Utility of Novel Rotational Load-Velocity Profiling Methods in Collegiate Softball Players.

ABSTRACT: Herring, CH, Beyer, KS, Redd, MJ, Stout, JR, and Fukuda, DH. Utility of novel rotational load-velocity profiling methods in collegiate softball players. J Strength Cond Res 38(1): 136-145, 2024-The purpose of this study was to determine the reliability of bat swing (BS) and rotational medicine ball throw (RMBT) load-velocity profiling (LVP) methods and explore relationships with batting performance in NCAA Division I softball players. Bat velocity was tracked with a swing sensor during the BS method, whereas an inertial measurement unit (IMU) tracked forearm velocity during the BS and RMBT methods. Intraclass correlation coefficients (ICC) were used for relative reliability, and coefficient of variation (CV) was used for absolute reliability. With the exception of theoretical maximum velocity (V0) using the average of top 2 peak velocities (PVavg) during the RMBT, no LVP variables were found to be reliable during the RMBT or BS method using the IMU (ICC ≤0.7; CV ≥15%). For the BS method with the swing sensor, all bat loads and V0 had acceptable reliability using peak velocity (PV) and PVavg (ICC >0.7; CV <15%), whereas all LVP variables were highly related between the multiple-load and two-load models when using PV and PVavg (r = 0.915-0.988; p < 0.01). There were significant relationships (r = 0.603-0.671; p < 0.05) between PV using the 0.99 Kg bat load and V0, and several in-game batting statistics. Practitioners may use the BS with the swing sensor as a rotational LVP assessment, although they should be cautious of aiming to improve batting performance in collegiate softball players based on the correlations reported until further research is performed.

Accumulated Workload Differences in Collegiate Women's Soccer: Starters versus Substitutes.

The purpose of this study was to estimate the workloads accumulated by collegiate female soccer players during a competitive season and to compare the workloads of starters and substitutes. Data from 19 college soccer players (height: 1.58 ± 0.06 m; body mass: 61.57 ± 6.88 kg) were extracted from global positioning system (GPS)/heart rate (HR) monitoring sensors to quantify workload throughout the 2019 competitive season. Total distance, distance covered in four speed zones, accelerations, and time spent in five HR zones were examined as accumulated values for training sessions, matches, and the entire season. Repeated-measures ANOVA and Student's t tests were used to determine the level of differences between starter and substitute workloads. Seasonal accumulated total distance (p < 0.001), sprints (≥19.00 km/h; p < 0.001), and high-speed distance (≥15.00 km/h; p = 0.005) were significantly greater for starters than substitutes. Accumulated training load (p = 0.08) and training load per minute played in matches (p = 0.08) did not differ between starters and substitutes. Substitutes had similar accumulated workload profiles during training sessions but differed in matches from starters. Coaches and practitioners should pursue strategies to monitor the differences in workload between starters and substitutes.

High-Risk Environmental Conditions Attenuates Performance Efficiency Index in NCAA DI Female Soccer Players.

The purpose of this study was to evaluate the effects of environmental conditions on running performance and performance efficiency index (Effindex). Performance data recorded using Polar Team Pro sensors from eight collegiate female soccer players in nine matches were analyzed during the 2019 competitive season. Effindex and running performance, including total distance covered (TDREL) and distance covered in five speed thresholds relative to minutes played, were examined for indications of fatigue with respect to environmental conditions, including ambient temperature and relative humidity. Matches were separated into three groups based on environmental conditions: Low-Risk (n = 2 matches), Moderate-Risk (n = 3 matches), or High-Risk (n = 4 matches). Speed thresholds were grouped as follows: walking (WALKREL), jogging (JOGREL), low-speed running (LSRREL), high-speed running (HSRREL), and sprinting (SPRINTREL). A significant effect was observed for TDREL in all environmental conditions (η2 = 0.614). TDREL was significantly lower in the High-Risk (p = 0.002; 95.32 ± 12.04 m/min) and Moderate-Risk conditions (p = 0.004; 94.85 ± 9.94 m/min) when compared to Low-Risk (105.61 ± 9.95 m/min). WALKREL (p = 0.005), JOGREL (p = 0.005) LSRREL (p = 0.001), HSRREL (p = 0.035), SPRINTREL (p = 0.017), and Effindex (p = 0.0004) were significantly greater in Low-Risk conditions when compared to Moderate-Risk conditions. WALKREL (p = 0.005), HSRREL (p = 0.029), SPRINTREL (p = 0.005), and Effindex (p = 0.0004) were significantly greater in Low-Risk conditions when compared to High-Risk conditions. High-Risk environmental conditions may result in adverse performance in female collegiate soccer players.

KRas-transformed epithelia cells invade and partially dedifferentiate by basal cell extrusion.

Metastasis is the main cause of carcinoma-related death, yet we know little about how it initiates due to our inability to visualize stochastic invasion events. Classical models suggest that cells accumulate mutations that first drive formation of a primary mass, and then downregulate epithelia-specific genes to cause invasion and metastasis. Here, using transparent zebrafish epidermis to model simple epithelia, we can directly image invasion. We find that KRas-transformation, implicated in early carcinogenesis steps, directly drives cell invasion by hijacking a process epithelia normally use to promote death-cell extrusion. Cells invading by basal cell extrusion simultaneously pinch off their apical epithelial determinants, endowing new plasticity. Following invasion, cells divide, enter the bloodstream, and differentiate into stromal, neuronal-like, and other cell types. Yet, only invading KRasV12 cells deficient in p53 survive and form internal masses. Together, we demonstrate that KRas-transformation alone causes cell invasion and partial dedifferentiation, independently of mass formation.

Tensiomyographic Responses to Warm-Up Protocols in Collegiate Male Soccer Athletes.

The mechanical properties of knee flexors and extensors in 15 collegiate male soccer players following different warm-up protocols [small-sided games (SSG), dynamic (DYN), and plyometric (PLY)] were evaluated. Tensiomyography (TMG) was used to assess contraction time (Tc), delay time (Td) and maximal displacement (Dm) of the rectus femoris (RF) and biceps femoris (BF) of both legs before and after each warm-up, while countermovement jump height variables, 20 m sprint, t-test and sit-and-reach were measured following the warm-ups. TMG was analyzed using a three-way [condition × time × leg] ANOVA, while performance variables were analyzed with a repeated measures ANOVA. Main effects of time were observed for BF-Tc (p = 0.035), RF-Td (p < 0.001), and BF-Td, (p = 0.008), and a main effect of condition was seen for RF-Tc (p = 0.038). Moreover, participants' 20 m sprint improved following SSG (p = 0.021) compared to DYN and PLY. Sit-and-reach was greater following PLY (p = 0.021). No significant interactions were noted for the measured TMG variables. Warm-up-specific improvements were demonstrated in sprint speed and flexibility following SSG and PLY, respectively. The present study revealed changes in certain TMG measures following the warm-ups that suggest enhanced response of lower leg muscles regardless of specific activities used.

Effect of somatic maturity on the aerobic and anaerobic adaptations to sprint interval training.

The purpose of this study was to assess the maturity-related differences in the aerobic and anaerobic adaptations to sprint interval training (SIT) among youth male athletes. Twenty-seven youth male athletes were assessed for years from peak height velocity (PHV) and classified into prepubescent (PRE, n = 7, years from PHV = -2.21 ± 0.47 years), peripubescent (PERI, n = 10, years from PHV = 0.25 ± 0.88 years), and postpubescent (POST, n = 10, years from PHV = 2.81 ± 0.50 years) groups based on their years from estimated peak height velocity. Participants completed a ramp exercise protocol on a cycle ergometer to determine maximal aerobic power, maximal oxygen consumption (VO2peak ), and fatigue thresholds. Following baseline, all participants completed a 4-week SIT program that consisted of eight total training sessions. During each session, participants completed repeated 20-s sprints on a cycle ergometer against a resistance of 7.5% of body mass. The number of sprints per sessions increased from four in session 1 to seven in session 7, with four sprints in session 8. Peak and mean power from sessions 1 and 8 were recorded. All participants completed a post-testing ramp exercise protocol that mirrored baseline. Maximal aerobic power increased (p < .001) across all groups from baseline (212.61 ± 57.45 W) to post-testing (223.24 ± 58.90 W); however, VO2peak only increased in POST (3.31 ± 0.43 to 3.54 ± 0.43 L min-1 , p = .003). Similarly, GET, VT, and RCP increased in POST, with no changes in PRE or PERI. In terms of anaerobic performance, PERI and POST had significant increases in peak and mean power. POST improved aerobic and anaerobic performance following SIT, while PERI only experienced improvements in anaerobic performance. Conversely, PRE had no changes in aerobic or anaerobic performance. The adaptations to SIT appear to be influenced by the somatic maturity status.

Dynamic post-activation potentiation protocol improves rowing performance in experienced female rowers.

Post-activation potentiation likely acutely improves power-based performance; however, few studies have demonstrated improved endurance performance. Forty collegiate female rowers performed isometric potentiating (ISO), dynamic potentiating (DYN) and control (CON) warm-up protocols on a rowing ergometer, followed by a three-minute all-out test to evaluate their total distance, peak power, mean power, critical power, anaerobic working capacity (W') and stroke rate. Fifteen-second splits were also analysed. ISO consisted of 5 × 5-second static muscle actions with the ergometer handle rendered immovable with a nylon strap, while DYN consisted of 2 × 10-second all-out rowing bouts, separated by a 2-minute rest interval. The participants were divided into high and low experience groups by median experience level (3.75 years) for statistical analysis. Significant differences (DYN > CON; p < 0.05) were found for distance (+5.6 m), mean power (+5.9 W) and W' (+1561.6 J) for more experienced rowers (n = 19) and no differences for less experienced rowers (n = 18). Mean power in DYN was significantly greater than CON and ISO in the 15-30, 30-45, 45-60 and 60-75 second intervals independent of experience level. These results suggest that DYN may benefit experienced female rowers and that these strategies might benefit a greater power output over shorter distances regardless of experience.

Maturity-Related Differences in Systemic Pulmonary and Localized Fatigue Threshold Among Youth Male Athletes.

PURPOSE: To examine the reliability and the maturity-related differences of fatigue thresholds (FTs) among youth males. METHODS: Twenty-nine youth males (11-17 y) completed 2 ramp exercise tests on a cycle ergometer. Systemic FTs were calculated from gas exchange and ventilation variables. Localized FTs were calculated from electromyography and near-infrared spectroscopy of the vastus lateralis. All FTs were determined using the maximal distance method and expressed relative to maximal oxygen consumption. All participants were grouped according to the number of years from peak height velocity into PRE- (< -1.5 y), PERI- (-1.5 to +1.5 y) and POST- (> +1.5 y) peak height velocity. Reliability was assessed with intraclass correlation coefficients, and differences between groups were assessed with analysis of variance and Cohen's d coefficients. RESULTS: Analysis of variance revealed significant group differences with PRE having significantly greater systemic pulmonary FTs than POST, while localized muscular FTs were significantly greater in PRE when compared with PERI and POST. All FTs exhibited excellent reliability (intraclass correlation coefficient > .75) in all maturity groups. CONCLUSION: Maturity status appears to influence the onset of FTs among youth male athletes, with FTs occurring later in younger athletes. Furthermore, all FTs were reliable measures regardless of maturity.

Resistance training does not induce uniform adaptations to quadriceps.

Resistance training may differentially affect morphological adaptations along the length of uni-articular and bi-articular muscles. The purpose of this study was to compare changes in muscle morphology along the length of the rectus femoris (RF) and vastus lateralis (VL) in response to resistance training. Following a 2-wk preparatory phase, 15 resistance-trained men (24.0 ± 3.0 y, 90.0 ± 13.8 kg, 174.9 ± 20.7 cm) completed pre-training (PRE) assessments of muscle thickness (MT), pennation angle (PA), cross-sectional area (CSA), and echo-intensity in the RF and VL at 30, 50, and 70% of each muscle's length; fascicle length (FL) was estimated from respective measurements of MT and PA within each muscle and region. Participants then began a high intensity, low volume (4 x 3-5 repetitions, 3min rest) lower-body resistance training program, and repeated all PRE-assessments after 8 weeks (2 d ∙ wk-1) of training (POST). Although three-way (muscle [RF, VL] x region [30, 50, 70%] x time [PRE, POST]) repeated measures analysis of variance did not reveal significant interactions for any assessment of morphology, significant simple (muscle x time) effects were observed for CSA (p = 0.002) and FL (p = 0.016). Specifically, average CSA changes favored the VL (2.96 ± 0.69 cm2, p < 0.001) over the RF (0.59 ± 0.20 cm2, p = 0.011), while significant decreases in average FL were noted for the RF (-1.03 ± 0.30 cm, p = 0.004) but not the VL (-0.05 ± 0.36 cm, p = 0.901). No other significant differences were observed. The findings of this study demonstrate the occurrence of non-homogenous adaptations in RF and VL muscle size and architecture following 8 weeks of high-intensity resistance training in resistance-trained men. However, training does not appear to influence region-specific adaptations in either muscle.

No Observable Relative Age Effects in Professional Surfers: a Constraints-Based Evaluation.

With the recent inclusion of surfing in the 2020 Olympic Games in Japan, there will be a number of surfing athletes vying for one of the twenty total available spots for their respective gender. The purpose was to evaluate relative age effects (RAEs) in surfing with consideration for specific developmental constraints. Elite competitive male surfers (n = 1590) were examined by birth month and subcategorized by competitive level, age groups, and geographical regions. The observed quarterly distribution was not significantly different (using χ2; p>0.05) from expected for the overall group or any of the subcategories. However, an odds ratio of 1.85 (90% confidence interval: 1.08-3.14) was calculated for being born in the first semester of the year compared to being born in the second semester between top 34 athletes and the rest of the field. Despite consideration for individual, environmental, and task constraints in this study, the sport of surfing does not appear to have any observable RAEs at the professional level. Thus, surfing appears to be one of the few sporting activities included in the Olympic Programme with limited RAEs.

A hyperactivating proinflammatory RIPK2 allele associated with early-onset osteoarthritis.

Osteoarthritis (OA) is a common debilitating disease characterized by abnormal remodeling of the cartilage and bone of the articular joint. Ameliorating therapeutics are lacking due to limited understanding of the molecular pathways affecting disease initiation and progression. Notably, although a link between inflammation and overt OA is well established, the role of inflammation as a driver of disease occurrence is highly disputed. We analyzed a family with dominant inheritance of early-onset OA and found that affected individuals harbored a rare variant allele encoding a significant amino acid change (p.Asn104Asp) in the kinase domain of receptor interacting protein kinase 2 (RIPK2), which transduces signals from activated bacterial peptidoglycan sensors through the NF-κB pathway to generate a proinflammatory immune response. Functional analyses of RIPK2 activity in zebrafish embryos indicated that the variant RIPK2104Asp protein is hyperactive in its signaling capacity, with augmented ability to activate the innate immune response and the NF-κB pathway and to promote upregulation of OA-associated genes. Further we show a second allele of RIPK2 linked to an inflammatory disease associated with arthritis also has enhanced activity stimulating the NF-κB pathway. Our studies reveal for the first time the inflammatory response can function as a gatekeeper risk factor for OA.

LEM2 recruits CHMP7 for ESCRT-mediated nuclear envelope closure in fission yeast and human cells.

Endosomal sorting complexes required for transport III (ESCRT-III) proteins have been implicated in sealing the nuclear envelope in mammals, spindle pole body dynamics in fission yeast, and surveillance of defective nuclear pore complexes in budding yeast. Here, we report that Lem2p (LEM2), a member of the LEM (Lap2-Emerin-Man1) family of inner nuclear membrane proteins, and the ESCRT-II/ESCRT-III hybrid protein Cmp7p (CHMP7), work together to recruit additional ESCRT-III proteins to holes in the nuclear membrane. In Schizosaccharomyces pombe, deletion of the ATPase vps4 leads to severe defects in nuclear morphology and integrity. These phenotypes are suppressed by loss-of-function mutations that arise spontaneously in lem2 or cmp7, implying that these proteins may function upstream in the same pathway. Building on these genetic interactions, we explored the role of LEM2 during nuclear envelope reformation in human cells. We found that CHMP7 and LEM2 enrich at the same region of the chromatin disk periphery during this window of cell division and that CHMP7 can bind directly to the C-terminal domain of LEM2 in vitro. We further found that, during nuclear envelope formation, recruitment of the ESCRT factors CHMP7, CHMP2A, and IST1/CHMP8 all depend on LEM2 in human cells. We conclude that Lem2p/LEM2 is a conserved nuclear site-specific adaptor that recruits Cmp7p/CHMP7 and downstream ESCRT factors to the nuclear envelope.

The effect of HMB ingestion on the IGF-I and IGF binding protein response to high intensity military training.

OBJECTIVE: Insulin-like growth factor-I (IGF-I) is a metabolic and anabolic biomarker that has been proposed to reflect physiological adaptations resulting from multistressor environments. The bioactivity of IGF-I is regulated by seven different insulin-like growth factor binding proteins (IGFBPs) which act not only as carriers of IGF-1, but also function as a modulator of IGF-I availability and activity. Supplementing with ß-hydroxy-ß-methylbutyrate (HMB) has been shown to enhance physiological outcomes associated with intense training, and has been reported to augment the IGF-1 response. The purpose of this study was to examine the effect of 23days of HMB supplementation on circulating levels of IGF-I and IGFBPs in combat soldiers during highly intense military training. METHODS: Thirteen male soldiers from an elite infantry unit volunteered to participate in this double-blind, parallel design study. Soldiers were provided 3g·day-1 of either HMB (n=6) or placebo (PL; n=7). During the study soldiers performed advanced military training with periods of restricted sleep and severe environmental stressors. Blood samples were obtained prior to (PRE) and approximately 18h following the final supplement consumption (POST). RESULTS: No significant differences were observed for circulating IGF-1 concentrations between HMB and PL (p=0.568). In addition, no differences were seen between the groups for IGFBP-1 (p=1.000), IGFBP-2 (p=0.855), IGFBP-3 (p=0.520), IGFBP-4 (p=0.103), IGFBP-5 (p=0.886), or IGFBP-6 (p=0.775). A significant difference was noted between HMB (169.9±23.0ng·ml-1) and PL (207.2±28.0ng·ml-1) for IGFBP-7 at POST (p=0.042). CONCLUSIONS: Although the results of this study do not support the influence of HMB supplementation on circulating concentrations of IGF-1 or IGFBPs1-6 during high intensity military training, it does present initial evidence that it may lower circulating IGFBP-7 concentrations. This may provide some indication of a reduced stress response, but further investigation on the physiological role of IGFBP-7 and military training is needed.

Player Selection Bias in National Football League Draftees.

Beyer, KS, Fukuda, DH, Redd, MJ, Stout, JR, and Hoffman, JR. Player selection bias in National Football League draftees. J Strength Cond Res 30(11): 2965-2971, 2016-Relative age effects (RAEs) have been studied as a potential factor associated with player selection bias in numerous sports. However, little research has examined the role of RAEs among National Football League (NFL) draftees. The purpose of the current study was to determine the existence of RAEs in NFL draftees from the last 10 NFL drafts. Draftee birth dates were collected and divided into calendar and scholastic quarters (SQ1-SQ4). To determine the presence of RAEs in specific subsets, NFL draftees were grouped according to round drafted, position, level of conference play, and age at the time of the draft. Significant χ tests (p ≤ 0.05) comparing observed birth-date distributions vs. the expected birth-date distribution from the general population were followed up by calculating the standardized residual for each quarter (z > ±2.0 indicating significance). Overall, no RAEs were seen when birth-date distribution was assessed using calendar quarters (p = 0.47), but more draftees were born in SQ2 (December-February) than expected (p < 0.01; z = +2.2). Significantly more draftees were born in SQ2 than expected for middle-round draftees (p = 0.01; z = +2.4), skill positions (p = 0.03; z = +2.3), Power Five college draftees (p < 0.01; z = +2.6), and early draftees (p < 0.01; z = +3.1). However, reverse RAEs were seen among late draftees, with fewer draftees being born in SQ2 (z = -3.6) and more being born in SQ4 (June-August; z = +2.6) than expected. In contrast to previous research, the current study observed significant RAEs in NFL draftees from the last 10 years. This player selection bias should be considered when evaluating long-term athlete development models in American football.

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.

Developing and mature human granulocytes express ELP 6 in the cytoplasm.

BACKGROUND: c3orf75 is a conserved open reading frame within the human genome and has recently been identified as the Elongator subunit, ELP6 [1]. The Elongator enzyme complex has diverse roles, including translational control, neuronal development, cell migration and tumorigenicity [2]. OBJECTIVE: To identify genes expressed early in human eosinophil development. METHODS: Eosinophilopoiesis was investigated by gene profiling of IL-5 stimulated CD34+ cells; ELP6 mRNA is upregulated. A monoclonal antibody was raised to the recombinant protein predicted by the open reading frame. RESULTS: ELP6 transcripts are upregulated in a human tissue culture model of eosinophil development during gene profiling experiments. Transcripts are expressed in most tissue types, as shown by reverse-transcriptase PCR. Western blot experiments show that human ELP6 is a 30 kDa protein expressed in the bone marrow, as well as in many other tissues. Flow cytometry experiments of human bone marrow mononuclear cells show that ELP6 is expressed intracellularly, in developing and mature human neutrophils, eosinophils and monocytes. CONCLUSIONS: ELP6 is expressed intracellularly in developing and mature granulocytes and monocytes but not in lymphocytes and erythrocytes.

Use of zebrafish to probe the divergent virulence potentials and toxin requirements of extraintestinal pathogenic Escherichia coli.

Extraintestinal pathogenic E. coli (ExPEC) cause an array of diseases, including sepsis, neonatal meningitis, and urinary tract infections. Many putative virulence factors that might modulate ExPEC pathogenesis have been identified through sequencing efforts, epidemiology, and gene expression profiling, but few of these genes have been assigned clearly defined functional roles during infection. Using zebrafish embryos as surrogate hosts, we have developed a model system with the ability to resolve diverse virulence phenotypes and niche-specific restrictions among closely related ExPEC isolates during either localized or systemic infections. In side-by-side comparisons of prototypic ExPEC isolates, we observed an unexpectedly high degree of phenotypic diversity that is not readily apparent using more traditional animal hosts. In particular, the capacity of different ExPEC isolates to persist and multiply within the zebrafish host and cause disease was shown to be variably dependent upon two secreted toxins, alpha-hemolysin and cytotoxic necrotizing factor. Both of these toxins appear to function primarily in the neutralization of phagocytes, which are recruited in high numbers to sites of infection where they act as an essential host defense against ExPEC as well as less virulent E. coli strains. These results establish zebrafish as a valuable tool for the elucidation and functional analysis of both ExPEC virulence factors and host defense mechanisms.

Analysis of WASp function during the wound inflammatory response--live-imaging studies in zebrafish larvae.

Wiskott-Aldrich syndrome protein (WASp) is haematopoietically restricted, and is the causative protein underlying a severe human disorder that can lead to death due to immunodeficiency and haemorrhaging. Much is known about the biochemistry of WASp and the migratory capacity of WASp-defective cells in vitro, but in vivo studies of immune-cell behaviour are more challenging. Using the translucency of zebrafish larvae, we live-imaged the effects of morpholino knockdown of WASp1 (also known as Was) on leukocyte migration in response to a wound. In embryos at 22 hours post-fertilisation, primitive macrophages were impaired in their migration towards laser wounds. Once a circulatory system had developed, at 3 days post-fertilisation, we observed significantly reduced recruitment of neutrophils and macrophages to ventral fin wounds. Cell-tracking studies indicated that fewer leukocytes leave the vessels adjacent to a wound and those that do exhibit impaired navigational capacity. Their cell morphology appears unaltered but their choice of leading-edge pseudopodia is more frequently incorrect, leading to impaired chemotaxis. We also identified two zebrafish mutants in WASp1 by TILLING, one of which was in the WIP-binding domain that is the hotspot for human lesions, and mutants exhibited the same deficiencies in wound inflammation and thrombus formation as WASp1 morphants.

In vivo analysis of zebrafish innate immunity.

Among vertebrate model species, the zebrafish embryo combines at an unprecedented level optical accessibility with easy genetic manipulation. As such, it is gaining recognition as a powerful model to study innate immunity. In this chapter, we provide a protocol for the generation of zebrafish embryos deficient in a protein of interest for innate immune signaling using antisense morpholino oligonucleotides, the systemic or local infection of these embryos with bacteria, and the assessment of various aspects of the following immune response with emphasis on microscopic observation. This example can be easily adapted to study the role of other genes, either knocked down or overexpressed, and in response to any other challenge, from purified microbial compounds to pathogenic viruses. This protocol is aimed at people not necessarily familiar with zebrafish biology and handling.