Comparative analyses of laying performance and follicular development characteristics between fat and lean broiler lines.

The deposition of high levels of fat in broiler breeder hens can have a profound impact on follicular development and laying performance. This study was formulated with the goal of comparing egg production and follicular development characteristics at different laying stages in the Northeast Agricultural University broiler lines divergently selected for abdominal fat content (NEAUHLF). The egg production was analyzed using the birds from the 19th to 24th generations of NEAUHLF; the follicular development characteristics were analyzed by hematoxylin-eosin staining and quantitative real-time polymerase chain reaction using the birds from the 24th generation of NEAUHLF. The results showed that the age at first egg of lean hens was significantly earlier than that of fat hens in this study. While no significant differences in total egg output from the first egg to 50 wk of age were noted when comparing these 2 chicken lines, lean hens laid more eggs from the first egg to 35 wk of age relative to fat hens, whereas fat hens laid more eggs from wk 36 to 42 and 43 to 50 relative to their lean counterparts. No differences in ovarian morphology and small yellow follicle (SYF) histological characteristics were noted when comparing these 2 chicken lines at 27 wk of age. At 35 and 52 wk of age, however, lean hens exhibited significantly lower ovarian weight, ovarian proportion values, numbers of hierarchical follicles, hierarchical follicle weight, and SYF granulosa layer thickness as compared to fat hens, together with a significant increase in the number of prehierarchical follicles relative to those in fat hens. Gene expression analyses suggested that follicle selection was impaired in the fat hens in the early laying stage, whereas both follicle selection and maturation were impaired in the lean hens in the middle and late laying stages. Overall, these data highlight that fat deposition in broiler hens can have a range of effects on follicular development and egg production that are laying stage-dependent.

HBP1 promotes chicken preadipocyte proliferation via directly repressing SOCS3 transcription.

Preadipocyte proliferation is an essential process in adipose development. During proliferation of preadipocytes, transcription factors play crucial roles. HMG-box protein 1 (HBP1) is an important transcription factor of cellular proliferation. However, the function and underlying mechanisms of HBP1 in the proliferation of preadipocytes remain unclear. Here, we found that the expression level of HBP1 decreased first and then increased during the proliferation of chicken preadipocytes. Knockout of HBP1 could inhibit the proliferation of preadipocytes, while overexpression of HBP1 could promote the proliferation of preadipocytes. ChIP-seq data showed that HBP1 had the unique DNA binding motif in chicken preadipocytes. By integrating ChIP-Seq and RNA-Seq, we revealed a total of 3 candidate target genes of HBP1. Furthermore, the results of ChIP-qPCR, RT-qPCR, luciferase reporter assay and EMSA showed that HBP1 could inhibit the transcription of suppressor of cytokine signaling 3 (SOCS3) by binding to its promoter. Moreover, we confirmed that SOCS3 can mediate the regulation of HBP1 on the proliferation of preadipocytes through RNAi and rescue experiments. Altogether, these data demonstrated that HBP1 directly targets SOCS3 to regulate chicken preadipocyte proliferation. Our findings expand the transcriptional regulatory network of preadipocyte proliferation, and they will be helpful in formulating a molecular breeding scheme to control excessive abdominal fat deposition and to improve meat quality in chickens.

Plasma obtained following murine hindlimb ischemic conditioning protects against oxidative stress in zebrafish models through activation of nrf2a and downregulation of duox.

Ischemia/reperfusion of organ systems in trauma patients with resuscitated hemorrhagic shock (HSR) contributes to tissue injury and organ dysfunction. Previous studies using a murine model of HSR showed that remote ischemic preconditioning (RIC) protected against organ injury and that the plasma was able to prevent neutrophil migration in a zebrafish tailfin-cut inflammation model. In this study, we hypothesized that RIC plasma inhibits neutrophil function through a decrease in reactive oxygen species (ROS) production via the upregulation of the transcription factor Nrf2 and downstream antioxidative genes. Plasma from mice subjected to RIC (4 cycles of 5-min hindlimb ischemia/reperfusion) was microinjected into zebrafish. The results show that RIC plasma caused a reduction of ROS generation in response to tail injury. In addition, RIC plasma protected the fish larvae in the survival studies when exposed to either H2O2 or LPS. Oxidative stress PCR Array showed that RIC plasma treatment led to upregulation of antioxidative related genes including hsp70, hmox1a, nqo1 as well as downregulation of duox, the producer of H2O2. To explore the role of nrf2 in RIC, RIC plasma from Nrf2 KO mice were injected to the zebrafish and showed no inhibitory effect on neutrophil migration. Moreover, knockdown of nrf2a attenuated the anti-inflammatory and protective effect of RIC plasma. The downregulation of duox and upregulation of hmox1a were confirmed to require the activation of nrf2a. Therefore, we show that the protective effect of RIC may be related to the elaboration of humoral factors which counter injury-induced ROS generation in a nrf2-dependent fashion.

Functional action biological image data screening method to reduce sports injury / Método de triagem de dados biológicos de ação funcional para reduzir lesões esportivas / Método de selección de datos de imágenes biológicas de acción funcional para reducir las lesiones deportivas

ABSTRACT 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<0.01). The OR values of the total athlete, male and female FMS total score positive groups were 25.85 (95%CI: 3.34∼200.23), 25.00 (95%CI: 2.36∼264.80), 14.22 (95%CI: 1.76∼114.92). Conclusions: Among Chinese rugby players, the total score of FMS has a strong correlation with non-contact sports injuries. The score is 13.5 for women and 15.5 for men. Level of evidence II; Therapeutic studies - investigation of treatment results.

RESUMO Introdução: O Teste de Movimento Funcional (FMS) é um método de avaliação dos padrões básicos de movimento do corpo humano, projetado por Gray Cook. Objetivo: Este artigo explora o valor da aplicação de dados de imagem biológica do teste de ação funcional (FMS) na avaliação do risco de lesões esportivas em jogadores de rúgbi chineses. Métodos: visando a seleção nacional de futebol e jogadores de futebol da província, o teste FMS padrão foi usado para coletar os dados e determinar o melhor limite para o escore total do FMS. Resultados: A área sob a curva ROC (AUC) dos atletas em geral, homens e mulheres, foi significativamente diferente da suposição de AUC = 0,5, que foi 0,780 (P = 0,000), 0,877 (P = 0,001), 0,7130 (P = 0,013); Os melhores pontos de corte para o escore total da FMS são 13,5 pontos, 15,5 pontos e 13,5 pontos, respectivamente. O teste do qui-quadrado mostrou que a prevalência do grupo positivo (a pontuação total da FMS foi menor do que o ponto de corte correspondente) foi significativamente maior do que a do grupo negativo (a pontuação total da FMS foi maior do que o ponto de corte correspondente) (P <0,01). Os valores de OR do total de atletas, homens e mulheres, grupos positivos de pontuação total de FMS foram 25,85 (IC 95%: 3,34 ∼ 200,23), 25,00 (IC 95%: 2,36 ∼ 264,80), 14,22 (IC 95%: 1,76 ∼ 114,92). Conclusões: Entre os jogadores de rúgbi chineses, a pontuação total da FMS tem uma forte correlação com lesões esportivas sem contato. A pontuação é de 13,5 para mulheres e 15,5 para homens. Nível de evidência II; Estudos terapêuticos- investigação dos resultados do tratamento.

RESUMEN Introducción: La prueba de movimiento funcional (FMS) es un método de evaluación de los patrones de movimiento básicos del cuerpo humano diseñado por Gray Cook. Objetivo: Este artículo explora el valor de la aplicación de los datos de imágenes biológicas de la prueba de acción funcional (FMS) en la evaluación del riesgo de lesiones deportivas de los jugadores de rugby chinos. Métodos: Tomando como objeto el equipo nacional de fútbol y los jugadores de fútbol provinciales, se utilizó la prueba estándar de FMS para recopilar los datos y determinar el mejor límite para la puntuación total de FMS. Resultados: El área bajo la curva ROC (AUC) de los atletas en general, hombres y mujeres fue significativamente diferente del supuesto de AUC = 0.5, que fue 0.780 (P = 0.000), 0.877 (P = 0.001), 0.7130 (P = 0,013); Los mejores puntos de corte correspondientes a la puntuación total de FMS son 13,5 puntos, 15,5 puntos y 13,5 puntos, respectivamente. La prueba de chi-cuadrado mostró que la prevalencia del grupo positivo (la puntuación total de FMS fue menor que el punto de corte correspondiente) fue significativamente más alta que la del grupo negativo (la puntuación total de FMS fue mayor que el punto de corte correspondiente) (P <0.01). Los valores de OR del total de atletas, hombres y mujeres, grupos positivos de puntuación total de FMS fueron 25,85 (95% CI: 3,34 ∼ 200,23), 25,00 (95% CI: 2,36 ∼ 264,80), 14,22 (95% CI: 1,76 ∼ 114,92). Conclusiones: Entre los jugadores de rugby chinos, la puntuación total de FMS tiene una fuerte correlación con las lesiones de deportes sin contacto. La puntuación es de 13,5 para las mujeres y 15,5 para los hombres. Nivel de evidencia II; Estudios terapéuticos-investigación de los resultados del tratamiento.

Protocol development for discovery of angiogenesis inhibitors via automated methods using zebrafish.

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(kdrl: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.

Bi-allelic GOT2 Mutations Cause a Treatable Malate-Aspartate Shuttle-Related Encephalopathy.

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.

Glutaminase Deficiency Caused by Short Tandem Repeat Expansion in GLS.

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.

Simultaneous ultra-high frequency photoacoustic microscopy and photoacoustic radiometry of zebrafish larvae in vivo.

With their optically transparent appearance, zebrafish larvae are readily imaged with optical-resolution photoacoustic (PA) microscopy (OR-PAM). Previous OR-PAM studies have mapped endogenous chromophores (e.g. melanin and hemoglobin) within larvae; however, anatomical features cannot be imaged with OR-PAM alone due to insufficient optical absorption. We have previously reported on the photoacoustic radiometry (PAR) technique, which can be used simultaneously with OR-PAM to generate images dependent upon the optical attenuation properties of a sample. Here we demonstrate application of the duplex PAR/PA technique for label-free imaging of the anatomy and vasculature of zebrafish larvae in vivo at 200 and 400 MHz ultrasound detection frequencies. We then use the technique to assess the effects of anti-angiogenic drugs on the development of the larval vasculature. Our results demonstrate the effectiveness of simultaneous PAR/PA for acquiring anatomical images of optically transparent samples in vivo, and its potential applications in assessing drug efficacy and embryonic development.

Histone acetyltransferase 7 (KAT7)-dependent intragenic histone acetylation regulates endothelial cell gene regulation.

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.

Development of a zebrafish sepsis model for high-throughput drug discovery.

Sepsis is a leading cause of death worldwide. Current treatment modalities remain largely supportive. Intervention strategies focused on inhibiting specific mediators of the inflammatory host response have been largely unsuccessful, a consequence of an inadequate understanding of the complexity and heterogeneity of the innate immune response. Moreover, the conventional drug development pipeline is time consuming and expensive and the low success rates associated with cell-based screens underline the need for whole organism screening strategies, especially for complex pathological processes. Here, we established an LPS-induced zebrafish endotoxemia model, which exhibits the major hallmarks of human sepsis including, edema and tissue/organ damage, increased vascular permeability and vascular leakage accompanied by an altered expression of cellular junction proteins, increased cytokine expression, immune cell activation and ROS production, reduced circulation and increased platelet aggregation. We tested the suitability of the model for phenotype-based drug screening using three primary readouts: mortality, vascular leakage, and ROS production. Preliminary screening identified fasudil, a drug known to protect against vascular leakage in murine models, as a lead hit thereby validating the utility of our model for sepsis drug screens. This zebrafish sepsis model has the potential to rapidly analyze sepsis associated pathologies and cellular processes in the whole organism, as well as to screen and validate large numbers of compounds that can modify sepsis pathology in vivo.

A Model of Excitotoxic Brain Injury in Larval Zebrafish: Potential Application for High-Throughput Drug Evaluation to Treat Traumatic Brain Injury.

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 < 0.0001) and mean velocity (p < 0.001) with 10 µM glutamate application as measured through automated 96-well plate behavioral analysis. Application of the NMDA receptor antagonist MK-801 (400 nM) or the calpain inhibitor, MDL-28170 (20 µM), resulted in significant recovery of locomotor function. A secA5-YFP transgenic line was used to visualize the localization of cell death due to glutamate exposure in vivo using confocal fluorescence microscopy. Our results indicate that zebrafish larvae exhibit responses to excitotoxic injury and pharmacotherapeutic intervention with pathophysiological relevance to mammalian excitotoxic brain injury. This system has potential to be applied as a high-throughput drug screening model to quickly identify candidate lead compounds for further evaluation.

Chromosome Condensation 1-Like (Chc1L) Is a Novel Tumor Suppressor Involved in Development of Histiocyte-Rich Neoplasms.

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.

Knockdown of ZNF403 inhibits cell proliferation and induces G2/M arrest by modulating cell-cycle mediators.

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.

Expression and characterization of recombinant Rhizopus oryzae lipase for enzymatic biodiesel production.

The Rhizopus oryzae lipase containing prosequence was expressed in Pichia pastoris. Recombinant lipase subunit showed a molecular mass of 32 kDa. The maximum activity of recombinant lipase obtained from Mut(s) recombinant was 90 IU/ml. The enzyme was stable in broad ranges of temperatures and pH, with the optimal temperature at 35 °C and pH 7.0. The crude recombinant R. oryzae lipase can be directly used for the transesterification of plant oils at high-water content of 60-100% (w/w) based on oil weight. The addition of 80% water to the transesterification systems resulted in the yield of methyl ester of 95%, 94% and 92% after 72 h using soybean oil, Jatropha curcas seed raw oil and Pistacia chinensis seed raw oil as raw material, respectively. These results indicate that the recombinant lipase is an effective biocatalyst for enzymatic biodiesel production.

Rosuvastatin, identified from a zebrafish chemical genetic screen for antiangiogenic compounds, suppresses the growth of prostate cancer.

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: 5.87 microM) 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.

Characterisation and biological activities of proanthocyanidins from the barks of Pinus massonian and Acacia mearnsii.

The biological activities and characterisation of proanthocyanidins (PAs) from the barks of Pinus massonian and Acacia mearnsii have been studied in our research. The free-radical scavenging activity of the PAs was measured by means of the DPPH method, and it was clear that the PA product had a strong radical scavenging ability. Furthermore, anti-tumour activities of PAs on different human cancer cells were investigated. The results indicated that PAs from the bark of A. mearnsii had better anti-tumour activities than those from the bark of P. massonian, and the PAs extracted from the ethyl acetate fraction had better anti-tumour activities than those from the water fraction. PAs from the bark of A. mearnsii in an ethyl acetate fraction (PAE) had an effective inhibition on human mammary cancer cells (MDA- MB-231) and human liver cancer cells (BEL-7402), a weak effect on human cervical cancer cells (Hela), but no effect on human lung cancer cells (A549); while the PAs from the bark of A. mearnsii in a water fraction (PAW) had weak effect on MDA- MB-231, Hela and A549, but no effect on BEL-7402. PAs from the bark of P. massonian in ethyl acetate fraction (PPE) had weak effect on Hela and BEL-7402; whereas the PAs from the bark of P. massonian in a water fraction (PPW) had a weak effect on Hela, but no effect on the other cells. PAs were characterised by HPLC- ESI-MS analysis and the PA dimers and trimers were identified, respectively.

Enhanced adaptive immunity in mice lacking the immunoinhibitory adaptor Hacs1.

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.

Effect of digital problem-based learning cases on student learning outcomes in ophthalmology courses.

OBJECTIVE: To assess the impact of digital problem-based learning (PBL) cases on student learning in ophthalmology courses. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

[Effect of epidermal growth factor on expression of cell cycle-regulatory proteins and proliferation of rabbit corneal epithelial cells].

OBJECTIVE: To investigate the effects of epidermal growth factor (EGF) on the proliferation of cultured rabbit corneal epithelial (RCE) cells and the expression of cell cycle-regulatory proteins in these cells. METHODS: It was an experimental study. Cultured RCE cells were exposed to EGF at different concentrations (0, 1, 5, 10, 25 and 50 microg/L) and different time (0, 2, 4, 6, 8 and 10 days). Cell morphologic changes were observed under a phase contrast microscope. Cell proliferation was measured using the WST-1 cell proliferation assay. The expression of cell cycle-regulatory proteins, cyclinD1, CDK4, p27, p21 and p18, was examined using Western blot analysis. The data was statistically analyzed with SPSS 11.5 software, difference between two groups and multiple groups was compared by t test and ANOVA, respectively. RESULTS: Treatment with EGF did not obviously change the cell morphology. EGF significantly induced the proliferation of RCE cells in a dose- and time-dependent manner. In cells treated with different concentrations of EGF, the proliferation rate of 5, 10, 25 and 50 microg/L EGF group at 72 h were 9.0%, 23.5%, 20.8% and 17.7%, respectively. The difference was statistically significant as compared with the control group (F = 45.48, P < 0.01). EGF at 10 microg/L showed maximally stimulating effect. After EGF treatment for 0, 24, 48 and 72 h, the expression of cyclinD1 and CDK4 proteins was markedly increased (cyclinD1/beta-actin ratio was 0.253, 0.591, 0.885 and 1.043; CDK4/beta-actin ratio was 0.422, 0.588, 0.804 and 1.241 at different periods, respectively). Furthermore, p27 protein expression was significantly inhibited by EGF (p27/beta-actin ratio was 0.225, 0.163, 0.107 and 0.082 after EGF treatment for 0, 24, 48 and 72 h, respectively). However, there was no detectable difference in p21 and p18 protein expression between EGF treatment and control groups (p21/beta-actin ratio was 0.432, 0.391, 0.407 and 0.329 and p18/beta-actin ratio was 0.268, 0.274, 0.231 and 0.309, respectively). CONCLUSIONS: This study suggests that EGF could induce RCE cell proliferation. EGF up-regulates cyclinD1 and CDK4 and down-regulates p27 in RCE cells. These changes may be the causes for EGF-induced proliferation of RCE.

Comparative analyses of genomic imprinting and CpG island-methylation in mouse Murr1 and human MURR1 loci revealed a putative imprinting control region in mice.

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.