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Introduction: The Functional Movement Test (FMS) is an evaluation method for the basic movement patterns of the human body that is designed by Gray Cook. Objective: This paper explores the application value of functional action test (FMS) biological image data in the risk assessment of sports injuries of Chinese rugby players. Methods: Taking the active national football team and provincial football players as the object, the standard FMS test is used to collect the data to determine the best deadline for the total FMS score. Results: The area under the ROC curve (AUC) of the overall athletes, men and women was significantly different from the assumption of AUC=0.5, which were 0.780 (P=0.000), 0.877 (P=0.001), 0.7130 (P=0.013); The best cutoff points corresponding to the total score of FMS are 13.5 points, 15.5 points, and 13.5 points, respectively. The chi-square test showed that the prevalence of the positive group (the total FMS score was less than the corresponding cutoff point) was significantly higher than the negative group (the total FMS score was greater than the corresponding cutoff point) (P

We report an inborn error of metabolism caused by an expansion of a GCA-repeat tract in the 5′ untranslated region of the gene encoding glutaminase (GLS) that was identified through detailed clinical and biochemical phenotyping, combined with whole-genome sequencing. The expansion was observed in three unrelated patients who presented with an early-onset delay in overall development, progressive ataxia, and elevated levels of glutamine. In addition to ataxia, one patient also showed cerebellar atrophy. The expansion was associated with a relative deficiency of GLS messenger RNA transcribed from the expanded allele, which probably resulted from repeat-mediated chromatin changes upstream of the GLS repeat. Our discovery underscores the importance of careful examination of regions of the genome that are typically excluded from or poorly captured by exome sequencing.

Early-infantile encephalopathies with epilepsy are devastating conditions mandating an accurate diagnosis to guide proper management. Whole-exome sequencing was used to investigate the disease etiology in four children from independent families with intellectual disability and epilepsy, revealing bi-allelic GOT2 mutations. In-depth metabolic studies in individual 1 showed low plasma serine, hypercitrullinemia, hyperlactatemia, and hyperammonemia. The epilepsy was serine and pyridoxine responsive. Functional consequences of observed mutations were tested by measuring enzyme activity and by cell and animal models. Zebrafish and mouse models were used to validate brain developmental and functional defects and to test therapeutic strategies. GOT2 encodes the mitochondrial glutamate oxaloacetate transaminase. GOT2 enzyme activity was deficient in fibroblasts with bi-allelic mutations. GOT2, a member of the malate-aspartate shuttle, plays an essential role in the intracellular NAD(H) redox balance. De novo serine biosynthesis was impaired in fibroblasts with GOT2 mutations and GOT2-knockout HEK293 cells. Correcting the highly oxidized cytosolic NAD-redox state by pyruvate supplementation restored serine biosynthesis in GOT2-deficient cells. Knockdown of got2a in zebrafish resulted in a brain developmental defect associated with seizure-like electroencephalography spikes, which could be rescued by supplying pyridoxine in embryo water. Both pyridoxine and serine synergistically rescued embryonic developmental defects in zebrafish got2a morphants. The two treated individuals reacted favorably to their treatment. Our data provide a mechanistic basis for the biochemical abnormalities in GOT2 deficiency that may also hold for other MAS defects.

Their optical clarity as larvae and embryos, small size, and high fecundity make zebrafish ideal for whole animal high throughput screening. A high-throughput drug discovery platform (HTP) has been built to perform fully automated screens of compound libraries with zebrafish embryos. A Tg(Flk1:EGFP) line, marking endothelial cell cytoplasm, was used in this work to help develop protocols and functional algorithms for the system, with the intent of screening for angiogenesis inhibitors. Indirubin 3’ Monoxime (I3M), a known angiogenesis inhibitor, was used at various concentrations to validate the protocols. Consistent with previous studies, a dose dependant inhibitory effect of I3M on angiogenesis was confirmed. The methods and protocols developed here could significantly increase the throughput of drug screens, while limiting human errors. These methods are expected to facilitate the discovery of novel anti-angiogenesis compounds and can be adapted for many other applications in which samples have a good fluorescent signal.

Although the functional role of chromatin marks at promoters in mediating cell-restricted gene expression has been well characterized, the role of intragenic chromatin marks is not well understood, especially in endothelial cell (EC) gene expression. Here, we characterized the histone H3 and H4 acetylation profiles of 19 genes with EC-enriched expression via locus-wide chromatin immunoprecipitation followed by ultra-high–resolution (5 bp) tiling array analysis in ECs versus non-ECs throughout their genomic loci. Importantly, these genes exhibit differential EC enrichment of H3 and H4 acetylation in their promoter in ECs versus non-ECs. Interestingly, VEGFR-2 and VEGFR-1 show EC-enriched acetylation across broad intragenic regions and are up-regulated in non-ECs by histone deacetylase inhibition. It is unclear which histone acetyltransferases (KATs) are key to EC physiology. Depletion of KAT7 reduced VEGFR-2 expression and disrupted angiogenic potential. Microarray analysis of KAT7-depleted ECs identified 263 differentially regulated genes, many of which are key for growth and angiogenic potential. KAT7 inhibition in zebrafish embryos disrupted vessel formation and caused loss of circulatory integrity, especially hemorrhage, all of which were rescued with human KAT7. Notably, perturbed EC-enriched gene expression, especially the VEGFR-2 homologs, contributed to these vascular defects. Mechanistically, KAT7 participates in VEGFR-2 transcription by mediating RNA polymerase II binding, H3 lysine 14, and H4 acetylation in its intragenic region. Collectively, our findings support the importance of differential histone acetylation at both promoter and intragenic regions of EC genes and reveal a previously underappreciated role of KAT7 and intragenic histone acetylation in regulating VEGFR-2 and endothelial function.

One of the main advantages of photoacoustic (PA) microscopy is its ability to generate high-resolution maps of vasculature due to strong endogenous absorption. In contrast the optical absorption of the gross anatomy surrounding the vasculature is negligible, and it is thus not readily imaged using PA. We have previously reported on a new imaging technique, termed Photoacoustic Radiometry (PAR), which derives contrast from the sample's optical attenuation properties (doi:10.1117/12.2212961). In this work, we use a dual PA and PAR technique to simultaneously acquire co-registered images of the vasculature and gross anatomy of the trunk of a zebrafish embryo from a single PA scan.

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Traumatic brain injury (TBI) is a leading cause of death and morbidity with no effective therapeutic treatments for secondary injury. Preclinical drug evaluation in rodent models of TBI is a lengthy process. In this regard, the zebrafish has numerous advantages to address the technical and time-dependent obstacles associated with drug evaluation. We developed a reproducible brain injury using glutamate excitoxicity in zebrafish larvae, a known initiator of delayed cell death in TBI. Glutamate challenge resulted in dose-dependent lethality over an 84-h observation period. We report significant decrease in locomotion (p

ZNF403, also known as GGNBP2 (gametogenetin binding protein 2), is a highly conserved gene implicated in spermatogenesis. However, the exact biological function of ZNF403 is not clear. In this study, we identified the role of ZNF403 in cell proliferation and cell-cycle regulation by utilizing short hairpin RNA (shRNA)-mediated knockdown. ZNF403-specific shRNA expressing helper-dependent adenoviral vector (HD-Ad-ZNF403-shRNA) was constructed and transduced human cell lines. ZNF403 mRNA and protein expression levels were inhibited as evidenced by real-time PCR and western blot analyses. Noticeably, we found that knockdown of ZNF403 expression suppressed cell proliferation compared to the non-target shRNA and vector controls. Furthermore, cell-cycle analysis demonstrated that downregulation of ZNF403 promoted G2/M cell-cycle arrest in a dose-dependent manner. Moreover, human cell-cycle real-time PCR array revealed that ZNF403 knockdown influenced the expression profile of genes in cell-cycle regulation. Among these genes, western blot analysis confirmed the protein up-regulation of p21 and down-regulation of MCM2 in response to the ZNF403 knockdown. Additionally, knockdown of ZNF403 also showed an anti-carcinogenetic effect on anchorage-independent growth by colony formation assay and tumor cell migration by wound-healing assay with human laryngeal cancer cell line Hep-2 cells. Altogether, our findings suggest an essential role of ZNF403 in cell proliferation and provide a new insight into the function of ZNF403 in regulating the G2/M cell-cycle transition.

Background Prostate cancer (PCa) is the most common malignancy in males in Western countries. Despite improvements in standard treatments such as surgery, radiotherapy, and chemotherapy, many patients still progress to advanced stages. Recent clinical trials have shown encouraging results regarding the application of angiogenic inhibitors in the treatment of angiogenesis-dependent diseases, paving the way for novel PCa therapies. Objective To identify new antiangiogenic compounds and examine their therapeutic potential in models of PCa. Design, setting, and participants We performed a chemical genetic screen in developing zebrafish embryos to identify small molecules inhibiting zebrafish angiogenesis. Transgenic Tg(flk1:EGFP) zebrafish embryos were used in the screening of the Spectrum Collection compound library. Subsequently, the antiangiogenic mechanism of an identified lead compound, rosuvastatin, was studied by conducting endothelial cell function assays and examining antitumor efficacy in a PCa xenograft mouse model. Measurements, results and limitations Seven lead compounds, including isorotenone, dihydromunduletone, aristolochic acid, simvastatin, mevastatin, lovastatin, and rosuvastatin, were identified to inhibit the growth of the zebrafish intersegmental vessels. Of these seven leads, rosuvastatin was further evaluated for its antiangiogenic mechanism and anticancer efficacy. Rosuvastatin decreased the viability of the human umbilical endothelial cells (HUVECs) (one-half inhibitory concentration: 5.87μM) by inducing G 1 phase arrest and promoting apoptosis. Moreover, rosuvastatin remarkably inhibited the migration of HUVECs and dose-dependently inhibited the HUVEC capillary-like tube formation in vitro. Furthermore, we demonstrated that rosuvastatin suppressed xenografted PPC-1 prostate tumors in nonobese diabetic severe combined immunodeficiency (NOD-SCID) mice associated with decreased microvessel density (MVD) and tumor cell apoptosis. Conclusions Collectively, our data suggest that rosuvastatin possesses antiangiogenic and antitumor activities and has therapeutic potential for the treatment of PCa. This study represents the first zebrafish antiangiogenic chemical genetic screen to identify a lead compound that targets cancer angiogenesis.

The typical electro-Fenton process requires electricity to produce H2O2 electrochemically on the cathode for generating powerful oxidants (e.g., hydroxyl radical) to degrade persistent organic pollutants. A new bioelectro-Fenton system in a two-chamber microbial fuel cell (MFC) was proposed using a Fe@Fe2O3/carbon felt composite cathode for simultaneous oxidation of wastewater at the anode and cathodic degradation of biorefractory pollutant by Fenton's reaction. Under the short-circuit conditions, it was found that the rate of Rhodamine B (RhB) decolorization, TOC removal and stoichiometric efficiency was significantly improved as compared with that found under close-circuit conditions (1000 Ω). The enhancement was determined to be caused by an increase in cathodic current density that was favorable for H2O2 production.

Hacs1, a SH3 and SAM domain-containing adaptor protein, is up-regulated by IL-4 in activated B cells and strongly expressed in dendritic cells. To elucidate the function of Hacs1 in immune regulation, we generated Hacs1(-/-) mice by deletion of the SH3 and SAM domains. Hacs1(-/-) mice were viable and fertile and had normal bone marrow B-cell development and normal splenic T- and B-cell populations. However, adult Hacs1(-/-) mice had increased peritoneal B1a cells (IgM(+)CD5(+)). On immunization with T-cell-independent antigen TNP-Ficoll, Hacs1(-/-) mice had increased production of anti-TNP IgM and IgG3. Purified splenic B cells from Hacs1(-/-) mice showed increased cell proliferation on BCR (B-cell receptor) stimulation. We further demonstrate that the Hacs1(-/-) B cells had increased global tyrosine phosphorylation, including tyrosine kinases Lyn and Akt. Both T-helper type 1 (T(h)1) and T-helper type 2 (T(h)2) humoral responses were enhanced in Hacs1(-/-) mice. In vitro bone marrow-derived Hacs1(-/-) dendritic cells showed increased IL-12 production on stimulation with ovalbumin (OVA). This study suggests that Hacs1 is an immunoinhibitory adaptor that might be a useful target for immune suppression therapy.-Wang, D., Stewart, A. K., Zhuang, L., Zhu, Y., Wang, Y., Shi, C., Keating, A., Slutsky, A., Zhang, H., Wen, X.-Y. Enhanced adaptive immunity in mice lacking the immunoinhibitory adaptor Hacs1.

Human chromosomal region 13q14 is a deletion hotspot in prostate cancer, multiple myeloma, and chronic lymphocytic leukemia. This region is believed to host multiple tumor suppressors. Chromosome Condensation 1-like (CHC1L) is located at 13q14, and found within the smallest common region of loss of heterozygosity in prostate cancer. Decreased expression of CHC1L is linked to pathogenesis and progression of both prostate cancer and multiple myeloma. However, there is no direct evidence for CHC1L’s putative tumor suppressing role in current literature. Presently, we describe the generation and characterization of Chc1L knockout mice. Chc1L -/- mice do not develop cancer at a young age, but bone marrow and spleen cells from 8–12 week-old mice display an exaggerated proliferative response. By approximately two years of age, knockout and heterozygote mice have a markedly increased incidence of tumorigenesis compared to wild-type controls, with tumors occurring mainly in the spleen, mesenteric lymph nodes, liver and intestinal tract. Histopathological analysis found that most heterozygote and knockout mice succumb to either Histiocytic Sarcoma or Histiocyte-Associated Lymphoma. Our study suggests that Chc1L is involved in suppression of these two histiocyte-rich neoplasms in mice and supports clinical data suggesting that CHC1L loss of function is an important step in the pathogenesis of cancers containing 13q14 deletion.

Human MURR1 is an orthologue of mouse Murr1 gene, which was previously reported to be imprinted only in adult brain with a maternal allele-predominant expression and to contain another imprinted gene, U2af1-rs1, in the first intron. Human MURR1 was found not to harbor the U2af1-rs1 orthologue and to be expressed biallelically in tissues, including adult brain. Three genes identified around Murr1 and their orthologues around MURR1 were expressed biallelically. These findings suggest that the mouse imprinting locus is limited to a small region and the introduction of U2af1-rs1 in mouse causes the imprinting of this locus. The CpG island (CGI) at U2af1-rs1 with maternal methylation was the only differentially methylated region among CGIs found in these loci. Detailed methylation analyses of the U2af1-rs1 CGI in germ cells led to identification of a region with oocyte-specific methylation. These results suggest that this region is the imprinting control region of the Murr1/U2af1-rs1 locus in mouse.

The mouse Murr1 gene contains an imprinted gene, U2af1-rs1, in its first intron. U2af1-rs1 shows paternal allele-specific expression and is transcribed in the direction opposite to that of the Murr1 gene. In contrast to a previous report of biallelic expression of Murr1 in neonatal mice, we have found that the maternal allele is expressed predominantly in the adult brain and also preferentially in other adult tissues. This maternal-predominant expression is not observed in embryonic and neonatal brains. In situ hybridization experiments that used the adult brain indicated that Murr1 gene was maternally expressed in neuronal cells in all regions of the brain. We analyzed the developmental change in the expression levels of both Murr1 and U2af1-rs1 in the brain and liver, and we propose that the maternal-predominant expression of Murr1 results from transcriptional interference of the gene by U2af1-rs1 through the Murr1 promoter region.

Human 11p15.5, as well as its orthologous mouse 7F4/F5, is known as the imprinting domain extending from IPL/Ipl to H19. OBPH1 and Obph1 are located beyond the presumed imprinting boundary on the IPL/Ipl side. We determined full-length cDNAs and complete genomic structures of both orthologues. We also investigated their precise imprinting and methylation status. The orthologues resembled each other in genomic structure and in the position of the 5' CpG island and were expressed ubiquitously. OBPH1 and Obph1 were predominantly expressed from the maternal allele only in placenta, with hypo- and not differentially methylated 5' CpG islands in both species. These results suggested that the imprinting domain would extend beyond the presumed imprinting boundary and that methylation of the 5' CpG island was not associated with the imprinting status in either species. It remains to be elucidated whether the gene is under the control of the KIP2/LIT1 subdomain or is regulated by a specific mechanism. Analysis of the precise genomic sequence around the region should help resolve this question.

The zebrafish has emerged as an excellent model for cardiovascular research thanks to its ex-utero and rapid embryonic development, its embryonic transparent nature, and its capacity to survive in the absence of a functional cardiovascular system during the first week of development, which enables functional characterization of mutations that would otherwise induce lethality in traditional murine models. The aim of this project is to identify and characterize novel genes involved in zebrafish cardiovascular development using mutagenic gene trapping, a technique that generates random insertional mutations across the genome. We use the well-defined RP2 gene-breaking transposon system, which not only mutates, but also fluorescently tags the trapped gene product(s) (Clark, Nat Methods, 2011). RP2 also introduces loxP sites into the mutant locus, which can be used for Cre-mediated phenotype rescue by microinjection of Cre recombinase, or by crossing to tissue-specific Cre lines. From over 3000 RP2-injected embryos, 141 fish showed germline transmission. Among them, 51 expressed strong fluorescence in different tissues, including the heart, vessels, notochord, central nervous system (CNS), and eyes. Three cardiovascular lines were selected for phenotypic characterization and functional studies. RP2#C2 strain expressed fluorescence in the heart valves, CNS, eyes and pectoral fin buds. Inverse PCR in RP2#C2 demonstrated a trapped gene at meis4.1a, which encodes a homeobox transcription factor that has not been previously studied in zebrafish. RP2#121 strain expressed strong fluorescence in cardiac and skeletal muscles. RP2#91 strain showed expression in the vasculature and demonstrated a trapped gene at pdgfra. Homozygous RP2#91 mutants showed severe defects in the heart, blood flow, and other body parts including the head and musculature. We are currently creating a panel of tissue-specific Cre lines targeting tissues such as cardiomyocytes, endothelial cells and smooth muscle cells for spatiotemporal rescuing of the mutant phenotype. The generated zebrafish protein-trap lines are invaluable tools to annotate gene function, dissect the molecular mechanisms of cardiovascular development, and potentially serve as disease models.

Introduction/rationale: Current anti-diabetic drug treatments have a variety of adverse side effects. Identifying functional foods with anti-diabetic properties may be the key to preventing and managing T2DM while minimizing side effects. Studies found that Pck-1 gene is often down-regulated by anti-diabetic drugs, as its enzyme is responsible for catalyzing the rate-limiting step of gluconeogenesis. In our study, we screened for functional foods that can down-regulate the expression of the Pck-1 gene. The newly emerged zebrafish (Danio rerio) is aptly suited for in vivo nutrigenomic screening due to its large clutch size and conservation of molecular pathways with humans, including those involved in glucose regulation. Methods: We exposed zebrafish larve (Tg(Pck-1:luc), a luminescent reporter line for Pck-1), at 4 days post fertilization (dpf) to extracts from forty functional fruits and vegetables. The level of Pck-1 expression was quantified by luminescence at 6 dpf. Extracts which resulted in a lower reading of luminescence were interpreted as functional foods that could successfully down-regulate Pck-1 expression, and therefore may be potential therapeutic for T2DM. We further validated our results using a fluorescent reporter line for Pck-1 (Pck-1:eGFP). Results: We completed toxicity assays on the functional food extracts to determine the optimal concentrations and conditions for nutrigenomic screening. Preliminary results show that bamboo and cherry extracts significantly reduced Pck-1 gene expression by two fold (p Conclusion and Discussion: We identified two functional food extracts that successfully reduced Pck-1 expression in zebrafish models. Increasing the intake of these two functional foods may regulate blood glucose levels in T2DM patients. Further research elucidating the active ingredient in these foods is required. The Pck-1 zebrafish model can be employed for further nutrigenomic screening and/or for drug discovery purposes.

Introduction: Angiogenic sprouting occurs in the presence of high levels of exogenous pro-angiogenesis factors. Pathological over expression of pro-angiogenesis factors leads to excessive tumor growth. Inhibition of pro-angiogenesis factors can restrict tumor growth. But, prolonged inhibition can lead to the activation of alternate pro-angiogenesis pathways. The objective of this research is to discover compounds that inhibit multiple pro-angiogenesis factors and increase efficacy in the treatment of cancerous tumors using zebrafish vascular development as a model in a high throughput setting. Methods: The NIH Clinical Collections Libraries I and II will be screened with a fully automated high throughput drug screening platform (HTS). Compounds that cause abnormal vascular phenotypes will be identified on Tg(flk1:EGFP) embryos. The lead compounds will then be analyzed with 3-D confocal imaging, studies on cultured human umbilical vein endothelial cells (HUVECs), and animal cancer models. Results: The HTS was constructed and made operational. A protocol for anti-angiogenesis screening with Tg(flk1:EGFP) zebrafish has been developed and validated with indirubin 3’ monoxime (I3M), a known angiogenesis inhibitor. Pixel counting (to quantify the overall vascular network) and ISV counting (to count the number of ISVs and give an indication of overall vascular morphology) programs were created to provide readouts from confocal images taken within the HTS. Normative parameters, including several endothelial cell counts and various vessel length measurements, were characterised at the 1 and 2 days post fertilization (dpf) stages using 3-D confocal imaging. These values will be compared to values found in the presence of lead compounds to validate them as angiogenesis inhibitors. Conclusions & Future Work: The framework for a robust drug discovery process has been put in place. The next step is to screen the NIH Clinical Collections Libraries I and II and analyze the efficacy and mechanism of lead compounds. Based on data from previous manual screens, 5-10 lead compounds are expected to be identified. These compounds will have the potential to be developed as repurposed anti-angiogenesis compounds and improve therapeutics for cancerous tumors.

Hardware Trojan (HT) has emerged as an impending security threat to hardware systems. However, conventional functional tests fail to detect HT since Trojans are triggered by rare events. Most of the existing side-channel based HT detection techniques just simply compare and analyze circuit's parameters and offer no signal calibration or error correction properties, so they suffer from the challenge and interference of large process variations (PV) and noises in modern nanotechnology which can completely mask Trojan's contribution to the circuit. This paper presents a novel HT detection method based on subspace technique which can detect tiny HT characteristics under large PV and noises. First, we formulate the HT detection problem as a weak signal detection problem, and then we model it as a feature extraction model. After that, we propose a novel subspace HT detection technique based on time domain constrained estimator. It is proved that we can distinguish the weak HT from variations and noises through particular subspace projections and reconstructed clean signal analysis. The reconstructed clean signal of the proposed algorithm can also be used for accurate parameter estimation of circuits, e.g. power estimation. The proposed technique is a general method for related HT detection schemes to eliminate noises and PV. Both simulations on benchmarks and hardware implementation validations on FPGA boards show the effectiveness and high sensitivity of the new HT detection technique. Published version

Mouse chromosome 7F4/F5, where the imprinting domain is located, is syntenic to human 11p15.5, the locus for Beckwith-Wiedemann syndrome. The domain is thought to consist of the two subdomains Kip2 (p57kip2)/Lit1 and Igf2/H19. Because DNA methylation is believed to be a key factor in genomic imprinting, we performed large-scale DNA methylation analysis to identify thecis-element crucial for the regulation of the Kip2/Lit1 subdomain. Ten CpG islands (CGIs) were found, and these were located at the promoter sites, upstream of genes, and within intergenic regions. Bisulphite sequencing revealed that CGIs 4, 5, 8, and 10 were differentially methylated regions (DMRs). CGIs 4, 5, and 10 were methylated paternally in somatic tissues but not in germ cells. CGI8 was methylated in oocyte and maternally in somatic tissues during development. Parental-specific DNase I hypersensitive sites (HSSs) were found near CGI8. These data indicate that CGI8, called DMR-Lit1, is not only the region for gametic methylation but might also be the imprinting control region (ICR) of the subdomain.

Here we identified a novel transcript in mouse that is transcribed from the portions of two independent genes, U2af1-rs1 and Murr1, and we designated it U2mu. The U2af1-rs1 gene is located in the intron and transcribed in the opposite direction from the Murr1 gene on the proximal region of mouse chromosome 11. The U2mu cDNA sequence is derived from three genomic regions - an intron of the Murr1 gene, an antisense sequence of U2af1-rs1 gene, and the last exon of Murr1 gene - in the order of 5' to 3'. The U2mu transcript of 2.8 kb is expressed ubiquitously in adult mice. It is transcribed biallelically, and is not imprinted, in neonatal and adult mice.

Human chromosome 13q14 is one of the hotspots of chromosomal deletion in prostate cancer, multiple myeloma, and chronic lymphocytic leukemia. Chromosome Condensation 1-like (CHC1L) is an uncharacterized gene in this region. CHC1L is found within the smallest common region of loss of heterozygosity in prostate cancer, and its decreased expression is linked to pathogenesis and progression of both prostate cancer and multiple myeloma. In the present study, we describe the generation and characterization of Chc1L gene knockout mice. Knockout mice develop normally and display an exaggerated proliferative response to LPS stimulation of cultured bone marrow and spleen cells at ten weeks of age. At approximately two years of age, both homozygous and heterozygous mice have markedly increased incidence of tumorigenesis, 80% and 56% respectively, compared to wild-type mice, 26%. The tumors are distributed most commonly to the spleen, mesenteric lymph nodes, liver and small intestine. The morphologic and immunophenotypic features of the tumor cells are consistent with those of tissue histiocytes, and most Chc1L knockout and heterozygote mice succumb to either Histiocytic Sarcoma or Histiocyte-Associated Lymphoma. These data provide the first direct evidence that Chc1L is a tumor suppressor gene involved in suppression of histiocyte-rich neoplasms. Citation Format: David Spillane, Ding Yan Wang, Changxin Shi, Youdong Wang, Anthony Gramolini, Keith Stewart, Susan Newbigging, Mingyao Liu, Xiao-Yan Wen. Chc1L is a tumor suppressor involved in development of histiocyte-rich neoplasms. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr C41.

To assess the impact of digital problem-based learning (PBL) cases on student learning in ophthalmology courses.Ninety students were randomly divided into 3 classes (30 students per class). The first class studied under a didactic model. The other 2 classes were divided into 6 groups (10 students per group) and received PBL teaching; 3 groups studied via cases presented in digital form and the others studied via paper-form cases. The results of theoretical and case analysis examinations were analyzed using the chi(2) test. Student performance on the interval practice was analyzed using the Kruskal-Wallis test. Questionnaires were used to evaluate student and facilitator perceptions.Students in the digital groups exhibited better performance in the practice procedures according to tutorial evaluations compared with the other groups (P.05). The 2 PBL classes had significantly higher mean results of theoretical and case analysis examinations (P.001), but there was no significant difference between the 2 PBL classes. Ninety-three percent of students in the digital groups (vs 73% in the paper groups) noted that the cases greatly stimulated their interest.Introducing PBL into ophthalmology could improve educational quality and effectiveness. Digital PBL cases stimulate interest and motivate students to further improve diagnosis and problem-handling skills.

Platelet adhesion and aggregation are critical events in thrombosis. Bioactive phospholipid LPA (lysophosphatidic acid) has been identified as an important agonist for platelet aggregation. Plasma LPA can be generated from phospholipid substrates by phospholipase A1 (PLA1) and phospholipase D (PLD). We previously identified two novel PLA1 enzymes, designated lpdl (lpd lipase) and lpdlr (lpdl related lipase). Together with phosphatidylserine phospholipase A1 (PS-PLA1), these three phospholipases form a unique PLA1 lipase subfamily. Phospholipids are important structural components of cellular membranes. Recent studies demonstrate that lipid raft microdomains on the platelet membrane contribute to platelet activation. The gathering of membrane lipid rafts is necessary for ADP-mediated platelet activation. Disruption of lipid raft results in a reduction of ADP-induced platelet aggregation. In addition, it is reported that alteration of membrane lipid composition also affects the function of platelet β3 integrin (GPIIbIIIa). Thus, phospholipases may play an important role in platelet function. However, the roles of lpdl and lpdlr in platelet activation and thrombosis are unknown. To study the function of lpdl and lpdlr, we have recently knocked out both lpdl and lpdlr genes in mice by deleting their exons I and II, including their translation start codon ATG. RT-PCR confirmed that neither lpdl nor lpdlr gene is expressed in homozygous lpdl−/− or lpdlr−/− mice but expressed in wild-type (WT) tissues, indicating successful knockout of these phospholipases. Next, optical platelet aggregometry was used to assess platelet aggregation in platelet rich plasma (PRP). Platelet aggregation was induced with 5, 10 and 20 μM ADP in pooled PRP (3×108 platelets/mL) from age- and sex-matched WT, heterozygous and homozygous mice. Our results demonstrated that both the lpdl−/− and lpdlr−/− mice have decreased platelet aggregation after ADP stimulation. We further studied thrombus formation in lpdl−/− mice using an ex vivo perfusion chamber model on collagen type I-coated rectangular glass microcapillary tubes. Our preliminary data showed that both platelet adhesion and aggregation were impaired in the lpdl−/− mice. We are repeating these experiments and are examining lpdlr−/− mice using the same perfusion chamber model. In vivo thrombosis models with intravital microscopy and the mechanisms of how lpdl and lpdlr enzymes regulate platelet activation will also be investigated. These studies could potentially identify a novel pathway for regulation of platelet function, which may lead to development of new diagnostic and/or therapeutic methods for atherothrombosis. (Drs. D. Wang and A. Reheman contribute equally to this work)

HACS1 is a SH3 (Src homology 3) and SAM (sterile alpha motif) domain containing adaptor protein that is expressed in activated B and dendritic cells, is up-regulated by Interleukin 4 in the process of B cell activation and likely serves to dampen the immune response. To elucidate the function of HACS1, we generated HACS1 gene knockout mice by deletion of the SH3 and SAM domains. HACS1−/− mice were viable and fertile and had normal bone marrow B cell development and normal splenic T and B cell populations. However, adult HACS1−/− mice had increased numbers of peritoneal B1 cells (IgM+CD5+). Upon LPS plus BCR or TCR stimulations, splenic cells from HACS1−/− mice demonstrated an upregulation of activation markers with increased intensity of CD23 expression or increased population of CD69 positive cells. Purified B220+ splenic B cells from HACS1−/− mice showed increased cell proliferation upon BCR stimulation. Both T helper type 1 (Th1) and T helper type 2 (Th2) humoral responses were enhanced in HACS1−/− mice upon immunization with T cell-dependent antigen NP-KLH, which resulted in increased production of interferon-gamma (IFN-γ), anti-NP IgG2a and IL-4, as well as anti-NP IgG1 and IgE. Upon immunization with T cell-independent antigens such as TNP-LPS and TNP-Ficoll, HACS1−/− mice had increased production of anti-TNP IgM and IgG3 as compared to normal controls. The in vitro maturation of bone marrow-derived dentritic cells from HACS1−/− mice was similar to wild-type mice but HACS1−/− dentritic cells showed increased IL-12 production upon stimulation with anti-CD40 or LPS. There was no significant difference in antigen uptake by cultured dentritic cells from non-immunized wild-type or knockout mice. However, in immunized mice, an increase in antigen uptake in HACS1−/− dentritic cells was observed. We further demonstrate that the HACS1−/− B cells had increased tyrosine phosphorylation in the resting state. As deletion of the SH3 & SAM domains of HACS1 appears to generate a hypersensitive immune response, our results collectively support that HACS1 negatively regulates adaptive immunity.

The zebrafish has emerged as an important model for studying vascular development and angiogenesis. Advantages over other models include rapid embryonic development and optical clarity of the embryos. Abnormal angiogenesis is linked to over 70 health conditions and inhibition of angiogenesis is an excellent target for cancer therapy (including hematopoietic malignancies) as tumor growth requires new blood vessels. In this study, we performed a chemical genetic screen in developing zebrafish embryos to identify compounds that modulate zebrafish vascular development and angiogenesis. A zebrafish transgenic line with the Flk1 promoter controlling the GFP (green fluorescent protein) reporter was used. The entire vascular network of the Flk1:GFP fish was marked by GFP and could be visualized under fluorescence microscopy. Screens were performed in 96 well plates with three embryos/well. The outer membranes of healthy zebrafish embryos expressing green fluorescent vasculature were removed by enzymatic digestion with protease at the 16–18 somite stages. Small molecules/compounds were then added to the water at concentrations of 0.1 μM, 1 μM, and 5 μM for 72 hrs. After 24, 48 and 72 hrs of exposure to the compound, the embryos were visually inspected for viability, gross morphological defects, heartbeating rate and circulation. The known angiogenic inhibitor, PD173074 was used as control. An initial screening of 780 compounds in a small molecule compound library identified four small molecules with potent activities in inhibiting zebrafish vascular development. Notably, 1–5 μM of Hit #1 inhibited the growth of cranial vessels and disrupted vascular patterning, which resulted in uneven spacing between intersegmental vessels. While Hit #2 and #5 also inhibited the growth of cranial vessels, the vascular patterning remained unaffected, however, the drug-treated embryos had weak or missing intersegmental vessels. Fish embryos treated by Hit #3 had an enlarged heart and thinner vessels. Interestingly, this screen also identified one compound (Hit #4) with pro-angiogenic activity. Embryos treated with Hit #4 had increased numbers of intersegmental vessels. Hit #2 is a known inhibitor of c-Jun N-terminal kinase and angiogenesis (SP600125), was recently reported to inhibit the proliferation and migration of human endothelial cells in vitro as well as inhibit solid tumor growth in mice. This observation lends validity to the zebrafish screen and the potential utility of the other hits. Experiments are currently in progress to investigate the detailed time course of the angiogenic inhibition and the potential molecular mechanism. Studies of these angiogenic inhibitors may lead to the development of potent anti-cancer drugs while the pro-angiogenic compound may prove useful in facilitating tissue/organ regeneration. We conclude that the zebrafish model is likely to yield valuable information regarding vasculogenesis and its manipulation.