Secretion of the Pasteurella leukotoxin by Escherichia coli

Abstract Nucleic acid sequence analysis has indicated that the leukotoxin determinant from Pasteurella haemolytica is related to the hemolysin determinant from E. coli . The cloning and expression in E. coli of the lktCA genes has been previously reported, but the existence of leukotoxin secretory genes equivalent to hlyBD has not been documented. In this report we demonstrate that a 4.0 kb segment of P. haemolytica genomic DNA distal to the lktA gene, when expressed in trans to the previous cloned lktCA genes, allow the synthesis and secretion of active leukotoxin from E. coli . Complementation analysis using the cloned hlyB and hlyD genes indicates that this secretory locus derived from P. haemolytica contains two genes which we designate, by analogy, lktB and lktD .     

Cerebrospinal Fluid Levels of Amyloid Precursor Protein Are Associated with Ventricular Size in Post-Hemorrhagic Hydrocephalus of Prematurity

Background

Neurological outcomes of preterm infants with post-hemorrhagic hydrocephalus (PHH) remain among the worst in infancy, yet there remain few instruments to inform the treatment of PHH. We previously observed PHH-associated elevations in cerebrospinal fluid (CSF) amyloid precursor protein (APP), neural cell adhesion molecule-L1 (L1CAM), neural cell adhesion molecule-1 (NCAM-1), and other protein mediators of neurodevelopment.

Objective

The objective of this study was to examine the association of CSF APP, L1CAM, and NCAM-1 with ventricular size as an early step toward developing CSF markers of PHH.

Methods

CSF levels of APP, L1CAM, NCAM-1, and total protein (TP) were measured in 12 preterm infants undergoing PHH treatment. Ventricular size was determined using cranial ultrasounds. The relationships between CSF APP, L1CAM, and NCAM-1, occipitofrontal circumference (OFC), volume of CSF removed, and ventricular size were examined using correlation and regression analyses.

Results

CSF levels of APP, L1CAM, and NCAM-1 but not TP paralleled treatment-related changes in ventricular size. CSF APP demonstrated the strongest association with ventricular size, estimated by frontal-occipital horn ratio (FOR) (Pearson R = 0.76, p = 0.004), followed by NCAM-1 (R = 0.66, p = 0.02) and L1CAM (R = 0.57,p = 0.055). TP was not correlated with FOR (R = 0.02, p = 0.95).

Conclusions

Herein, we report the novel observation that CSF APP shows a robust association with ventricular size in preterm infants treated for PHH. The results from this study suggest that CSF APP and related proteins at once hold promise as biomarkers of PHH and provide insight into the neurological consequences of PHH in the preterm infant.     

Incidence and nature of testicular toxicity findings in pharmaceutical development

BACKGROUND : Testicular toxicity (TT) is a sporadic and challenging issue in pharmaceutical drug development. Efforts to develop TT screening assays or biomarkers have been overshadowed by consortium efforts to predict drug‐induced toxicities such as hepatic injury, which are encountered more frequently. METHODS : To gauge the current state of the field and to prioritize future TT activities, the International Life Sciences Institute‐Health and Environmental Sciences Institute Developmental and Reproductive Toxicology (DART) Technical Committee sponsored a survey to better understand the incidence and nature of TT findings encountered during drug development. RESULTS : Highlights from the 16 survey respondents include: (1) Although preclinical TT was encountered relatively infrequently, half of the participants observed repeated problems with TT during pharmaceutical development, (2) despite control measures such as use of sexually mature animals to diminish confounding effects of spurious lesions, interpretation of TT remains a challenge, (3) “traditional” evaluation tools such as hormonal monitoring and newer approaches such as ‐omics are utilized to investigate testicular changes, and (4) an understanding of the risk and relevance of TT findings is achieved through joint consideration of factors such as species specificity, potential mode of action, and safety margins. CONCLUSIONS : TT remains a relatively uncommon but persistent challenge in pharmaceutical development. Although current preclinical TT approaches appear to be effective in limiting the occurrence of pharmaceutical candidate attrition in clinical trials, improved biomarker or screening platforms would allow companies to identify TT at an earlier stage, thus decreasing the time and resources expended on safety evaluation of pharmaceutical candidates. Birth Defects Res (Part B) 92: 511–525, 2011. © 2011 Wiley Periodicals, Inc.     

Distinct expression patterns of CD69 in mucosal and systemic lymphoid tissues in primary SIV infection of rhesus macaques

Although the intestinal tract plays a major role in early human immunodeficiency virus (HIV) infection, the role of immune activation and viral replication in intestinal tissues is not completely understood. Further, increasing evidence suggests the early leukocyte activation antigen CD69 may be involved in the development or regulation of important T cell subsets, as well as a major regulatory molecule of immune responses. Using the simian immunodeficiency virus (SIV) rhesus macaque model, we compared expression of CD69 on T cells from the intestine, spleen, lymph nodes, and blood of normal and SIV-infected macaques throughout infection. In uninfected macaques, the majority of intestinal lamina propria CD4+ T cells had a memory (CD95+) phenotype and co-expressed CD69, and essentially all intestinal CCR5+ cells co-expressed CD69. In contrast, systemic lymphoid tissues had far fewer CD69+ T cells, and many had a naïve phenotype. Further, marked, selective depletion of intestinal CD4+CD69+ T cells occurred in early SIV infection, and this depletion persisted throughout infection. Markedly increased levels of CD8+CD69+ T cells were detected after SIV infection in virtually all tissues, including the intestine. Further, confocal microscopy demonstrated selective, productive infection of CD3+CD69+ T cells in the intestine in early infection. Combined, these results indicate CD69+CD4+ T cells are a major early target for viral infection, and their rapid loss by direct infection may have profound effects on intestinal immune regulation in HIV infected patients.     

Gene expression analysis uncovers novel hedgehog interacting protein (HHIP) effects in human bronchial epithelial cells

Hedgehog interacting protein (HHIP) was implicated in chronic obstructive pulmonary disease (COPD) by genome-wide association studies (GWAS). However, it remains unclear how HHIP contributes to COPD pathogenesis. To identify genes regulated by HHIP, we performed gene expression microarray analysis in a human bronchial epithelial cell line (Beas-2B) stably infected with HHIP shRNAs. HHIP silencing led to differential expression of 296 genes; enrichment for variants nominally associated with COPD was found. Eighteen of the differentially expressed genes were validated by real-time PCR in Beas-2B cells. Seven of 11 validated genes tested in human COPD and control lung tissues demonstrated significant gene expression differences. Functional annotation indicated enrichment for extracellular matrix and cell growth genes. Network modeling demonstrated that the extracellular matrix and cell proliferation genes influenced by HHIP tended to be interconnected. Thus, we identified potential HHIP targets in human bronchial epithelial cells that may contribute to COPD pathogenesis.     

A biofilm model developed to investigate survival and disinfection of Mycobacterium mucogenicum in potable water

Water in healthcare environments can be a source for healthcare-associated infections (HAI). However, information on the exposure risk to opportunistic pathogens in potable water distribution systems (PWDS) is lacking. Laboratory studies characterizing the interaction of opportunistic pathogens with biofilms are needed to understand their role in water systems within healthcare facilities. A stable, repeatable, PWDS multi-species biofilm model comprising Sphingomonas paucimobilis, Methylobacterium sp., Delftia acidovorans, and Mycobacterium mucogenicum was developed in the CDC Biofilm Reactor (CBR), reaching 6 log₁₀ CFU cm^?2 within 6 days. The model was used to investigate the interaction of the opportunistic pathogen M. mucogenicum with the other species, and to determine the efficacy of monochloramine (NH₂Cl) as a disinfectant against 2-week-old biofilms. Addition of 1 or 2 mg l^?1 NH₂Cl resulted in the same or an increased log density of viable M. mucogenicum in the biofilm while inactivating some of the Proteobacteria. Although M. mucogenicum preferentially resided in the biofilm, NH₂Cl exposure caused release of viable M. mucogenicum from the biofilm into the water. Additional studies with this model should determine if sodium hypochlorite has a comparative effect and if other nontuberculous mycobacteria (NTM) respond to NH₂Cl similarly.     

Juveniles Are More Resistant to Warming than Adults in 4 Species of Antarctic Marine Invertebrates

Juvenile stages are often thought to be less resistant to thermal challenges than adults, yet few studies make direct comparisons using the same methods between different life history stages. We tested the resilience of juvenile stages compared to adults in 4 species of Antarctic marine invertebrate over 3 different rates of experimental warming. The species used represent 3 phyla and 4 classes, and were the soft-shelled clam Laternula elliptica, the sea cucumber Cucumaria georgiana, the sea urchin Sterechinus neumayeri, and the seastar Odontaster validus. All four species are widely distributed, locally abundant to very abundant and are amongst the most important in the ecosystem for their roles. At the slowest rate of warming used (1°C 3 days⁻¹) juveniles survived to higher temperatures than adults in all species studied. At the intermediate rate (1°C day⁻¹) juveniles performed better in 3 of the 4 species, with no difference in the 4^th, and at the fastest rate of warming (1°C h⁻¹) L. elliptica adults survived to higher temperatures than juveniles, but in C. georgiana juveniles survived to higher temperatures than adults and there were no differences in the other species. Oxygen limitation may explain the better performance of juveniles at the slower rates of warming, whereas the loss of difference between juveniles and adults at the fastest rate of warming suggests another mechanism sets the temperature limit here.     

Gene Expression Signature of Normal Cell-of-Origin Predicts Ovarian Tumor Outcomes

The potential role of the cell-of-origin in determining the tumor phenotype has been raised, but not adequately examined. We hypothesized that distinct cells-of-origin may play a role in determining ovarian tumor phenotype and outcome. Here we describe a new cell culture medium for in vitro culture of paired normal human ovarian (OV) and fallopian tube (FT) epithelial cells from donors without cancer. While these cells have been cultured individually for short periods of time, to our knowledge this is the first long-term culture of both cell types from the same donors. Through analysis of the gene expression profiles of the cultured OV/FT cells we identified a normal cell-of-origin gene signature that classified primary ovarian cancers into OV-like and FT-like subgroups; this classification correlated with significant differences in clinical outcomes. The identification of a prognostically significant gene expression signature derived solely from normal untransformed cells is consistent with the hypothesis that the normal cell-of-origin may be a source of ovarian tumor heterogeneity and the associated differences in tumor outcome.     

Scleral Micro-RNA Signatures in Adult and Fetal Eyes

Introduction

In human eyes, ocular enlargement/growth reflects active extracellular matrix remodeling of the outer scleral shell. Micro-RNAs are small non-coding RNAs that regulate gene expression by base pairing with target sequences. They serve as nodes of signaling networks. We hypothesized that the sclera, like most tissues, expresses micro-RNAs, some of which modulate genes regulating ocular growth. In this study, the scleral micro-RNA expression profile of rapidly growing human fetal eyes was compared with that of stable adult donor eyes using high-throughput microarray and quantitative PCR analyses.

Methods

Scleral samples from normal human fetal (24 wk) and normal adult donor eyes were obtained (n=4 to 6, each group), and RNA extracted. Genome-wide micro-RNA profiling was performed using the Agilent micro-RNA microarray platform. Micro-RNA target predictions were obtained using Microcosm, TargetScan and PicTar algorithms. TaqMan® micro-RNA assays targeting micro-RNAs showing either highest significance, detection, or fold differences, and collagen specificity, were applied to scleral samples from posterior and peripheral ocular regions (n=7, each group). Microarray data were analyzed using R, and quantitative PCR data with 2^-deltaCt methods.

Results

Human sclera was found to express micro-RNAs, and comparison of microarray results for adult and fetal samples revealed many to be differentially expressed (p<0.01, min p= 6.5x10¹¹). Specifically, fetal sclera showed increased expression of mir-214, let-7c, let-7e, mir-103, mir-107, and mir-98 (1.5 to 4 fold changes, p<0.01). However, no significant regionally specific differences .i.e., posterior vs. peripheral sclera, were observed for either adult or fetal samples.

Conclusion

For the first time, micro-RNA expression has been catalogued in human sclera. Some micro-RNAs show age-related differential regulation, higher in the sclera of rapidly growing fetal eyes, consistent with a role in ocular growth regulation. Thus micro-RNAs represent potential targets for ocular growth manipulation, related to myopia and/or other disorders such as scleral ectasia.     

Discovery of 5-phenoxy-1,3-dimethyl-1H-pyrazole-4-carboxamides as potent agonists of TGR5 via sequential combinatorial libraries

Optimization of a high-throughput screening hit led to the discovery of a new series of 5-phenoxy-1,3-dimethyl-1H-pyrazole-4-carboxamides as highly potent agonists of TGR5. This novel chemotype was rapidly developed through iterative combinatorial library synthesis. It was determined that in vitro agonist potency correlated with functional activity data from human peripheral blood monocytes.