A Preliminary Exploration of Concussion and Strength Performance in Youth Ice Hockey Players.

The objective of this study was to describe the effect of concussion on upper and lower body strength in children and youth athletes. The participant group was made up of 178 unique male and female ice hockey players (ages 8-14 years). Using a 3-year prospective longitudinal research design, baseline and post-concussion data on hand grip strength, jump tests, and leg maximal voluntary contraction were collected. Using a linear mixed-effects model, no significant differences were found when comparing the baseline strength performance of individuals who went on to experience a concussion and those who did not. When accounting for sex, multiple concussions, and ongoing changes in strength associated with age, weaker hand grip scores were found following concussion while participants were still symptomatic. Lower squat jump heights were achieved while participants were symptomatic as well as when they were no longer self-reporting symptoms associated with concussion. This study represents an initial step towards better understanding strength performance following concussion that may limit the on and off ice performance of youth ice hockey players, as well as predispose youth to subsequent injuries.

Age and competition level on injuries in female ice hockey.

The objective of this study was to describe the number, types and locations of known injuries occurring across different age categories and levels of competition in female ice hockey within the Ontario Women's Hockey Association from 2004/05 to 2007/08. We further examined under which aforementioned factors and combination of factors an unusually high or low number of injuries was recorded. Secondary analysis of anonymized injury data was conducted. The most common known injury type was strain/sprain, followed by concussion while the most frequent injury location was head/face/mouth. Analysis of deviance indicated that a significantly higher than expected number of sprain/strain, concussion and laceration injuries were recorded compared to all other injury types. In addition, there were a higher number of injuries recorded at the AA level compared to all other levels of competition. Finally, the age categories of Peewee, Midget and Intermediate within the AA level of competition, as well as Senior/Adult within the Houseleague level of competition also recorded a significantly higher number of injuries compared to other combinations of descriptive factors. Further research with female youth is needed to better understand the high number of injuries, including concussions, reported overall.

Late fetal hematopoietic failure results from ZBTB11 deficiency despite abundant HSC specification.

Hematopoiesis produces diverse blood cell lineages to meet the basal needs and sudden demands of injury or infection. A rapid response to such challenges requires the expansion of specific lineages and a prompt return to balanced steady-state levels, necessitating tightly coordinated regulation. Previously we identified a requirement for the zinc finger and broad complex, tramtrak, bric-a-brac domain-containing 11 (ZBTB11) transcription factor in definitive hematopoiesis using a forward genetic screen for zebrafish myeloid mutants. To understand its relevance to mammalian systems, we extended these studies to mice. When Zbtb11 was deleted in the hematopoietic compartment, embryos died at embryonic day (E) 18.5 with hematopoietic failure. Zbtb11 hematopoietic knockout (Zbtb11hKO) hematopoietic stem cells (HSCs) were overabundantly specified from E14.5 to E17.5 compared with those in controls. Overspecification was accompanied by loss of stemness, inability to differentiate into committed progenitors and mature lineages in the fetal liver, failure to seed fetal bone marrow, and total hematopoietic failure. The Zbtb11hKO HSCs did not proliferate in vitro and were constrained in cell cycle progression, demonstrating the cell-intrinsic role of Zbtb11 in proliferation and cell cycle regulation in mammalian HSCs. Single-cell RNA sequencing analysis identified that Zbtb11-deficient HSCs were underrepresented in an erythroid-primed subpopulation and showed downregulation of oxidative phosphorylation pathways and dysregulation of genes associated with the hematopoietic niche. We identified a cell-intrinsic requirement for Zbtb11-mediated gene regulatory networks in sustaining a pool of maturation-capable HSCs and progenitor cells.

Examining the neural impact of pediatric concussion: a scoping review of multimodal and integrative approaches using functional and structural MRI techniques.

PURPOSE OF REVIEW: This study presents the findings from a scoping review of recent, original research investigating changes in brain structure and/or function following pediatric concussion or mild traumatic brain injury (mTBI) using MRI and functional MRI techniques. RECENT FINDINGS: Our scoping review identified only five studies, two of which were focused specifically on sports-related concussion. A common finding across studies was that traditional structural methods such as anatomical T1, T2, and even susceptibility-weighted MRI failed to reveal abnormalities in brain structure following pediatric concussion/mTBI. Although data suggest alterations in brain function associated with concussion, correlation with changes in performance is inconsistently found, possibly because of the use of compensatory cerebral mechanisms or alternate pathways while the brain is still dysfunctional. SUMMARY: In conclusion, the literature describing neuroimaging investigations of pediatric concussion is too scarce to allow the formulation of definitive conclusions regarding the impact of concussion on the developing brain. There is a dire need for longitudinal, multisite investigations focused on a wider age range and recovery period.

Measurement of head impacts in youth ice hockey players.

Despite growing interest in the biomechanical mechanisms of sports-related concussion, ice hockey and the youth sport population has not been studied extensively. The purpose of this pilot study was: 1) to describe the biomechanical measures of head impacts in youth minor ice hockey players; and, 2) to investigate the influence of player and game characteristics on the number and magnitude of head impacts. Data was collected from 13 players from a single competitive Bantam boy's (ages 13-14 years) AAA ice hockey team using telemetric accelerometers implanted within the players' helmets at 27 ice hockey games. The average linear acceleration, rotational acceleration, Gadd Severity Index and Head Injury Criterion of head impacts were recorded. A significantly higher number of head impacts per player per game were found for wingers when compared to centre and defense player positions (df=355, t=3.087, p=0.00218) and for tournament games when compared to regular season and playoff games (df=355, t=2.641, p=0.086). A significant difference in rotational acceleration according to player position (F2,1812=4.9551, p=0.0071) was found. This study is an initial step towards a greater understanding of head impacts in youth ice hockey.

Frontline Science: Dynamic cellular and subcellular features of migrating leukocytes revealed by in vivo lattice lightsheet microscopy.

Neutrophil and macrophage (Mϕ) migration underpin the inflammatory response. However, the fast velocity, multidirectional instantaneous movement, and plastic, ever-changing shape of phagocytes confound high-resolution intravital imaging. Lattice lightsheet microscopy (LLSM) captures highly dynamic cell morphology at exceptional spatiotemporal resolution. We demonstrate the first extensive application of LLSM to leukocytes in vivo, utilizing optically transparent zebrafish, leukocyte-specific reporter lines that highlighted subcellular structure, and a wounding assay for leukocyte migration. LLSM revealed details of migrating leukocyte morphology, and permitted intricate, volumetric interrogation of highly dynamic activities within their native physiological setting. Very thin, recurrent uropod extensions must now be considered a characteristic feature of migrating neutrophils. LLSM resolved trailing uropod extensions, demonstrating their surprising length, and permitting quantitative assessment of cytoskeletal contributions to their evanescent form. Imaging leukocytes in blood vessel microenvironments at LLSM's spatiotemporal resolution displayed blood-flow-induced neutrophil dynamics and demonstrated unexpected leukocyte-endothelial interactions such as leukocyte-induced endothelial deformation against the intravascular pressure. LLSM of phagocytosis and cell death provided subcellular insights and uncovered novel behaviors. Collectively, we provide high-resolution LLSM examples of leukocyte structures (filopodia lamellipodia, uropod extensions, vesicles), and activities (interstitial and intravascular migration, leukocyte rolling, phagocytosis, cell death, and cytoplasmic ballooning). Application of LLSM to intravital leukocyte imaging sets the stage for transformative studies into the cellular and subcellular complexities of phagocyte biology.

Meta-Analysis of Grainyhead-Like Dependent Transcriptional Networks: A Roadmap for Identifying Novel Conserved Genetic Pathways.

: The Drosophilagrainyhead (grh) and vertebrate Grainyhead-like (Grhl) transcription factors are among the most critical genes for epithelial development, maintenance and homeostasis, and are remarkably well conserved from fungi to humans. Mutations affecting grh/Grhl function lead to a myriad of developmental and adult onset epithelial disease, such as aberrant skin barrier formation, facial/palatal clefting, impaired neural tube closure, age-related hearing loss, ectodermal dysplasia, and importantly, cancers of epithelial origin. Recently, mutations in the family member GRHL3 have been shown to lead to both syndromic and non-syndromic facial and palatal clefting in humans, particularly the genetic disorder Van Der Woude Syndrome (VWS), as well as spina bifida, whereas mutations in mammalian Grhl2 lead to exencephaly and facial clefting. As transcription factors, Grhl proteins bind to and activate (or repress) a substantial number of target genes that regulate and drive a cascade of transcriptional networks. A multitude of large-scale datasets have been generated to explore the grh/Grhl-dependent transcriptome, following ablation or mis-regulation of grh/Grhl-function. Here, we have performed a meta-analysis of all 41 currently published grh and Grhl RNA-SEQ, and microarray datasets, in order to identify and characterise the transcriptional networks controlled by grh/Grhl genes across disparate biological contexts. Moreover, we have also cross-referenced our results with published ChIP and ChIP-SEQ datasets, in order to determine which of the critical effector genes are likely to be direct grh/Grhl targets, based on genomic occupancy by grh/Grhl genes. Lastly, to interrogate the predictive strength of our approach, we experimentally validated the expression of the top 10 candidate grhl target genes in epithelial development, in a zebrafish model lacking grhl3, and found that orthologues of seven of these (cldn23,ppl, prom2, ocln, slc6a19, aldh1a3, and sod3) were significantly down-regulated at 48 hours post-fertilisation. Therefore, our study provides a strong predictive resource for the identification of putative grh/grhl effector target genes.

A GCSFR/CSF3R zebrafish mutant models the persistent basal neutrophil deficiency of severe congenital neutropenia.

Granulocyte colony-stimulating factor (GCSF) and its receptor (GCSFR), also known as CSF3 and CSF3R, are required to maintain normal neutrophil numbers during basal and emergency granulopoiesis in humans, mice and zebrafish. Previous studies identified two zebrafish CSF3 ligands and a single CSF3 receptor. Transient antisense morpholino oligonucleotide knockdown of both these ligands and receptor reduces neutrophil numbers in zebrafish embryos, a technique widely used to evaluate neutrophil contributions to models of infection, inflammation and regeneration. We created an allelic series of zebrafish csf3r mutants by CRISPR/Cas9 mutagenesis targeting csf3r exon 2. Biallelic csf3r mutant embryos are viable and have normal early survival, despite a substantial reduction of their neutrophil population size, and normal macrophage abundance. Heterozygotes have a haploinsufficiency phenotype with an intermediate reduction in neutrophil numbers. csf3r mutants are viable as adults, with a 50% reduction in tissue neutrophil density and a substantial reduction in the number of myeloid cells in the kidney marrow. These csf3r mutants are a new animal model of human CSF3R-dependent congenital neutropenia. Furthermore, they will be valuable for studying the impact of neutrophil loss in the context of other zebrafish disease models by providing a genetically stable, persistent, reproducible neutrophil deficiency state throughout life.

Frontotemporal correlates of impulsivity and machine learning in retired professional athletes with a history of multiple concussions.

The frontotemporal cortical network is associated with behaviours such as impulsivity and aggression. The health of the uncinate fasciculus (UF) that connects the orbitofrontal cortex (OFC) with the anterior temporal lobe (ATL) may be a crucial determinant of behavioural regulation. Behavioural changes can emerge after repeated concussion and thus we used MRI to examine the UF and connected gray matter as it relates to impulsivity and aggression in retired professional football players who had sustained multiple concussions. Behaviourally, athletes had faster reaction times and an increased error rate on a go/no-go task, and increased aggression and mania compared to controls. MRI revealed that the athletes had (1) cortical thinning of the ATL, (2) negative correlations of OFC thickness with aggression and task errors, indicative of impulsivity, (3) negative correlations of UF axial diffusivity with error rates and aggression, and (4) elevated resting-state functional connectivity between the ATL and OFC. Using machine learning, we found that UF diffusion imaging differentiates athletes from healthy controls with significant classifiers based on UF mean and radial diffusivity showing 79-84 % sensitivity and specificity, and 0.8 areas under the ROC curves. The spatial pattern of classifier weights revealed hot spots at the orbitofrontal and temporal ends of the UF. These data implicate the UF system in the pathological outcomes of repeated concussion as they relate to impulsive behaviour. Furthermore, a support vector machine has potential utility in the general assessment and diagnosis of brain abnormalities following concussion.

Experimental approaches to studying the nature and impact of splicing variation in zebrafish.

From a fixed number of genes carried in all cells, organisms create considerable diversity in cellular phenotype through differential regulation of gene expression. One prevalent source of transcriptome diversity is alternative pre-mRNA splicing, which is manifested in many different forms. Zebrafish models of splicing dysfunction due to mutated spliceosome components provide opportunity to link biochemical analyses of spliceosome structure and function with whole organism phenotypic outcomes. Drawing from experience with two zebrafish mutants: cephalophonus (a prpf8 mutant, isolated for defects in granulopoiesis) and caliban (a rnpc3 mutant, isolated for defects in digestive organ development), we describe the use of glycerol gradient sedimentation and native gel electrophoresis to resolve components of aberrant splicing complexes. We also describe how RNAseq can be employed to examine relatively rare alternative splicing events including intron retention. Such experimental approaches in zebrafish can promote understanding of how splicing variation and dysfunction contribute to phenotypic diversity and disease pathogenesis.

Unique sequences in the guinea pig glucocorticoid receptor induce constitutive transactivation and decrease steroid sensitivity.

Previous attempts to characterize the structural determinants required for binding of cortisol by the glucocorticoid receptor (GR) have proved difficult since almost all modifications of the ligand binding domain (LBD) of GRs either eliminate or greatly decrease steroid binding. The guinea pig, a New World hystricomorph with a phylogeny the subject of recent dispute, is corticoresistant due to a GR that has diminished affinity for dexamethasone. The guinea pig GR has been cloned, and sequencing has identified many unique amino acid substitutions in the LBD. Using a domain-swap approach, the cloned guinea pig GR LBD was substituted for the human GR LBD in a human GR expression vector. Dexamethasone response curves for these constructs show that the cortisol resistance observed in the guinea pig in vivo is conferred in vitro by the guinea pig GR LBD. In addition, the guinea pig GR LBD induces a high level of constitutive activity. This constitutive activity is not repressed by RU486 (1 microM) but is enhanced by the addition of 8-bromo-cAMP. One of the amino acid substitutions results in the loss of a cysteine, which in the human, rat, and mouse GR is the site of covalent attachment for dexamethasone-21-mesylate. This cysteine is replaced by a tryptophan residue in the guinea pig GR, the implications of which were examined by reciprocal mutation of the tryptophan to a cysteine in the guinea pig GR LBD, and the cysteine to a tryptophan in the human GR LBD.(ABSTRACT TRUNCATED AT 250 WORDS)

Determinants of specificity of transactivation by the mineralocorticoid or glucocorticoid receptor.

Glucocorticoids and mineralocorticoids have distinct in vivo roles despite close structural homology and similarities in vitro. Known mechanisms of specificity focus on factors extrinsic to the receptor; interactions that directly regulate the receptor to confer specificity are less well understood, particularly for the mineralocorticoid receptor (MR). To examine relative MR vs. glucocorticoid receptor (GR) function in a more physiological context, we compared transactivation by GR and MR in the standard experimental fibroblast CV-1 cell line, the renal epithelial LLC-PK1 line, and neuronal medullary raphe RN33B cells. Maximal transactivational activity mediated by MR, relative to that mediated by GR, is enhanced in both of these cell lines and is primarily conferred by an N-terminal-mediated enhancement of the MR response. In addition, the ligand concentration required for maximal transcriptional activity of the GR varies significantly between cell lines. This is independent of binding affinity or 11beta-hydroxysteroid dehydrogenase-mediated inactivation and may contribute to in vivo tissue-specific differences in responses to the GR. Although ligand binding affinity is clearly conferred by the LBD, receptor-specific variations between cell lines in transcriptional sensitivity to ligand appear, rather, to be associated with the N-terminus. These studies demonstrate that the specificity of the MR vs. the GR response may be mediated via unique cellular factors, as well as suggesting a novel means of expanding the cellular response to cortisol.

Structural determinants of cortisol resistance in the guinea pig glucocorticoid receptor.

The guinea pig exhibits resistance to glucocorticoids in vivo which results from the guinea pig glucocorticoid receptor (GR) having a lower affinity for cortisol than the human GR. Cloning of the guinea pig GR has revealed that the amino acid sequence of the ligand-binding domain (LBD) differs from the human GR at 24 residues. The present study confirms that the decreased sensitivity and binding affinity of the guinea pig GR are conferred in vitro by the LBD. Further, the substitutions in the LBD do not confer altered relative steroid sensitivity or selectivity compared with the human GR. The altered sensitivity and binding of dexamethasone are confined to the first third of the LBD, which contains 5 nonconservative substitutions in a region that is otherwise highly conserved across several species of GR. These residues, either alone or in combination, were targeted for site-directed mutagenesis in both the human and guinea pig LBD. Trans-activation studies with these mutant GR failed to exclusively implicate or exclude any of the residues in the observed resistance. Rather, the changes, with 1 exception, caused a decrease in sensitivity, suggesting that critical intramolecular interactions involving at least 4 of these residues determine the correct conformation of this region. Recent molecular modeling of the GR LBD structure suggests that although the above region is not part of the core ligand-binding pocket, it is required to maintain the conformation of the binding pocket.

Steroid receptor isoforms: exception or rule?

Since the first steroid receptor was cloned, it was quickly identified as one of many such receptors constituting a gene superfamily which has grown to include not only steroid receptors but also receptors for thyroid hormone, retinoic acid, 1,25-dihydroxyvitamin D3 as well as a number of less traditional ligands, including farnesoids and fatty acids. Interestingly, these receptors are far outnumbered by the 'orphan' receptors for which ligands are still being sought. The original cloning of nuclear receptors, although sometimes identifying more than one receptor form, led to the general premise that each ligand has its cognate receptor through which signal is transduced to the transcriptional machinery. Regulation of this process was found to occur at the level of receptor expression, ligand availability, and more recently, through post-translational modifications of the receptor and interaction of a variety of coactivators/corepressors with the receptor protein. The continuing identification of more than a single form for many of the receptors directed the attention of a number of investigators toward defining possible roles for these 'extras'. This review examines the different forms of nuclear receptor gene family members and how they may provide an additional level of regulation.

Molecular cloning and sequencing of a guinea-pig pro-opiomelanocortin cDNA.

The guinea-pig has high levels of circulating cortisol. Though adrenocorticotropin (ACTH) levels are similar to those in other mammals, guinea-pig ACTH has been reported to have a single amino-acid substitution which results in increased bioactivity of the peptide. Pro-opiomelanocortin (POMC) is the precursor for ACTH, gamma-melanocyte-stimulating hormone (gamma-MSH) and the endogenous opioid peptide beta-endorphin. Both to confirm this substitution in guinea-pig ACTH and to establish whether other non-conservative substitutions occur elsewhere in the precursor we cloned guinea-pig POMC. The guinea-pig alanine for proline substitution at position 24 of ACTH was confirmed. Potentially significant mutations were also identified in gamma-MSH and beta-endorphin. A similar pattern of POMC mRNA expression was obtained for guinea-pig and rat as determined by Northern analysis and in situ hybridization. Southern blot analysis indicated that guinea-pig POMC is a single-copy gene. Cloning and sequencing of guinea-pig POMC thus further demonstrate the divergence of the New World hystricomorph peptides from those in New World primates, and underscore the differences observed in other endocrine axes in the guinea-pig.

The molecular basis of RU486 resistance in the Tammar Wallaby, Macropus eugenii.

RU486 acts as a potent anti-progestin in humans but does not antagonise progesterone action in the chicken or hamster reflecting a substitution in the ligand binding domain (LBD) of cysteine for glycine in both the chicken and the hamster progesterone receptor (PR), at the position corresponding to codon 722 of the human PR. The tammar wallaby, Macropus eugenii, is also resistant to the effects of RU486. Cloning of a partial cDNA of the PR in the tammar wallaby reveals a glycine to alanine substitution (gly 722 in the human PR), as well as a glutamine to histidine substitution two amino acids upstream of this alanine residue. Both the glycine and glutamine residues are substituted in all three resistant species. These substitutions are also found in the mineralocorticoid receptor, which also does not bind RU486, and suggest an important role for these residues in the formation of the 11-beta pocket of the receptor, which accommodates the bulky side-chains of 11-beta substituted steroids.

Cortisol resistance and the guinea pig glucocorticoid receptor.

The guinea pig has been employed as a model to study the structure/function relationships of the glucocorticoid receptor (GR), and to determine the regions of the receptor important for binding hormone and antihormone. Guinea pigs have high levels of circulating cortisol and GR with a approximately 20-fold lower affinity for dexamethasone than mouse GR. Cloning and sequencing of guinea pig GR has identified 24 amino acid changes in the ligand-binding domain (LBD) compared to the human GR. By substituting the guinea pig GR LBD for the human LBD in a human GR expression vector we have shown in cotransfection studies that guinea pig GR LBD confers glucocorticoid resistance as observed in vivo. In initial studies guinea pig GR LBD appeared constitutively active; in subsequent studies to determine which of the 24 amino acid changes present in the guinea pig GR LBD conferred resistance, it became apparent that the guinea pig LBD (LBD delta), amplified by PCR for subcloning into the human GR expression vector, contained a single adenine deletion in the hinge region within ten bases of the PCR primer. This single base deletion resulted in a frameshift bringing a stop codon into frame one codon after the deletion. While this now clearly accounts for the observed constitutive activity, since it is known that C-terminally truncated steroid receptors exhibit constitutive activation such a truncation is more difficult to reconcile with the repeatedly demonstrable hormone dose-response curves obtained with this guinea pig GR LBD delta.(ABSTRACT TRUNCATED AT 250 WORDS)

Immune priming: mothering males modulate immunity.

The transfer of immunity from mother to offspring is widespread in animals. The father's contribution to this is usually negligible. However, in a sex-role reversed pipefish where fathers do the mothering, fathers make an important immune priming contribution, too.

Neutrophil-delivered myeloperoxidase dampens the hydrogen peroxide burst after tissue wounding in zebrafish.

Prompt neutrophil arrival is critical for host defense immediately after injury [1-3]. Following wounding, a hydrogen peroxide (H(2)O(2)) burst generated in injured tissues is the earliest known leukocyte chemoattractant [4]. Generating this tissue-scale H(2)O(2) gradient uses dual oxidase [4] and neutrophils sense H(2)O(2) by a mechanism involving the LYN Src-family kinase [5], but the molecular mechanisms responsible for H(2)O(2) clearance are unknown [6]. Neutrophils carry abundant amounts of myeloperoxidase, an enzyme catalyzing an H(2)O(2)-consuming reaction [7, 8]. We hypothesized that this neutrophil-delivered myeloperoxidase downregulates the high tissue H(2)O(2) concentrations that follow wounding. This was tested in zebrafish using simultaneous fluorophore-based imaging of H(2)O(2) concentrations and leukocytes [4, 9-11] and a new neutrophil-replete but myeloperoxidase-deficient mutant (durif). Leukocyte-depleted zebrafish had an abnormally sustained wound H(2)O(2) burst, indicating that leukocytes themselves were required for H(2)O(2) downregulation. Myeloperoxidase-deficient zebrafish also had abnormally sustained high wound H(2)O(2) concentrations despite similar numbers of arriving neutrophils. A local H(2)O(2)/myeloperoxidase interaction within wound-recruited neutrophils was demonstrated. These data demonstrate that leukocyte-delivered myeloperoxidase cell-autonomously downregulates tissue-generated wound H(2)O(2) gradients in vivo, defining a new requirement for myeloperoxidase during inflammation. Durif provides a new animal model of myeloperoxidase deficiency closely phenocopying the prevalent human disorder [7, 12, 13], offering unique possibilities for investigating its clinical consequences.