The biomechanics of concussion for ice hockey head impact events

The purpose of this research was to conduct reconstructions of concussive and non-concussive impacts in ice hockey to determine the biomechanics and thresholds of concussive injury in ice hockey. Videos of concussive and non-concussive impacts in an elite professional ice hockey league in North America were reconstructed using physical and finite element model methods. Eighty concussive and 45 non-concussive events were studied. Logistic regressions indicate significant thresholds for concussion for linear/rotational acceleration and CSDM_10%. Impacts in ice hockey were mostly long duration events, longer than 15?ms. These results have significant implications for helmet standards and development to prevent concussion.     

Intestinal alkaline phosphatase detoxifies lipopolysaccharide and prevents inflammation in zebrafish in response to the gut microbiota

Vertebrates harbor abundant lipopolysaccharide (LPS) in their gut microbiota. Alkaline phosphatases can dephosphorylate and detoxify the endotoxin component of LPS. Here, we show that expression of the zebrafish intestinal alkaline phosphatase (Iap), localized to the intestinal lumen brush border, is induced during establishment of the gut microbiota. Iap-deficient zebrafish are hypersensitive to LPS toxicity and exhibit the excessive intestinal neutrophil influx characteristic of wild-type zebrafish exposed to LPS. Both of these Iap mutant phenotypes are dependent on Myd88 and Tumor Necrosis Factor Receptor (Tnfr), proteins also involved in LPS sensitivity in mammals. When reared germ-free, the intestines of Iap-deficient zebrafish are devoid of neutrophils. Together, these findings demonstrate that the endogenous microbiota establish the normal homeostatic level of neutrophils in the zebrafish intestine through a process involving Iap, Myd88, and Tnfr. Thus, by preventing inflammatory responses, Iap plays a crucial role in promoting mucosal tolerance to resident gut bacteria.     

Regeneration of sponges in ecological context: is regeneration an integral part of life history and morphological strategies?

Sponges, simple and homogeneous relative to other animals, are particularly adept at regeneration. Although regeneration may appear to be obviously beneficial, and many specific advantages to regeneration of lost portions have been demonstrated, comparisons of regeneration among species of sponges have consistently revealed substantial differences in style (i.e., relative rates of reconstituting surface features, infilling depressions, regaining lost primary substratum), and overall time course, raising questions about adaptive significance of variations in patterns of regeneration. Do sponges simply regenerate as quickly as possible, given constraints imposed by skeletal construction, morphology, or other traits that are determined primarily by evolutionary heritage? Does allocation of energy or materials impose trade-offs between regeneration versus competing processes such as growth or reproduction? Is regeneration time-course and style an integral part of coherent life history and morphological strategies? One approach to answering these questions is to compare regeneration among species that represent a spectrum of higher taxa within the demosponges as well as different growth forms and life-history strategies. Because detailed ecological studies of sponges have tended to focus on small sets of species of the same growth form, community-wide comparisons have been hampered. Data on growth rate, colonization, mortality, susceptibility to predation, and competitive ability have recently been accumulated for species of sponges typical of the Caribbean mangrove prop-root community. Experimentally generated wounds in individuals of 13 of these species allow comparison of the timing and style of regeneration among sponge species that span a range of life histories and growth forms. The species chosen represent four orders of the class Demospongiae, and include four sets of congeneric species, allowing distinction of patterns related to life history and morphology from those determined by shared evolutionary heritage.     

Dr. Janie Merkel is interviewed by Ryan Blum and Janice Friend

Dr. Janie Merkel is the director of Yale’s Chemical Genomics Screening Facility, a high-throughput screening laboratory that is part of the Yale University Center for Genomics and Proteomics. The Screening Facility connects Yale researchers with industry-quality robotic machinery and a diverse group of compound libraries, which have been used successfully to link therapeutic targets with potential therapies.     

Dual expression system suitable for high-throughput fluorescence-based screening and production of soluble proteins

Many studies that aim to characterize the proteome structurally or functionally require the production of pure protein in a high-throughput format. We have developed a fast and flexible integrated system for cloning, protein expression in Escherichia coli, solubility screening and purification that can be completely automated in a 96-well microplate format. We used recombination cloning in custom-designed vectors including (i) a (His)(6) tag-encoding sequence, (ii) a variable solubilizing partner gene, (iii) the DNA sequence corresponding to the TEV protease cleavage site, (iv) the gene (or DNA fragment) of interest, (v) a suppressible amber stop codon, and (vi) an S.tag peptide-encoding sequence. First, conditions of bacterial culture in microplates (250 microL) were optimized to obtain expression and solubility patterns identical to those obtained in a 1-L flask (100-mL culture). Such conditions enabled the screening of various parameters in addition to the fusion partners (E. coli strains, temperature, inducer...). Second, expression of fusion proteins in amber suppressor strains allowed quantification of soluble and insoluble proteins by fluorescence through the detection of the S.tag. This technique is faster and more sensitive than other commonly used methods (dot blots, Western blots, SDS-PAGE). The presence of the amber suppressor tRNA was shown to affect neither the expression pattern nor the solubility of the target proteins. Third, production of the most interesting soluble fusion proteins, as detected by our screening method, could be performed in nonsuppressor strains. After cleavage with the TEV protease, the target proteins were obtained in a native form with a unique additional N-terminal glycine.     

De novo ICAM-1 synthesis in the mouse lung: model of assessment of protein expression in lungs

Because most studies addressing the regulatory mechanisms of intercellular adhesion molecule (ICAM)-1 expression have used cultured endothelial cells, we set out to develop an isolated mouse lung preparation to study gene and protein expression in its proper cellular context in the organ. Lungs from CD1 mice were isolated and perfused (2 ml/min, 37 degrees C) with a recirculating volume of RPMI 1640 solution supplemented with 3 g/100 ml albumin. Lungs maintained their isogravimetric state for 4 h. Tumor necrosis factor (TNF-alpha; 2,000 U/ml) was added to the perfusate for 0.5, 1, 2, or 3.5 h to induce ICAM-1 expression or lungs received no treatment (control). After quick-freezing the lungs using liquid nitrogen at different time points, the prepared tissue homogenates were analyzed for ICAM-1 protein expression by Western blotting and NF-kappaB activation by electrophoretic mobility shift assay. TNF-alpha caused a progressive increase in NF-kappaB activity after 0.5 h and ICAM-1 protein expression two- to threefold of basal after 2 h. Untreated lungs expressed a low and constant level of ICAM-1 between 0 and 3.5 h. TNF-alpha failed to induce NF-kappaB activation and ICAM-1 expression in lungs of NADPH oxidase-deficient mice lacking p47(phox). We disaggregated mouse lungs using collagenase and stained the cells for ICAM-1 and VE-cadherin (used as an endothelial marker) to assess the in situ endothelial-specific expression of ICAM-1. We observed that TNF-alpha challenge resulted in increased ICAM-1 expression in endothelial cells freshly isolated from lungs. These data show the role of NADPH oxidase-derived oxidant signaling in the mechanism of NF-kappaB activation and ICAM-1 expression in mouse lung endothelial cells. Moreover, the general method presented herein has potential value in assessing mechanisms of gene and protein expression in the isolated-perfused mouse lung model.     

Sponge systematics by starfish: predators distinguish cryptic sympatric species of Caribbean fire sponges, Tedania ignis and Tedania klausi n. sp. (Demospongiae, Poecilosclerida)

Tedania ignis (Duchassaing and Michelotti, 1864), the fire sponge, is common throughout the tropical western Atlantic, and is a popular species for studies of ecology, larval biology, and chemistry. T. ignis is readily consumed by seagrass-dwelling starfish, and so finding sponges similar to this species in a seagrass meadow provoked closer scrutiny. A variety of ecological, morphological, and molecular traits consistently and unambiguously distinguish T. ignis from a cryptic sympatric congener, here described as Tedania klausi, n. sp. Starfish that consume T. ignis reject T. klausi, and angelfish consume T. klausi less quickly. In Belize, T. ignis individuals transplanted to a seagrass meadow inhabited by T. klausi were consumed by starfish, and individuals of T. klausi transplanted to a mangrove-lined creek in which T. ignis flourishes, died. In Panama, many individuals of T. klausi were diseased in May 2004, while adjacent individuals of T. ignis were unaffected. Spicule types are the same in the two forms, and sizes overlap; but within individuals, the relative sizes of styles and tylotes differ in a pattern that distinguishes the two forms. Comparison of DNA sequences for mitochondrial cytochrome c oxidase subunit I (COI) revealed that 8 single nucleotide mutations consistently differ between the two forms regardless of habitat (seagrass vs. mangrove) and geographically separated sites (Belize vs. Panama).     

Hydrolysis of α-chloro-substituted 2- and 4-pyridones: rate enhancement by zwitterionic structure

The hydrolysis of α-chloro-N-methyl-4-pyridone was found to be more than five times faster than that of α-chloro-N-methyl-2-pyridone. Structural studies of 2- and 4-pyridones have revealed the higher polarity and greater extent of zwitterionic content in 4-pyridone. The results are thus consistent with the hypothesis that polarization and higher zwitterionic content in the heterocyclic structures enhances the rate of hydrolysis in α-substituted pyridone and uracil derivatives.