Evaluation of the Antifungal Activities of Photorhabdus akhurstii and Its Secondary Metabolites against Phytopathogenic Colletotrichum gloeosporioides.

Colletotrichum gloeosporioides is a phytopathogenic fungus that causes devastating losses in strawberries without effective countermeasures. Members of the genus Photorhabdus exhibit antimicrobial capability and have been found to have the potential for use as biocontrol agents against C. gloeosporioides. Photorhabdus species exhibit two phase variations with a differentiated composition of secondary metabolites designated to each phase. In this study, Photorhabdus akhurstii sp. nov. 0813-124 exhibited phase I (PL1) and phase II (PL2); however, only PL1 displayed distinct inhibition of C. gloeosporioides in the confrontation assay. We identified the bioactive ingredients of P. akhurstii sp. nov. 0813-124 to be glidobactin A and cepafungin I, with MIC values lower than 1.5 and 2.0 µg/mL, respectively. Furthermore, we revealed the biosynthetic gene cluster (BGC) of corresponding bioactive molecules through genomics analysis and determined its expression level in PL1 and PL2. The expression of glidobactin BGC in PL1 increased rapidly within 24 h, while PL2 was eventually stimulated after 60 h. In summary, we demonstrated that P. akhurstii sp. nov. 0813-124 could potentially be used as a biocontrol agent or part of a natural product repertoire for combating C. gloeosporioides.

Investigating the Reciprocal Interrelationships among the Ruminal Microbiota, Metabolome, and Mastitis in Early Lactating Holstein Dairy Cows.

Mastitis in dairy cow significantly affects animal performance, ultimately reducing profitability. The reciprocal interrelationships among ruminal microbiota, metabolome, and mastitis combining early inflammatory factors (serum proinflammatory cytokines) in lactating dairy cows has not been explored, thus, this study evaluated these reciprocal interrelationships in early lactating Holstein dairy cows to identify potential microbial biomarkers and their relationship with ruminal metabolites. The ruminal fluid was sampled from 8 healthy and 8 mastitis cows for the microbiota and metabolite analyses. The critical ruminal microbial biomarkers and metabolites related to somatic cell counts (SCC) and serum proinflammatory cytokines were identified by the linear discriminant analysis effect size (LEfSe) algorithm and Spearman's correlation analysis, respectively. The SCC level and proinflammatory cytokines positively correlated with Sharpea and negatively correlated with Ruminococcaceae UCG-014, Ruminococcus flavefaciens, and Treponema saccharophilum. Furthermore, the metabolites xanthurenic acid, and 1-(1H-benzo[d]imidazol-2-yl) ethan-1-ol positively correlated with microbial biomarkers of healthy cows, whereas, xanthine, pantothenic acid, and anacardic acid were negatively correlated with the microbial biomarkers of mastitis cows. In conclusion, Ruminococcus flavefaciens and Treponema saccharophilum are potential strains for improving the health of dairy cows. The current study provides a novel perspective to assist in targeting the ruminal microbiota with preventive/therapeutic strategies against inflammatory diseases in the future.

The Rumen Specific Bacteriome in Dry Dairy Cows and Its Possible Relationship with Phenotypes.

Most microbiome studies of dairy cows have investigated the compositions and functions of rumen microbial communities in lactating dairy cows. The importance of the relationships among hosts, microbiota, diet composition, and milk production remains unknown in dry dairy cows. Thus, in the present study, the composition of the rumen microbiome in cows from three dairy farms was investigated to identify core bacteria contributing to various physiological roles during rumen fermentation in dry dairy cows. The results indicated that ruminal fluid in dry dairy cows from different regional farms had core rumen microbiota that could be clearly distinguished from that of cows of the other farms. Further identification of key microorganisms associated with each farm revealed that Prevotella, Methanobrevibacter, Pseudobutyrivibrio, Ruminococcus, Bacteroides, and Streptococcus were major contributors. Spearman's correlation indicated that the abundance of genera such as Prevotella and Ruminococcus in dry dairy cows could indicate milk yield in the previous lactating period. Functional pathway analysis of the rumen bacterial communities demonstrated that amino acid metabolism and carbohydrate metabolism were the major pathways. Our findings provide knowledge of the composition and predicted functions of rumen microbiota in dry dairy cows from regional farms, which underscore the importance of the relationships among hosts, microbiota, diet composition, and milk production.

Down-regulation of ATF1 leads to early neuroectoderm differentiation of human embryonic stem cells by increasing the expression level of SOX2.

We reveal by high-throughput screening that activating transcription factor 1 (ATF1) is a novel pluripotent regulator in human embryonic stem cells (hESCs). The knockdown of ATF1 expression significantly up-regulated neuroectoderm (NE) genes but not mesoderm, endoderm, and trophectoderm genes. Of note, down-regulation or knockout of ATF1 with short hairpin RNA (shRNA), small interfering RNA (siRNA), or clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) was sufficient to up-regulate sex-determining region Y-box (SOX)2 and paired box 6 (PAX6) expression under the undifferentiated or differentiated conditions, whereas overexpression of ATF1 suppressed NE differentiation. Endogenous ATF1 was spontaneously down-regulated after d 1-3 of neural induction. By double-knockdown experiments, up-regulation of SOX2 was critical for the increase of PAX6 and SOX1 expression in shRNA targeting Atf1 hESCs. Using the luciferase reporter assay, we identified ATF1 as a negative transcriptional regulator of Sox2 gene expression. A novel function of ATF1 was discovered, and these findings contribute to a broader understanding of the very first steps in regulating NE differentiation in hESCs.-Yang, S.-C., Liu, J.-J., Wang, C.-K., Lin, Y.-T., Tsai, S.-Y., Chen, W.-J., Huang, W.-K., Tu, P.-W. A., Lin, Y.-C., Chang, C.-F., Cheng, C.-L., Lin, H., Lai, C.-Y., Lin, C.-Y., Lee, Y.-H., Chiu, Y.-C., Hsu, C.-C., Hsu, S.-C., Hsiao, M., Schuyler, S. C., Lu, F. L., Lu, J. Down-regulation of ATF1 leads to early neuroectoderm differentiation of human embryonic stem cells by increasing the expression level of SOX2.

Expected Sonographic Appearance of the Spleen in Children and Young Adults With Sickle Cell Disease: An Update.

OBJECTIVES: To update the imaging literature regarding spleen appearances in young patients with sickle cell disease (SCD). METHODS: We conducted a retrospective study and included 112 patients age 0 to 21 years with SCD who had at least 1 abdominal sonogram at our institution between 1999 and 2011. Radiologic findings were compared between risk groups by χ(2) analysis. Findings were correlated with other imaging modalities when available. RESULTS: In our cohort, 35.7% of patients had autosplenectomy, and 8.0% had undergone surgical splenectomy. Only 5.0% of individuals age 0 to 5 years had autosplenectomy. In those who had not undergone surgical splenectomy or autosplenectomy, 76.2% had echogenic spleens, heterogeneous-appearing spleens, or both, and patients with the homozygous sickle cell anemia (HbSS) genotype were more likely to have an abnormal spleen echo texture. Patients treated with transfusions had echogenic spleens and had a higher frequency of splenic regeneration nodules. Most patients (80%) with splenomegaly did not require surgical splenectomy after 5.7 years of follow-up. CONCLUSIONS: Twenty years ago, children with HbSS SCD were expected to have autosplenectomy by age 5 years. There have been changes in the radiologic appearance of the spleen in patients with SDC, likely due to improved supportive care and the use of acute and chronic transfusion therapy. We found that autosplenectomy is rare by age 5 years, and during childhood and adolescence, the spleen typically appears echogenic, heterogeneous, or both, depending on disease severity.

Implications of radiologic-pathologic correlation for gallbladder disease in children and young adults with sickle cell disease.

The purpose of this study is to describe gallbladder imaging findings in patients with sickle cell disease, and to determine how they correspond with occurrence of complications, need for cholecystectomy, and surgical pathology. This study is IRB approved and HIPAA compliant. Informed consent requirements were waived. We reviewed records of 77 children with sickle cell disease ages 0-18 years at the time of their first gallbladder imaging study. Demographics, hospital courses, and radiologic and pathologic reports were collected. Two pediatric radiologists independently and retrospectively reviewed the imaging studies. Statistical analysis was performed using kappa statistic, chi-squared test, and ANOVA F-test. Continuous variables were described with mean, median, variance, and range. Patients who underwent cholecystectomy (N = 25) were more likely than the patients who did not undergo cholecystectomy (N = 52) to have gallstones or sludge (100 versus 36.5 %, p = <0.0001) or other gallbladder or biliary abnormality (70.8 versus 1.9 %, p = <0.0001). Patients who did not undergo cholecystectomy more frequently had normal-appearing gallbladders and biliary tracts (63.5 versus 0 %, p = <0.0001). Ninety-two percent of patients with cholecystectomy had chronic cholecystitis on pathology, and 96 % had a complication, including chronic cholecystitis and sequelae of biliary obstruction. Young patients with sickle cell disease, cholelithiasis, and any other biliary imaging abnormality will almost certainly require cholecystectomy, and many will experience complications. The most common surgical pathologic diagnosis in this group is chronic cholecystitis, which has a variable radiologic appearance. Our findings support recommendations to perform elective cholecystectomy for children and young adults with sickle cell disease and cholelithiasis or gallbladder sludge.

Novel strategies for improving physical function.

BACKGROUND: The number of elderly people is growing worldwide, and a substantial proportion of them report difficulty with mobility and in performing activities of daily living. The demographic trends have created enormous interest in development of function-promoting anabolic therapies. At present, androgens and myostatin inhibitors are the two leading classes of function-promoting anabolic therapies that are the most advanced in the drug development process. However, clinical development has been slowed by vexing trial design issues. CONCLUSIONS: Clinical investigators, regulatory agencies and the National Institutes of Health must continue working toward a consensus about the indications for study, efficacy outcomes and what differences in functional outcomes are needed to determine sample size estimates.

Blockade of PAR1 signaling with cell-penetrating pepducins inhibits Akt survival pathways in breast cancer cells and suppresses tumor survival and metastasis.

Protease-activated receptor 1 (PAR1) is a G protein-coupled receptor that is not expressed in normal breast epithelia but is up-regulated in invasive breast carcinomas. In the present study, we found that matrix metalloprotease-1 (MMP-1) robustly activates the PAR1-Akt survival pathway in breast carcinoma cells. This process is blocked by a cell-penetrating lipopeptide "pepducin," P1pal-7, which is a potent inhibitor of cell viability in breast carcinoma cells expressing PAR1. Both a MMP-1 inhibitor and P1pal-7 significantly promote apoptosis in breast tumor xenografts and inhibit metastasis to the lungs by up to 88%. Dual therapy with P1pal-7 and Taxotere inhibits the growth of MDA-MB-231 xenografts by 95%. Consistently, biochemical analysis of xenograft tumors treated with P1pal-7 or MMP-1 inhibitor showed attenuated Akt activity. Ectopic expression of constitutively active Akt rescues breast cancer cells from the synergistic cytotoxicity of P1pal-7 and Taxotere, suggesting that Akt is a critical component of PAR1-dependent cancer cell viability. Together, these findings indicate that blockade of MMP1-PAR1 signaling may provide a benefit beyond treatment with Taxotere alone in advanced, metastatic breast cancer.

Genetic disruption of myostatin reduces the development of proatherogenic dyslipidemia and atherogenic lesions in Ldlr null mice.

OBJECTIVE: Insulin-resistant states, such as obesity and type 2 diabetes, contribute substantially to accelerated atherogenesis. Null mutations of myostatin (Mstn) are associated with increased muscle mass and decreased fat mass. In this study, we determined whether Mstn disruption could prevent the development of insulin resistance, proatherogenic dyslipidemia, and atherogenesis. RESEARCH DESIGN AND METHODS: C57BL/6 Ldlr(-/-) mice were cross-bred with C57BL/6 Mstn(-/-) mice for >10 generations to generate Mstn(-/-)/Ldlr(-/-) double-knockout mice. The effects of high-fat/high-cholesterol diet on body composition, plasma lipids, systemic and tissue-specific insulin sensitivity, hepatic steatosis, as well as aortic atheromatous lesion were characterized in Mstn(-/-)/Ldlr(-/-) mice in comparison with control Mstn(+/+)/Ldlr(-/-) mice. RESULTS: Compared with Mstn(+/+)/Ldlr(-/-) controls, Mstn(-/-)/ Ldlr(-/-) mice were resistant to diet-induced obesity, and had greatly improved insulin sensitivity, as indicated by 42% higher glucose infusion rate and 90% greater muscle [(3)H]-2-deoxyglucose uptake during hyperinsulinemic-euglycemic clamp. Mstn(-/-)/Ldlr(-/-) mice were protected against diet-induced hepatic steatosis and had 56% higher rate of hepatic fatty acid beta-oxidation than controls. Mstn(-/-)/Ldlr(-/-) mice also had 36% lower VLDL secretion rate and were protected against diet-induced dyslipidemia, as indicated by 30-60% lower VLDL and LDL cholesterol, free fatty acids, and triglycerides. Magnetic resonance angiography and en face analyses demonstrated 41% reduction in aortic atheromatous lesions in Ldlr(-/-) mice with Mstn deletion. CONCLUSIONS: Inactivation of Mstn protects against the development of insulin resistance, proatherogenic dyslipidemia, and aortic atherogenesis in Ldlr(-/-) mice. Myostatin may be a useful target for drug development for prevention and treatment of obesity and its associated type 2 diabetes and atherosclerosis.

Airway epithelial STAT3 is required for allergic inflammation in a murine model of asthma.

The STAT3 transcription factor is critical for cytokine signaling and the acute phase response, but its role in allergic asthma is largely undefined. To investigate the role of STAT3 in mediating allergic inflammation, we used chemical and genetic approaches to inactivate STAT3 in the airway epithelium of mice. In a murine model of chronic asthma, we demonstrate that the administration of house dust mite (HDM) leads to robust STAT3 activation in the airway epithelium, smooth muscle, and immune cells in the lungs of C57BL/6 mice. To investigate the role of STAT3 in HDM-induced airway inflammation, a conditional knockout of STAT3 in the airway epithelium was generated, e-STAT3-/-. We determined that e-STAT3-/- mice had a significant decrease in HDM-induced airway eosinophilia, lung Th2 accumulation, and chemokines compared with wild-type animals. Importantly, the e-STAT3-/- mice had a significant decrease in airway hyperresponsiveness to methacholine. The administration of two STAT kinase inhibitors diminished STAT3 activation and markedly abrogated the HDM-induced lung inflammation. These findings suggest that STAT3 acts as a novel epithelial regulator of the allergic response by altering Th2 cell recruitment and effector function, and thus, targeting this molecule may provide the basis for a novel asthma therapy.

Inhibition of the Src and Jak kinases protects against lipopolysaccharide-induced acute lung injury.

The cascade of cellular and molecular pathways mediating acute lung injury is complex and incompletely defined. Although the Src and Jak family of kinases is upregulated in LPS-induced murine lung injury, their role in the development of lung injury is unknown. Here we report that systemic inhibition of these kinases using specific small molecule inhibitors (PP2, SU6656, tyrphostin A1) significantly attenuated LPS-induced lung injury, as determined by histologic and capillary permeability assays. These inhibitors blocked LPS-dependent cytokine and chemokine production in the lung and in the serum. In contrast, lung-targeted inhibition of these kinases in the airway epithelium via adenoviral-mediated gene transfer of dominant negative Src or of suppressor of cytokine signaling (SOCS-1) disrupted lung cytokine production but had no effect on systemic cytokine production or lung vascular permeability. Mice were significantly protected from lethal LPS challenge by the small molecule inhibitors of Jak and Src kinase. Importantly, this protection was still evident even when the inhibitors were administered 6 hours after LPS challenge. Taken together, these observations suggest that Jak and Src kinases participate in acute lung injury and verify the potential of this class of selective tyrosine kinase inhibitors to serve as novel therapeutic agents for this disease.