Sea anemone model has a single Toll-like receptor that can function in pathogen detection, NF-κB signal transduction, and development

In organisms from insects to vertebrates, Toll-like receptors (TLRs) are primary pathogen detectors that activate downstream pathways, specifically those that direct expression of innate immune effector genes. TLRs also have roles in development in many species. The sea anemone Nematostella vectensis is a useful cnidarian model to study the origins of TLR signaling because its genome encodes a single TLR and homologs of many downstream signaling components, including the NF-κB pathway. We have characterized the single N. vectensis TLR (Nv-TLR) and demonstrated that it can activate canonical NF-κB signaling in human cells. Furthermore, we show that the intracellular Toll/IL-1 receptor (TIR) domain of Nv-TLR can interact with the human TLR adapter proteins MAL and MYD88. We demonstrate that the coral pathogen Vibrio coralliilyticus causes a rapidly lethal disease in N. vectensis and that heat-inactivated V. coralliilyticus and bacterial flagellin can activate a reconstituted Nv-TLR–to–NF-κB pathway in human cells. By immunostaining of anemones, we show that Nv-TLR is expressed in a subset of cnidocytes and that many of these Nv-TLR–expressing cells also express Nv-NF-κB. Additionally, the nematosome, which is a Nematostella-specific multicellular structure, expresses Nv-TLR and many innate immune pathway homologs and can engulf V. coralliilyticus. Morpholino knockdown indicates that Nv-TLR also has an essential role during early embryonic development. Our characterization of this primitive TLR and identification of a bacterial pathogen for N. vectensis reveal ancient TLR functions and provide a model for studying the molecular basis of cnidarian disease and immunity.IOS-1354935 - National Science Foundation (NSF); GRFP - National Science Foundation (NSF); GRFP - National Science Foundation (NSF); 1262934 - National Science Foundation (NSF); 2014-BSP - Arnold and Mabel Beckman Foundatio

Body composition changes during 8 weeks of military training are not accurately captured by circumference-based assessments

In 1981, the US military adopted body fat standards to promote physical readiness and prevent obesity. Separate circumference-based equations were developed for women and men. Both predictive equations were known to underestimate %BF. However, it was not known how well these abdominal circumference-based methods tracked changes in %BF. This study examined the validity of the circumference-based %BF equations for assessing changes in %BF in young adult recruits during Army Basic Combat Training (BCT). Dual-energy X-ray absorptiometry (DXA) and circumference-based measures of %BF were obtained in women (n = 481) and men (n = 926) at the start (pre-BCT) and end (post-BCT) of 8 weeks of BCT. Repeated-measure ANOVAs were used to assess differences between DXA and circumference pre-BCT and for the change during BCT. Pre-BCT, circumferences underestimated %BF relative to DXA, with mean errors of −6.0% ± 4.4% for women and −6.0% ± 3.5% for men (both p < 0.01), and no difference between sexes was observed (p = 0.77). DXA detected a −4.0% ± 2.4% and −3.3% ± 2.8% change in %BF for women and men in response to BCT, respectively (both p < 0.01), whereas circumference estimates of %BF indicated a 0.0% ± 3.3% (p = 0.86) change in women and a −2.2% ± 3.3% (p < 0.01) change in men (sex difference by technique p < 0.01). In conclusion, circumference-based measures underestimated %BF at the start of BCT in both sexes as compared to DXA. Circumference measures underestimated changes in %BF during BCT in men and did not detect changes in women. These findings suggest that circumference-based %BF metrics may not be an appropriate tool to track changes in body composition during short duration training

Comparison of CRISPR and adenovirus-mediated Myd88 knockdown in RAW 264.7 cells and responses to lipopolysaccharide stimulation

Genomic manipulation offers the possibility for novel therapies in lieu of medical interventions in use today. The ability togenetically restore missing inflammatory genes will have a monumental impact on our current immunotherapy treatments. This study compared the efficacy of two different genetic manipulation techniques: clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9) transfection to adenoviral transduction to determine which method would provide the most transient and stable knockdown of myeloid differentiation primary response 88 (MyD88). MyD88 is a major regulator of nuclear factor kappa light chain enhancer of activated B cells (NFκB) pathway in Raw 264.7 macrophages. Following genetic manipulation, cells were treated for 24 h with Lipopolysaccharide (LPS) to stimulate the inflammatory pathway. Confirmation of knockdown was determined by western immunoblotting and quantification of band density. Both CRISPR/Cas9 and adenoviral transduction produced similar knockdown efficiency (~64% and 60%, respectively) in MyD88 protein 48 h post adenoviral transduction. NFκB phosphorylation was increased in CRISPR/Cas9-mediated MyD88 knockdown and control cells, but not in adenovirus-mediated MyD88 knockdown cells, following LPS administration. CRISPR/Cas9-mediated MyD88 knockdown macrophages treated with LPS for 24 h showed a 65% reduction in tumor necrosis factor alpha (TNFα) secretion, and a 67% reduction in interleukin-10 (IL-10) secretion when compared to LPS-stimulated control cells (P ≤ 0.01 for both). LPS did not stimulate TNFα or IL-10 secretion in adenovirus-mediated control or MyD88 knockdown cells. These data demonstrate that Raw 264.7 macrophages maintain responsiveness to inflammatory stimuli following CRISPR/Cas9-mediated reductions in MyD88, but not following adenovirus-mediated MyD88 knockdown