Identification and characterization of a spore-like morphotype in chronically starved Mycobacterium avium subsp. paratuberculosis cultures

Mycobacteria are able to enter into a state of non-replication or dormancy, which may result in their chronic persistence in soil, aquatic environments, and permissive hosts. Stresses such as nutrient deprivation and hypoxia provide environmental cues to enter a persistent state; however, a clear definition of the mechanism that mycobacteria employ to achieve this remains elusive. While the concept of sporulation in mycobacteria is not novel, it continues to spark controversy and challenges our perceptions of a non-replication. We investigated the potential role of sporulation in one-year old broth cultures of Mycobacterium subsp. paratuberculosis (MAP). We show that dormant cultures of MAP contain a mix of vegetative cells and a previously unknown morphotype resembling a spore. These spore-like structures can be enriched for using sporulating media. Furthermore, purified MAP spore forms survive exposure to heat, lysozyme and proteinase K. Heat-treated spores are positive for MAP 16SrRNA and IS900. MAP spores display enhanced infectivity as well as maintain acid-fast characteristics upon germination in a well-established bovine macrophage model. This is the first study to demonstrate a new MAP morphotype possessing spore-like qualities. Data suggest that sporulation may be a viable mechanism by which MAP accomplishes persistence in the host and/or environment. Thus, our current understanding of mycobacterial persistence, pathogenesis, epidemiology and rational drug and vaccine design may need to be reevaluated.     

Alternaria-Induced Release of IL-18 from Damaged Airway Epithelial Cells: An NF-κB Dependent Mechanism of Th2 Differentiation?

Background

A series of epidemiologic studies have identified the fungus Alternaria as a major risk factor for asthma. The airway epithelium plays a critical role in the pathogenesis of allergic asthma. These reports suggest that activated airway epithelial cells can produce cytokines such as IL-25, TSLP and IL-33 that induce Th2 phenotype. However the epithelium-derived products that mediate the pro-asthma effects of Alternaria are not well characterized. We hypothesized that exposure of the airway epithelium to Alternaria releasing cytokines that can induce Th2 differentiation.

Methodology/Principal Finding

We used ELISA to measure human and mouse cytokines. Alternaria extract (ALT-E) induced rapid release of IL-18, but not IL-4, IL-9, IL-13, IL-25, IL-33, or TSLP from cultured normal human bronchial epithelial cells; and in the BAL fluids of naïve mice after challenge with ALT-E. Both microscopic and FACS indicated that this release was associated with necrosis of epithelial cells. ALT-E induced much greater IL-18 release compared to 19 major outdoor allergens. Culture of naïve CD4 cells with rmIL-18 induced Th2 differentiation in the absence of IL-4 and STAT6, and this effect was abrogated by disrupting NF- κB p50 or with a NEMO binding peptide inhibitor.

Conclusion/Significance

Rapid and specific release of IL-18 from Alternaria-exposed damaged airway epithelial cells can directly initiate Th2 differentiation of naïve CD4⁺ T-cells via a unique NF-κB dependent pathway.     

PET imaging of herpes simplex virus type 1 thymidine kinase (HSV1-tk) or mutant HSV1-sr39tk reporter gene expression in mice and humans using [18F]FHBG

The herpes simplex virus type 1 thymidine kinase (HSV1-tk) positron emission tomography (PET) reporter gene (PRG) or its mutant HSV1-sr39tk are used to investigate intracellular molecular events in cultured cells and to image intracellular molecular events and cell trafficking in living subjects. The expression of these PRGs can be imaged using 18F- or 124I-radiolabeled acycloguanosine or pyrimidine analog PET reporter probes (PRPs). This protocol describes the procedures for imaging HSV1-tk or HSV1-sr39tk PRG expression in living subjects with the acycloguanosine analog 9-4-[18F]fluoro-3-(hydroxymethyl)butyl]guanine ([18F]FHBG). [18F]FHBG is a high-affinity substrate for the HSV1-sr39TK enzyme with relatively low affinity for mammalian TK enzymes, resulting in improved detection sensitivity. Furthermore, [18F]FHBG is approved by the US Food and Drug Administration as an investigational new imaging agent and has been shown to detect HSV1-tk transgene expression in the liver tumors of patients. MicroPET imaging of each small animal can be completed in approximately 1.5 h, and each patient imaging session takes approximately 3 h.     

Effect of light quality on the C-phycoerythrin production in marine cyanobacteria Pseudanabaena sp. isolated from Gujarat coast, India

The isolated cyanobacterium containing biopigments like chlorophyll-a, phycoerythrin, phycocyanin, and carotenoid was cultured under different quality of light modes to ascertain biomass and pigment productivity. On the basis of 16S rRNA gene sequence, the isolate was identified as Pseudanabaena sp. Maximum biomass concentration obtained in white-, blue-, and green-light was 0.82, 0.94, and 0.89 g/L, respectively. It was observed that maximum phycoerythrin production was in green light (39.2 mg/L), ensued by blue light (32.2 mg/L), while phycocyanin production was maximum in red light (10.9 mg/L). In yellow light, pigment production as well as the growth rate gradually declined after 12 days. Carotenoid production decreased in blue-, white-, and red-light after 15 days, while in green light it had increased gradually. The present communication suggests that Pseudanabaena sp. can be used for commercial production of phycoerythrin when grown under green light.     

Quinazolinones as γ-secretase modulators

Synthesis, SAR and evaluation of styrenyl quinazolinones as novel gamma secretase modulators are presented in this communication. Starting from literature and in-house leads we evaluated a range of quinazolinones which showed good modulation of γ-secretase activity.     

Analysis of a Panel of 48 Cytokines in BAL Fluids Specifically Identifies IL-8 Levels as the Only Cytokine that Distinguishes Controlled Asthma from Uncontrolled Asthma,and Correlates Inversely with FEV1

We sought to identify cells and cytokines in bronchoalveolar lavage (BAL) fluids that distinguish asthma from healthy control subjects and those that distinguish controlled asthma from uncontrolled asthma. Following informed consent, 36 human subjects were recruited for this study. These included 11 healthy control subjects, 15 subjects with controlled asthma with FEV₁≥80% predicted and 10 subjects with uncontrolled asthma with FEV₁ <80% predicted. BAL fluid was obtained from all subjects. The numbers of different cell types and the levels of 48 cytokines were measured in these fluids. Compared to healthy control subjects, patients with asthma had significantly more percentages of eosinophils and neutrophils, IL-1RA, IL-1α, IL-1β, IL-2Rα, IL-5, IL-6, IL-7, IL-8, G-CSF, GROα (CXCL1), MIP-1β (CCL4), MIG (CXCL9), RANTES (CCL5) and TRAIL in their BAL fluids. The only inflammatory markers that distinguished controlled asthma from uncontrolled asthma were neutrophil percentage and IL-8 levels, and both were inversely correlated with FEV₁. We examined whether grouping asthma subjects on the basis of BAL eosinophil % or neutrophil % could identify specific cytokine profiles. The only differences between neutrophil-normal asthma (neutrophil≤2.4%) and neutrophil-high asthma (neutrophils%>2.4%) were a higher BAL fluid IL-8 levels, and a lower FEV₁ in the latter group. By contrast, compared to eosinophil-normal asthma (eosinophils≤0.3%), eosinophil-high asthma (eosinophils>0.3%) had higher levels of IL-5, IL-13, IL-16, and PDGF-bb, but same neutrophil percentage, IL-8, and FEV₁. Our results identify neutrophils and IL-8 are the only inflammatory components in BAL fluids that distinguish controlled asthma from uncontrolled asthma, and both correlate inversely with FEV₁.     

Genome-Wide Analysis of Copy Number Variation Identifies Candidate Gene Loci Associated with the Progression of Non-Alcoholic Fatty Liver Disease

Between 10 and 25% of individuals with non-alcoholic fatty liver disease (NAFLD) develop hepatic fibrosis leading to cirrhosis and hepatocellular carcinoma (HCC). To investigate the molecular basis of disease progression, we performed a genome-wide analysis of copy number variation (CNV) in a total of 49 patients with NAFLD [10 simple steatosis and 39 non-alcoholic steatohepatitis (NASH)] and 49 matched controls using high-density comparative genomic hybridization (CGH) microarrays. A total of 11 CNVs were found to be unique to individuals with simple steatosis, whilst 22 were common between simple steatosis and NASH, and 224 were unique to NASH. We postulated that these CNVs could be involved in the pathogenesis of NAFLD progression. After stringent filtering, we identified four rare and/or novel CNVs that may influence the pathogenesis of NASH. Two of these CNVs, located at 13q12.11 and 12q13.2 respectively, harbour the exportin 4 (XPO4) and phosphodiesterase 1B (PDE1B) genes which are already known to be involved in the etiology of liver cirrhosis and HCC. Cross-comparison of the genes located at these four CNV loci with genes already known to be associated with NAFLD yielded a set of genes associated with shared biological processes including cell death, the key process involved in ‘second hit’ hepatic injury. To our knowledge, this pilot study is the first to provide CNV information of potential relevance to the NAFLD spectrum. These data could prove invaluable in predicting patients at risk of developing NAFLD and more importantly, those who will subsequently progress to NASH.     

Patient-specific modeling of dyssynchronous heart failure: A case study

The development and clinical use of patient-specific models of the heart is now a feasible goal. Models have the potential to aid in diagnosis and support decision-making in clinical cardiology. Several groups are now working on developing multi-scale models of the heart for understanding therapeutic mechanisms and better predicting clinical outcomes of interventions such as cardiac resynchronization therapy. Here we describe the methodology for generating a patient-specific model of the failing heart with a myocardial infarct and left ventricular bundle branch block. We discuss some of the remaining challenges in developing reliable patient-specific models of cardiac electromechanical activity, and identify some of the main areas for focusing future research efforts. Key challenges include: efficiently generating accurate patient-specific geometric meshes and mapping regional myofiber architecture to them; modeling electrical activation patterns based on cellular alterations in human heart failure, and estimating regional tissue conductivities based on clinically available electrocardiographic recordings; estimating unloaded ventricular reference geometry and material properties for biomechanical simulations; and parameterizing systemic models of circulatory dynamics from available hemodynamic measurements.     

Ultrasonic radiation induced lipid peroxidation in liposomal membrane

Summary Ultrasonic radiation produced a dose dependent linear increase in lipid peroxidation (MDA formation) in the liposomal membrane. The yield of MDA was significantly inhibited by butylated hydroxytoluene (BHT), the antioxidant, sodium formate, the OH radical scavenger, and EDTA, the metal ion chelator. Ascorbic acid at low concentration increased the ultrasonic induced MDA formation while high concentrations inhibited lipid peroxidation. A mechanism of ultrasound induced lipid peroxidation is suggested.