Plans for 1916 demonstration clubs

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Development of a Multiplex Real-Time PCR Assay for the detection of ruminant DNA

The U.S. Food and Drug Administration (FDA) has previously validated a real-time PCR-based assay that is currently being used by the FDA and several state laboratories as the official screening method. Due to several shortcomings to the assay, a multiplex real-time PCR assay (MRTA) to detect three ruminant species (bovine, caprine, and ovine) was developed using a lyophilized bead design. The assay contained two primer or probe sets: a "ruminant" set to detect bovine-, caprine-, and ovine-derived materials and a second set to serve as an internal PCR control, formatted using a lyophilized bead design. Performance of the assay was evaluated against stringent acceptance criteria developed by the FDA's Center for Veterinary Medicine's Office of Research. The MRTA for the detection of ruminant DNA passed the stringent acceptance criteria for specificity, sensitivity, and selectivity. The assay met sensitivity and reproducibility requirements by detecting 30 of 30 complete feed samples fortified with meals at 0.1 % (wt/wt) rendered material from each of the three ruminant species. The MRTA demonstrated 100 % selectivity (0.0 % false positives) for negative controls throughout the assessment period. The assay showed ruggedness in both sample selection and reagent preparation. Second and third analyst trials confirmed the quality of the written standard operating procedure with consistency of results. An external laboratory participating in a peer-verification trial demonstrated 100 % specificity in identifying bovine meat and bone meal, while exhibiting a 0.03 % rate of false positives. The assay demonstrated equal levels of sensitivity and reproducibility compared with the FDA's current validated real-time PCR assay. The assay detected three prohibited species in less than 1.5 h of total assay time, a significant improvement over the current real-time assay. These results demonstrated this assay's suitability for routine regulatory use both as a primary screening tool and as a confirmatory test

6-Thioguanine blocks SARS-CoV-2 replication by inhibition of PLpro

The emergence of SARS-CoV-2 has led to a global health crisis that, in addition to vaccines and immunomodulatory therapies, calls for the identification of antiviral therapeutics. The papain-like protease (PLpro) activity of nsp3 is an attractive drug target as it is essential for viral polyprotein cleavage and for deconjugation of ISG15, an antiviral ubiquitin-like protein. We show here that 6-Thioguanine (6-TG), an orally available and widely available generic drug, inhibits SARS-CoV-2 replication in Vero-E6 cells with an EC50 of approximately 2 μM. 6-TG also inhibited PLpro-catalyzed polyprotein cleavage and de-ISGylation in cells and inhibited proteolytic activity of the purified PLpro domai

Hypoxia and oxidative stress in breast cancer: Oxidative stress: its effects on the growth, metastatic potential and response to therapy of breast cancer

Reactive oxygen species (ROS) damage DNA, but the role of ROS in breast carcinoma may not be limited to the mutagenic activity that drives carcinoma initiation and progression. Carcinoma cells in vitro and in vivo are frequently under persistent oxidative stress. In the present review, we outline potential causes of oxygen radical generation within carcinoma cells and explore the possible impact of oxidative stress on the clinical outcome of breast carcinoma

Zebrafish Kidney Phagocytes Utilize Macropinocytosis and Ca2+-Dependent Endocytic Mechanisms

Background: The innate immune response constitutes the first line of defense against invading pathogens and consists of a variety of immune defense mechanisms including active endocytosis by macrophages and granulocytes. Endocytosis can be used as a reliable measure of selective and non-selective mechanisms of antigen uptake in the early phase of an immune response. Numerous assays have been developed to measure this response in a variety of mammalian and fish species. The small size of the zebrafish has prevented the large-scale collection of monocytes/macrophages and granulocytes for these endocytic assays. Methodology/Principal Findings: Pooled zebrafish kidney hematopoietic tissues were used as a source of phagocytic cells for flow-cytometry based endocytic assays. FITC-Dextran, Lucifer Yellow and FITC-Edwardsiella ictaluri were used to evaluate selective and non-selective mechanisms of uptake in zebrafish phagocytes. Conclusions/Significance: Zebrafish kidney phagocytes characterized as monocytes/macrophages, neutrophils and lymphocytes utilize macropinocytosis and Ca 2+-dependant endocytosis mechanisms of antigen uptake. These cells do not appear to utilize a mannose receptor. Heat-killed Edwardsiella ictaluri induces cytoskeletal interactions for internalization in zebrafish kidney monocytes/macrophages and granulocytes. The proposed method is easy to implement and should prove especially useful in immunological, toxicological and epidemiological research

Super Resolution Microscopy Reveals that Caveolin-1 Is Required for Spatial Organization of CRFB1 and Subsequent Antiviral Signaling in Zebrafish

10.1371/journal.pone.0068759PLoS ONE87-POLN

A High-Throughput Screen for Tuberculosis Progression

One-third of the world population is infected with Mycobacterium tuberculosis and multi-drug resistant strains are rapidly evolving. The noticeable absence of a whole organism high-throughput screening system for studying the progression of tuberculosis is fast becoming the bottleneck in tuberculosis research. We successfully developed such a system using the zebrafish Mycobacterium marinum infection model, which is a well-characterized model for tuberculosis progression with biomedical significance, mimicking hallmarks of human tuberculosis pathology. Importantly, we demonstrate the suitability of our system to directly study M. tuberculosis, showing for the first time that the human pathogen can propagate in this vertebrate model, resulting in similar early disease symptoms to those observed upon M. marinum infection. Our system is capable of screening for disease progression via robotic yolk injection of early embryos and visual flow screening of late-stage larvae. We also show that this system can reliably recapitulate the standard caudal vein injection method with a throughput level of 2,000 embryos per hour. We additionally demonstrate the possibility of studying signal transduction leading to disease progression using reverse genetics at high-throughput levels. Importantly, we use reference compounds to validate our system in the testing of molecules that prevent tuberculosis progression, making it highly suited for investigating novel anti-tuberculosis compounds in vivo

Local Ca2+ Entry Via Orai1 Regulates Plasma Membrane Recruitment of TRPC1 and Controls Cytosolic Ca2+ Signals Required for Specific Cell Functions

Store-operated Ca2+ entry (SOCE) has been associated with two types of channels: CRAC channels that require Orai1 and STIM1 and SOC channels that involve TRPC1, Orai1, and STIM1. While TRPC1 significantly contributes to SOCE and SOC channel activity, abrogation of Orai1 function eliminates SOCE and activation of TRPC1. The critical role of Orai1 in activation of TRPC1-SOC channels following Ca2+ store depletion has not yet been established. Herein we report that TRPC1 and Orai1 are components of distinct channels. We show that TRPC1/Orai1/STIM1-dependent ISOC, activated in response to Ca2+ store depletion, is composed of TRPC1/STIM1-mediated non-selective cation current and Orai1/STIM1-mediated ICRAC; the latter is detected when TRPC1 function is suppressed by expression of shTRPC1 or a STIM1 mutant that lacks TRPC1 gating, STIM1(684EE685). In addition to gating TRPC1 and Orai1, STIM1 mediates the recruitment and association of the channels within ER/PM junctional domains, a critical step in TRPC1 activation. Importantly, we show that Ca2+ entry via Orai1 triggers plasma membrane insertion of TRPC1, which is prevented by blocking SOCE with 1 µM Gd3+, removal of extracellular Ca2+, knockdown of Orai1, or expression of dominant negative mutant Orai1 lacking a functional pore, Orai1-E106Q. In cells expressing another pore mutant of Orai1, Orai1-E106D, TRPC1 trafficking is supported in Ca2+-containing, but not Ca2+-free, medium. Consistent with this, ICRAC is activated in cells pretreated with thapsigargin in Ca2+-free medium while ISOC is activated in cells pretreated in Ca2+-containing medium. Significantly, TRPC1 function is required for sustained KCa activity and contributes to NFκB activation while Orai1 is sufficient for NFAT activation. Together, these findings reveal an as-yet unidentified function for Orai1 that explains the critical requirement of the channel in the activation of TRPC1 following Ca2+ store depletion. We suggest that coordinated regulation of the surface expression of TRPC1 by Orai1 and gating by STIM1 provides a mechanism for rapidly modulating and maintaining SOCE-generated Ca2+ signals. By recruiting ion channels and other signaling pathways, Orai1 and STIM1 concertedly impact a variety of critical cell functions that are initiated by SOCE

Mycobacteria counteract a TLR-mediated nitrosative defense mechanism in a zebrafish infection model.

Pulmonary tuberculosis (TB), caused by the intracellular bacterial pathogen Mycobacterium tuberculosis (Mtb), is a major world health problem. The production of reactive nitrogen species (RNS) is a potent cytostatic and cytotoxic defense mechanism against intracellular pathogens. Nevertheless, the protective role of RNS during Mtb infection remains controversial. Here we use an anti-nitrotyrosine antibody as a readout to study nitration output by the zebrafish host during early mycobacterial pathogenesis. We found that recognition of Mycobacterium marinum, a close relative of Mtb, was sufficient to induce a nitrosative defense mechanism in a manner dependent on MyD88, the central adaptor protein in Toll like receptor (TLR) mediated pathogen recognition. However, this host response was attenuated by mycobacteria via a virulence mechanism independent of the well-characterized RD1 virulence locus. Our results indicate a mechanism of pathogenic mycobacteria to circumvent host defense in vivo. Shifting the balance of host-pathogen interactions in favor of the host by targeting this virulence mechanism may help to alleviate the problem of infection with Mtb strains that are resistant to multiple drug treatments