Despite the growing importance of bioinformatics in molecular diagnostics, not all medical laboratory sciences (MLS) programs provide instruction in this field. We developed and assessed a virtual ...laboratory learning unit to introduce basic bioinformatics concepts and tools to MLS students.
The unit included a video tutorial, written instructions for the online laboratory activity, and a postactivity review video. The effectiveness of the instruction was evaluated using preassessment and postassessment questions, performance of the online tasks, and a survey assessing the students' attitudes toward the learning unit.
A prototype of the module was tested with 32 graduate and undergraduate students. Modifications were made based on the pilot test results and student feedback, and the refined version was subsequently evaluated with a different group of 20 undergraduate students. The participants responded favorably to the learning unit and successfully achieved the learning objectives, gaining familiarity with fundamental bioinformatics concepts and terminology, effectively employing basic computational tools, and developing an appreciation for the field.
Our learning unit is a promising tool for introducing MLS students to the field of bioinformatics. As an open educational resource, it has the potential to be integrated into molecular biology education for MLS programs anywhere.
Abstract
Objectives
The implementation of nucleic acid testing in laboratory medicine has revolutionized clinical diagnosis. Unfortunately, incorporation of these technologies in less developed ...countries remains a challenge. Despite Romania’s recent economic growth, the country is in dire need of medical and laboratory staff trained in modern technologies. The aim of the study was to develop a curriculum that could easily be delivered to laboratory professionals in Romania and to pilot test the effectiveness of the training in increasing their understanding of molecular tests.
Methods
The program was developed in accordance with the US Centers for Disease Control and Prevention’s (CDC’s) quality training standards. It was offered to 50 laboratory professionals and consisted of online, asynchronous lectures and optional synchronous review sessions. Training effectiveness was evaluated using CDC guidelines based on pre- and postassessment questions answered anonymously.
Results
Forty-two people participated in the program, and 32 (81%) completed the training successfully. Based on 16 participants’ self-assessment, the course was successful in improving learners’ overall knowledge of molecular diagnostics—specifically, their understanding of molecular techniques and how to interpret results. Those participants were highly satisfied with the overall training.
Conclusions
The piloted platform presented here is promising and can be a foundation for future larger-scale studies in countries with developing health systems.
Yersinia pseudotuberculosis is a foodborne pathogenic bacterium that causes acute gastrointestinal illness, but its mechanisms of infection are incompletely described. We examined how host cell ...sterol composition affected Y. pseudotuberculosis uptake. To do this, we depleted or substituted cholesterol in human MDA-MB-231 epithelial cells with various alternative sterols. Decreasing host cell cholesterol significantly reduced pathogen internalization. When host cell cholesterol was substituted with various sterols, only desmosterol and 7-dehydrocholesterol supported internalization. This specificity was not due to sterol dependence of bacterial attachment to host cells, which was similar with all sterols studied. Because a key step in Y. pseudotuberculosis internalization is interaction of the bacterial adhesins invasin and YadA with host cell β1 integrin, we compared the sterol dependence of wildtype Y. pseudotuberculosis internalization with that of Δinv, ΔyadA, and ΔinvΔyadA mutant strains. YadA deletion decreased bacterial adherence to host cells, whereas invasin deletion had no effect. Nevertheless, host cell sterol substitution had a similar effect on internalization of these bacterial deletion strains as on the wildtype bacteria. The ΔinvΔyadA double mutant adhered least to cells and so was not significantly internalized. The sterol structure dependence of Y. pseudotuberculosis internalization differed from that of endocytosis, as monitored using antibody-clustered β1 integrin and previous studies on other proteins, which had a more permissive sterol dependence. This study suggests that agents could be designed to interfere with internalization of Yersinia without disturbing endocytosis.
Serotonin (5-HT) modulates synaptic efficacy in the nervous system of vertebrates and invertebrates. In the nematode Caenorhabditis elegans, many behaviors are regulated by 5-HT levels, which are in ...turn regulated by the presence or absence of food. Here, we show that both food and 5-HT signaling modulate chemosensory avoidance response of octanol in C. elegans, and that this modulation is both rapid and reversible. Sensitivity to octanol is decreased when animals are off food or when 5-HT levels are decreased; conversely, sensitivity is increased when animals are on food or have increased 5-HT signaling. Laser microsurgery and behavioral experiments reveal that sensory input from different subsets of octanol-sensing neurons is selectively used, depending on stimulus strength, feeding status, and 5-HT levels. 5-HT directly targets at least one pair of sensory neurons, and 5-HT signaling requires the Gα protein GPA-11. Glutamatergic signaling is required for response to octanol, and the GLR-1 glutamate receptor plays an important role in behavioral response off food but not on food. Our results demonstrate that 5-HT modulation of neuronal activity via G protein signaling underlies behavioral plasticity by rapidly altering the functional circuitry of a chemosensory circuit.
, the causative agent of plague, evolved from the closely related pathogen
During its emergence,
is believed to have acquired its unique pathogenic characteristics through numerous gene gains/losses, ...genomic rearrangements, and single nucleotide polymorphism (SNP) changes. One such SNP creates a single amino acid variation in the DNA binding domain of PhoP, the response regulator in the PhoP/PhoQ two-component system.
and the basal human-avirulent strains of
harbor glycines at position 215 of PhoP, whereas the modern human-virulent strains (e.g., KIM and CO92) harbor serines at this residue. Since PhoP plays multiple roles in the adaptation of
to stressful host conditions, we tested whether this amino acid substitution affects PhoP activity or the ability of
to survive in host environments. Compared to the parental KIM6+ strain carrying the modern allele of
(
), a derivative carrying the basal allele (
) exhibited slightly defective growth under a low-Mg
condition and decreased transcription of a PhoP target gene,
, as well as an ∼8-fold increase in the susceptibility to the antimicrobial peptide polymyxin B. The
strain showed no apparent defect in flea colonization, although a
-null mutant showed decreased flea infectivity in competition experiments. Our results suggest that the amino acid variation at position 215 of PhoP causes subtle changes in the PhoP activity and raise the possibility that the change in this residue have contributed to the evolution of increased virulence in
acquired a single nucleotide polymorphism (SNP) in
when the highly human-virulent strains diverged from less virulent basal strains, resulting in an amino acid substitution in the DNA binding domain of the PhoP response regulator. We show that
carrying the modern
allele has an increased ability to induce the PhoP-regulated
gene and resist antimicrobial peptides compared to an isogenic strain carrying the basal allele. Given the important roles PhoP plays in host adaptation, the results raise an intriguing possibility that this amino acid substitution contributed to the evolution of increased virulence in
Additionally, we present the first evidence that
confers a survival fitness advantage to
inside the flea midgut.
The transcriptional activator PhoP is important for survival of Yersinia pestis in macrophage phagosomes. However, the phagosomes inhabited by Y. pestis have not been well characterized, and the ...mechanism by which PhoP promotes bacterial survival in these vacuoles is not fully understood. Lysosomal tracers, as well as antibodies to late endosomal or lysosomal proteins, were used in conjunction with confocal or electron microscopy to study the trafficking of phagosomes containing phoP⁺ or phoP mutant Y. pestis strains or latex beads in J774A.1 macrophages. Phagosomes containing phoP⁺ or phoP mutant Y. pestis acquired lysosomal markers to the same degree that phagosomes containing latex beads acquired these markers after 1.5 h of infection, showing that nascent phagosomes containing Y. pestis fuse with lysosomes irrespective of the phoP genotype. Similar results were obtained when phagosomes containing viable or dead phoP⁺ Y. pestis cells or beads were analyzed at 8 h postinfection, indicating that the Y. pestis vacuole does not become secluded from the lysosomal compartment. However, only viable phoP⁺ bacteria induced the formation of spacious phagosomes at 8 h postinfection, suggesting that Y. pestis can actively direct the expansion of its vacuole. PhoP-regulated genes that are important for survival of Y. pestis in phagosomes were identified by Tn5-lacZ mutagenesis and oligonucleotide microarray analysis. Three such genes were identified, and the products of these genes are predicted to promote resistance to antimicrobial peptides (ugd and pmrK) or low-Mg²⁺ conditions (mgtC) found in phagosomes. Viable count assays carried out with Y. pestis ugd, mgtC, and ugd mgtC mutants revealed that the products of ugd and mgtC function independently to promote early survival of Y. pestis in macrophage phagosomes.
, the causative agent of plague, has a complex infectious cycle that alternates between mammalian hosts (rodents and humans) and insect vectors (fleas). Consequently, it must adapt to a wide range of ...host environments to achieve successful propagation.
PhoP is a response regulator of the PhoP/PhoQ two-component signal transduction system that plays a critical role in the pathogen's adaptation to hostile conditions. PhoP is activated in response to various host-associated stress signals detected by the sensor kinase PhoQ and mediates changes in global gene expression profiles that lead to cellular responses.
PhoP is required for resistance to antimicrobial peptides, as well as growth under low Mg
and other stress conditions, and controls a number of metabolic pathways, including an alternate carbon catabolism. Loss of
function in
causes severe defects in survival inside mammalian macrophages and neutrophils in vitro, and a mild attenuation in murine plague models in vivo, suggesting its role in pathogenesis. A
mutant also exhibits reduced ability to form biofilm and to block fleas in vivo, indicating that the gene is also important for establishing a transmissible infection in this vector. Additionally,
promotes the survival of
inside the soil-dwelling amoeba
, a potential reservoir while the pathogen is quiescent. In this review, we summarize our current knowledge on the mechanisms of PhoP-mediated gene regulation in
and examine the significance of the roles played by the PhoP regulon at each stage of the
life cycle.
Yersinia pestis is able to survive and replicate within murine macrophages. However, the mechanism by which Y. pestis promotes its intracellular survival is not well understood. To identify genes ...that are important for Y. pestis survival in macrophages, a library comprised of ∼31,500 Y. pestis KIM6+ transposon insertion mutants (input pool) was subjected to negative selection in primary murine macrophages. Genes underrepresented in the output pool of surviving bacteria were identified by transposon site hybridization to DNA oligonucleotide microarrays. The screen identified several genes known to be important for survival of Y. pestis in macrophages, including phoPQ and members of the PhoPQ regulon (e.g., pmrF). In addition, genes predicated to encode a glucose-1-phosphate uridylyltransferase (galU), a UDP-N-acetylglucosamine 2-epimerase (wecB) and a UDP-N-acetyl-D-mannosamine dehydrogenase (wecC) were identified in the screen. Viable-count assays demonstrated that a KIM6+ galU mutant and a KIM6+ wecBC mutant were defective for survival in murine macrophages. The galU mutant was studied further because of its strong phenotype. The KIM6+ galU mutant exhibited increased susceptibility to the antimicrobial peptides polymyxin B and cathelicidin-related antimicrobial peptide (CRAMP). Polyacrylamide gel electrophoresis demonstrated that the lipooligosaccharide (LOS) of the galU mutant migrated faster than the LOS of the parent KIM6+, suggesting the core was truncated. In addition, the analysis of LOS isolated from the galU mutant by mass spectrometry showed that aminoarabinose modification of lipid A is absent. Therefore, addition of aminoarabinose to lipid A and complete LOS core (galU), as well as enterobacterial common antigen (wecB and wecC), is important for survival of Y. pestis in macrophages.
Yersinia pestis, the causative agent of plague, is a facultative intracellular pathogen. Previous studies have indicated that the ability of Y. pestis to survive inside macrophages may be critical ...during the early stages of plague pathogenesis. To gain insights into the biology of intracellular Y. pestis and its environment following phagocytosis, we determined the genome-wide transcriptional profile of Y. pestis KIM5 replicating inside J774.1 macrophage-like cells using DNA microarrays. At 1.5, 4, and 8 h postinfection, a total of 801, 464, and 416 Y. pestis genes were differentially regulated, respectively, compared to the level of gene expression of control bacteria grown in tissue culture medium. A number of stress-response genes, including those involved in detoxification of reactive oxygen species, as well as several metabolic genes involved in macromolecule synthesis, were significantly induced in intracellular Y. pestis, consistent with the presence of oxidative stress and nutrient starvation inside Yersinia-containing vacuoles. A putative stress-induced operon consisting of y2313, y2315, and y2316 (y2313-y2316), and a previously unidentified open reading frame, orfX, was studied further on the basis of its high level of intracellular expression. Mutant strains harboring either deletion, Δy2313-y2316 or ΔorfX, exhibited diverse phenotypes, including reduced effector secretion by the type III secretion system, increased intracellular replication, and filamentous morphology of the bacteria growing inside macrophages. The results suggest a possible role for these genes in regulating cell envelope characteristics in the intracellular environment.
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