Tubulin Polymerization Promoting Protein Affects the Circadian Timing System in C57Bl/6 Mice

The circadian timing system (CTS) is a complex set of cyclic cellular mechanisms which serve to synchronize discrete cell groups across multiple organ systems to adapt the body’s physiology to a (roughly) 24-hour clock. Many genes and hormones have been shown to be strongly associated with the CTS, some of which include the genes 'Bmal1, Period1, Period2, Cryptochrome1', and 'Cryptochrome2', and the hormone melatonin. Previous data suggest that microtubule dynamics play an important role in melatonin function as it relates to the CTS in vitro, though this relationship has never been explored in vivo. The purpose of this study was to determine whether disruption of microtubule regulation in C57Bl/6 mice results in measurable changes to the CTS. To study the potential effects of microtubule dynamics on the CTS in vivo, we utilized a mouse model of microtubule instability, knocked out for the tubulin polymerization promoting protein gene ('Tppp' -/-), comparing them to their wild type (WT) littermates in three categories: locomotor activity (in light/dark and dark/dark photoperiods), serial clock gene expression, and serial serum melatonin concentration. These comparisons showed differences in all three categories, including significant differences in locomotor characteristics under dark/dark conditions. Our findings support and extend previous reports that microtubule dynamics are a modulator of circadian rhythm regulation likely through a mechanism involving melatonin induced phase shifting

Members of Marinobacter and Arcobacter Influence System Biogeochemistry During Early Production of Hydraulically Fractured Natural Gas Wells in the Appalachian Basin

Hydraulic fracturing is the prevailing method for enhancing recovery of hydrocarbon resources from unconventional shale formations, yet little is understood regarding the microbial impact on biogeochemical cycling in natural-gas wells. Although the metabolisms of certain fermentative bacteria and methanogenic archaea that dominate in later produced fluids have been well studied, few details have been reported on microorganisms prevelant during the early flowback period, when oxygen and other surface-derived oxyanions and nutrients become depleted. Here, we report the isolation, genomic and phenotypic characterization of Marinobacter and Arcobacter bacterial species from natural-gas wells in the Utica-Point Pleasant and Marcellus Formations coupled to supporting geochemical and metagenomic analyses of produced fluid samples. These unconventional hydrocarbon system-derived Marinobacter sp. are capable of utilizing a diversity of organic carbon sources including aliphatic and aromatic hydrocarbons, amino acids, and carboxylic acids. Marinobacter and Arcobacter can metabolize organic nitrogen sources and have the capacity for denitrification and dissimilatory nitrate reduction to ammonia (DNRA) respectively; with DNRA and ammonification processes partially explaining high concentrations of ammonia measured in produced fluids. Arcobacter is capable of chemosynthetic sulfur oxidation, which could fuel metabolic processes for other heterotrophic, fermentative, or sulfate-reducing community members. Our analysis revealed mechanisms for growth of these taxa across a broad range of salinities (up to 15% salt), which explains their enrichment during early natural-gas production. These results demonstrate the prevalence of Marinobacter and Arcobacter during a key maturation phase of hydraulically fractured natural-gas wells, and highlight the significant role these genera play in biogeochemical cycling for this economically important energy system

Evaluation of the Sublingual Route for Administration of Influenza H5N1 Virosomes in Combination with the Bacterial Second Messenger c-di-GMP

Avian influenza A H5N1 is a virus with pandemic potential. Mucosal vaccines are attractive as they have the potential to block viruses at the site of entry, thereby preventing both disease and further transmission. The intranasal route is safe for the administration of seasonal live-attenuated influenza vaccines, but may be less suitable for administration of pandemic vaccines. Research into novel mucosal routes is therefore needed. In this study, a murine model was used to compare sublingual administration with intranasal and intramuscular administration of influenza H5N1 virosomes (2 µg haemagglutinin; HA) in combination with the mucosal adjuvant (3′,5′)-cyclic dimeric guanylic acid (c-di-GMP). We found that sublingual immunisation effectively induced local and systemic H5N1-specific humoral and cellular immune responses but that the magnitude of response was lower than after intranasal administration. However, both the mucosal routes were superior to intramuscular immunisation for induction of local humoral and systemic cellular immune responses including high frequencies of splenic H5N1-specific multifunctional (IL-2+TNF-α+) CD4+ T cells. The c-di-GMP adjuvanted vaccine elicited systemic haemagglutination inhibition (HI) antibody responses (geometric mean titres ≥40) both when administered sublingually, intranasally and inramuscularly. In addition, salivary HI antibodies were elicited by mucosal, but not intramuscular vaccination. We conclude that the sublingual route is an attractive alternative for administration of pandemic influenza vaccines

A third generation vaccine for human visceral leishmaniasis and post kala azar dermal leishmaniasis : First-in-human trial of ChAd63-KH

BACKGROUND: Visceral leishmaniasis (VL or kala azar) is the most serious form of human leishmaniasis, responsible for over 20,000 deaths annually, and post kala azar dermal leishmaniasis (PKDL) is a stigmatizing skin condition that often occurs in patients after successful treatment for VL. Lack of effective or appropriately targeted cell mediated immunity, including CD8+ T cell responses, underlies the progression of VL and progression to PKDL, and can limit the therapeutic efficacy of anti-leishmanial drugs. Hence, in addition to the need for prophylactic vaccines against leishmaniasis, the development of therapeutic vaccines for use alone or in combined immuno-chemotherapy has been identified as an unmet clinical need. Here, we report the first clinical trial of a third-generation leishmaniasis vaccine, developed intentionally to induce Leishmania-specific CD8+ T cells. METHODS: We conducted a first-in-human dose escalation Phase I trial in 20 healthy volunteers to assess the safety, tolerability and immunogenicity of a prime-only adenoviral vaccine for human VL and PKDL. ChAd63-KH is a replication defective simian adenovirus expressing a novel synthetic gene (KH) encoding two Leishmania proteins KMP-11 and HASPB. Uniquely, the latter was engineered to reflect repeat domain polymorphisms and arrangements identified from clinical isolates. We monitored innate immune responses by whole blood RNA-Seq and antigen specific CD8+ T cell responses by IFNγ ELISPOT and intracellular flow cytometry. FINDINGS: ChAd63-KH was safe at intramuscular doses of 1x1010 and 7.5x1010 vp. Whole blood transcriptomic profiling indicated that ChAd63-KH induced innate immune responses characterized by an interferon signature and the presence of activated dendritic cells. Broad and quantitatively robust CD8+ T cell responses were induced by vaccination in 100% (20/20) of vaccinated subjects. CONCLUSION: The results of this study support the further development of ChAd63-KH as a novel third generation vaccine for VL and PKDL. TRIAL REGISTRATION: This clinical trial (LEISH1) was registered at EudraCT (2012-005596-14) and ISRCTN (07766359)

Increased Expression of RhoA in Epithelium and Smooth Muscle of Obese Mouse Models: Implications for Isoprenoid Control of Airway Smooth Muscle and Fibroblasts

The simultaneous rise in the prevalence of asthma and obesity has prompted epidemiologic studies that establish obesity as a risk factor for asthma. The alterations in cell signaling that explain this link are not well understood and warrant investigation so that therapies that target this asthma phenotype can be developed. We identified a significant increase in expression of the small GTPase RhoA in nasal epithelial cells and tracheal smooth muscle cells from leptin-deficient (ob/ob) mice compared to their wild-type counterparts. Since RhoA function is dependent on isoprenoid modification, we sought to determine the role of isoprenoid-mediated signaling in regulating the viability and proliferation of human airway smooth muscle cells (ASM) and normal human lung fibroblasts (NHLF). Inhibiting isoprenoid signaling with mevastatin significantly decreased the viability of ASM and NHLF. This inhibition was reversed by geranylgeranyl pyrophosphate (GGPP), but not farnesyl pyrophosphate (FPP), suggesting specificity to the Rho GTPases. Conversely, increasing isoprenoid synthesis significantly increased ASM proliferation and RhoA protein expression. RhoA expression is inherently increased in airway tissue from ob/ob mice, and obesity-entrained alterations in this pathway may make it a novel therapeutic target for treating airway disease in the obese population

Growth deficits in cystic fibrosis mice begin in utero prior to IGF-1 reduction.

Growth deficits are common in cystic fibrosis (CF), but their cause is complex, with contributions from exocrine pancreatic insufficiency, pulmonary complications, gastrointestinal obstructions, and endocrine abnormalities. The CF mouse model displays similar growth impairment despite exocrine pancreatic function and in the absence of chronic pulmonary infection. The high incidence of intestinal obstruction in the CF mouse has been suggested to significantly contribute to the observed growth deficits. Previous studies by our group have shown that restoration of the cystic fibrosis transmembrane conductance regulator (CFTR) in the intestinal epithelium prevents intestinal obstruction but does not improve growth. In this study, we further investigate growth deficits in CF and gut-corrected CF mice by assessing insulin-like growth factor 1 (IGF-1). IGF-1 levels were significantly decreased in CF and gut-corrected CF adult mice compared to wildtype littermates and were highly correlated with weight. Interestingly, perinatal IGF-1 levels were not significantly different between CF and wildtype littermates, even though growth deficits in CF mice could be detected late in gestation. Since CFTR has been suggested to play a role in water and nutrient exchange in the placenta through its interaction with aquaporins, we analyzed placental aquaporin expression in late-gestation CF and control littermates. While significant differences were observed in Aquaporin 9 expression in CF placentas in late gestation, there was no evidence of placental fluid exchange differences between CF and control littermates. The results from this study indicate that decreased IGF-1 levels are highly correlated with growth in CF mice, independent of CF intestinal obstruction. However, the perinatal growth deficits that are observed in CF mice are not due to decreased IGF-1 levels or differences in placenta-mediated fluid exchange. Further investigation is necessary to understand the etiology of early growth deficits in CF, as growth has been shown to be a significant factor in disease outcomes

A simplified sequence-based identification scheme for Bordetella reveals several putative novel species

The differentiation of Bordetella species, particularly those causing human infection, is problematic. We found that sequence analysis of an internal fragment of nrdA allowed differentiation of the currently named Bordetella species. Analysis of 107 "Bordetella" isolates recovered almost exclusively from human respiratory tract specimens identified several putative novel species

Placental aquaporin expression in CF mice.

<p><i>Aquaporin(Aqp) 1</i>,<i>3</i>,<i>8</i> and <i>9</i> expression was evaluated in placentas from e15 and e18 CF and control littermates. Only <i>Aqp9</i> at e18 was significantly different between placentas from CF and control littermates (* = P<0.005) Each measurement is an average of six mice completed in replicates (data represent mean±SEM).</p