia-mediated growth defects in the ΔprrAB mutant. Despite inducing genes encoding the F1F0 ATP synthase, the ΔprrAB mutant accumulates significantly less ATP during aerobic, exponential growth compared to the wild-type and complementation strains. Finally, the M. smegmatis ΔprrAB mutant exhibited growth impairment in media containing gluconeogenic carbon sources. M. tuberculosis mutants unable to utilize these substrates fail to establish chronic infection, suggesting that PrrAB may regulate Mtb central carbon metabolism in response to chronic infection. In conclusion, 1) prrAB is not universally essential in mycobacteria; 2) M. smegmatis PrrAB regulates genetic responsiveness to nutrient and oxygen stress; and 3) PrrAB may provide feed-forward control of the DosRS TCS and dormancy phenotypes. The data generated in these studies provide insight into the mycobacterial PrrAB TCS transcriptional regulon, PrrAB essentiality in Mtb, and how PrrAB may mediate stresses encountered by Mtb during the transition to chronic infection.

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Date Created
  • 2019

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MALDI-TOF MS as a rapid characterization tool for economically-relevant microalgae

Description

The ability of microalgae to be mass cultivated and harvested for production of pharmaceuticals, nutraceuticals, and biofuels has made microalgae a focal point of scientific investigation. However, negative impacts

The ability of microalgae to be mass cultivated and harvested for production of pharmaceuticals, nutraceuticals, and biofuels has made microalgae a focal point of scientific investigation. However, negative impacts on production are essentially inevitable due to the open design of many microalgae mass culture systems. This challenge generates a need for the consistent monitoring of microalgae cultures for health and the presence of contaminants, predators, and competitors. The techniques for monitoring microalgae cultures are generally time-intensive, labor-intensive, and expensive. The scope of this work was to evaluate the use of Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) as a viable alternative for the characterization of microalgae cultures. The studies presented here evaluated whether MALDI-TOF MS can be used to: 1) differentiate microalgae at the species and strain levels, 2) characterize simple mixtures of microalgae, 3) detect changes in a single microalgae culture over time, and 4) characterize growth phases of microalgae cultures. This research required the development of a MALDI-TOF MS microalgae analysis protocol for organism characterization. The results yielded in this research showed that MALDI-TOF MS was just as accurate, if not more so, than molecular techniques for the identification of microalgae at the species and strain levels during its logarithmic growth phase. Additionally, results suggest that MALDI-TOF MS is sensitive enough to characterize simple mixtures and detect changes in cultures over time. The data presented here suggests the next logical step is the development of protocols for the near-real time health monitoring of microalgae cultures and detection of contaminants using MALDI-TOF MS.

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Created

Date Created
  • 2016