Nanoparticles may be introduced into aquatic environments during production processes and also as a result of release following their use in various commercial formulations and biologic applications. ...Filter-feeding bivalve mollusks such as oysters are valuable model species for characterizing nanoparticle bioavailability and interactions with basic cellular processes. The adults release their gametes into the environment, so their embryos and larvae are also likely targets of nanoparticles. The purpose of these studies was to characterize the toxicity of metal nanoparticles on embryonic development of oysters,
Crassostrea virginica and to compare the relative sensitivity of embryos to adults. Newly-fertilized oyster embryos were exposed to silver nanoparticles (AgNP) and then the percent normal development after 48
h was assessed. Studies were conducted with adult oysters in which they were also exposed to AgNP for 48
h, and the effects on lysosomal destabilization were determined. The expression of metallothionein (MT) gene expression was also assessed in both embryos and adults. Adverse effects on embryonic development were observed at concentrations similar to those that caused both statistically and biologically significant effects on lysosomal destabilization of adults. Significant increases in MT mRNA levels were observed in both embryos and adult oysters, and MT levels were highly induced in embryos. While we do not know whether the toxicity and gene expression responses observed in this study were due to the nanoparticles themselves or the Ag ions that dissociated from the nanoparticles, these kinds of basic studies are essential for addressing the potential impacts of nanoengineered particles on fundamental cellular processes as well as aquatic organisms.
The interaction of Borrelia burgdorferi, the causative agent of Lyme borreliosis, with phagocytic cells induces the activation of NF-κB and the expression of proinflammatory cytokines including tumor ...necrosis factor alpha (TNF-α). B. burgdorferi-induced TNF-α production is also dependent on the activation of p38 mitogen-activated protein (MAP) kinase. The specific contribution of these signaling pathways to the response of phagocytic cells to the spirochete and the molecular mechanisms underlying this response remain unresolved. We now show that p38 MAP kinase activity regulates the transcriptional activation of NF-κB in response to spirochetal lysate stimulation of phagocytic cells. The regulation occurs at the nuclear level and is independent of the translocation of the transcription factor to the nucleus or its capacity to bind to specific DNA target sequences. In RAW264.7 cells, p38α MAP kinase regulates the phosphorylation of NF-κB RelA. p38 MAP kinase phosphorylates the nuclear kinase mitogen- and stress-activated protein kinase 1 (MSK1). MSK1 in turn phosphorylates the transcriptionally active subunit of NF-κB, RelA. The repression of MSK1 expression with small interfering RNA results in reduced RelA phosphorylation and a significant decrease in the production of TNF-α in response to B. burgdorferi lysates. Overall, these results clarify the contribution of the signaling pathways that are activated in response to the interaction of spirochetes with phagocytic cells to TNF-α production. Our results situate p38 MAP kinase activity as a central regulator of the phagocytic proinflammatory response through MSK1-mediated transcriptional activation of the transcription factor NF-κB.
The Lyme disease spirochete Borrelia burgdorferi is the only known human pathogen that directly activates invariant NKT (iNKT) cells. The number and activation kinetics of iNKT cells vary greatly ...among different strains of mice. We now report the role of the iNKT cell response in the pathogenesis of Lyme disease using C57BL/6 mice, a strain with optimal iNKT cell activation that is resistant to the development of spirochetal-induced inflammation. During experimental infection of B6 mice with B. burgdorferi, iNKT cells localize to the inflamed heart where they are activated by CD1d-expressing macrophages. Activation of iNKT cells in vivo results in the production of IFN-gamma, which we demonstrate ameliorates the severity of murine Lyme carditis by at least two mechanisms. First, IFN-gamma enhances the recognition of B. burgdorferi by macrophages, leading to increased phagocytosis of the spirochete. Second, IFN-gamma activation of macrophages increases the surface expression of CD1d, thereby facilitating further iNKT activation. Collectively, our data demonstrate that in the resistant background, B6, iNKT cells modulate the severity of murine Lyme carditis through the action of IFN-gamma, which appears to self-renew through a positive feedback loop during infection.
Vibrio vulnificus has proven difficult to culture from water or shellfish during winter months, which is attributed to the viable but nonculturable (VBNC) state. Because reactive oxygen species were ...found to be involved in the low temperature-induced entrance of
V. vulnificus into this state, we generated an
oxyR mutant which lacks catalase activity. This strain is nonculturable on solid media even at ambient temperature, due to the presence of H
2O
2 in such media. Low temperature incubation of the parent resulted in loss of catalase activity, making the cells H
2O
2 sensitive, and paralleling the loss of culturability (entry into the VBNC state). Thus, cells of
V. vulnificus in the VBNC state are likely exhibiting this response to low in situ temperature and only when the artificial condition of laboratory culture is attempted are the cells nonculturable due to cold-induced loss of catalase activity. To our knowledge, this is the first study providing direct evidence for the metabolic basis of nonculturability and the viable but nonculturable state.
Members of the testis-specific serine/threonine kinases (Tssk) family may have a role in sperm differentiation in the testis and/or fertilization. To gain insight into the functional relevance of ...these kinases, their expression was examined both at the mRNA and protein levels. Quantitative PCR analysis confirmed that all five Tssk mRNAs are almost exclusively expressed postmeiotically in the testis. Recombinant mouse and human Tssks were cloned and used for validation of an array of commercial and custom-made antibodies against Tssks. Immunolocalization in mouse testis, and in mouse and human sperm, showed that Tssk1, Tssk2, Tssk4 and Tssk6, but not Tssk3, were present in mouse sperm and in germ cells from mouse testis. TSSK1, TSSK2 and TSSK6 were also detected in human sperm, while TSSK3 was absent. In both mouse and human sperm, Tssk1 was partially soluble, while Tssk2, Tssk4 and Tssk6 were insoluble in non-ionic detergents. In vitro recombinant TSSK2 activity assays showed maximum enzymatic activity at 5 mM Mg(2+) and a Km for ATP of ∼10 µM. These, observations together with findings that the Tssk1/Tssk2 double knock-out as well as the Tssk6 null mice are sterile without presenting other detectable defects, suggest that these kinases could be used as targets for male contraception.
Arthrobacter phage Tokki is a siphovirus isolated from soil in River Falls, Wisconsin. The genome contains 57,652-bp encoding 98 proteins, of which 23 were assigned a function. Tokki’s genome ...structure and content is typical of other AU2 subcluster phages, except for the lack of an identifiable acetyltransferase.
Phage Culver, with a siphovirus morphology, was isolated using
CAG3. Culver is assigned to phage cluster CQ1 based on gene content similarity to actinobacteriophages. Notably, Culver is predicted to ...encode eight tRNAs, lysin A by two adjacent genes, and, unlike other CQ1 phages, two putative integrase genes.
Course-based research pedagogy involves positioning students as contributors to authentic research projects as part of an engaging educational experience that promotes their learning and persistence ...in science. To develop a model for assessing and grading students engaged in this type of learning experience, the assessment aims and practices of a community of experienced course-based research instructors were collected and analyzed. This approach defines four aims of course-based research assessment—(1) Assessing Laboratory Work and Scientific Thinking; (2) Evaluating Mastery of Concepts, Quantitative Thinking and Skills; (3) Appraising Forms of Scientific Communication; and (4) Metacognition of Learning—along with a set of practices for each aim. These aims and practices of assessment were then integrated with previously developed models of course-based research instruction to reveal an assessment program in which instructors provide extensive feedback to support productive student engagement in research while grading those aspects of research that are necessary for the student to succeed. Assessment conducted in this way delicately balances the need to facilitate students’ ongoing research with the requirement of a final grade without undercutting the important aims of a CRE education.
Background & aims: MicroRNAs (miRNAs) are members of a class of small noncoding functional RNAs that modulate gene regulation at the post‐transcriptional level in a sequence specific manner. miRNA ...dysfunction has been linked to the pathophysiology of human diseases including those resulting from viral infections. The objective of this study was to investigate changes in miRNA profiles that occur in hepatoma cells expressing hepatitis C virus (HCV) and identify anticorrelated mRNAs, which may be their regulatory targets.
Methods: Microarrays were used to perform global miRNA and mRNA expression analysis. Fold changes and pairwise statistics were computed for the resulting datasets. Hierarchical cluster and pathway analyses were performed to assess the degree of differential expression and identify regulatory networks. Bioinformatics tools were used to integrate mRNA profiling results with miRNA target predictions.
Results: Replication of the Con1 strain of HCV virus in hepatoma cells elicited extensive differential expression of both miRNAs and mRNAs. Forty‐three differentially expressed miRNAs (P≤0.001) were identified by microarray analysis in HCV expressing cells. Six thousand eight hundred and fifteen differentially expressed mRNAs (P≤0.05) were identified. Computational analyses revealed anticorrelated miRNA:mRNA pairs for each target prediction algorithm used. Pathway analysis generated a filtered pathway with 120 entities, including seven major regulators and nine major targets potentially under the control of at least 11 miRNAs.
Conclusions: The expression of a number of anticorrelated miRNAs:mRNA pairs are affected by the presence of HCV. These miRNAs and their putative targets are attractive candidates for being involved in the pathogenesis and/or progression of HCV‐induced chronic hepatitis.
The interaction of macrophages with infectious agents leads to the activation of several signaling cascades, including mitogen-activated protein (MAP) kinases, such as p38. We now demonstrate that ...p38 MAP kinase-mediated responses are critical components to the immune response to Borrelia burgdorferi. The pharmacological and genetic inhibition of p38 MAP kinase activity during infection with the spirochete results in increased carditis. In transgenic mice that express a dominant negative form of p38 MAP kinase specifically in macrophages, production of the invariant natural killer T (iNKT) cell—attracting chemokine MCP-1 and of the antigen-presenting molecule CD1d are significantly reduced. The expression of the transgene therefore results in the deficient infiltration of iNKT cells, their decreased activation, and a diminished production of interferon γ (IFN-γ), leading to increased bacterial burdens and inflammation. These results show that p38 MAP kinase provides critical checkpoints for the protective immune response to the spirochete during infection of the heart.
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