Evaluation of live attenuated influenza a virus h6 vaccines in mice and ferrets

Avian influenza A virus A/teal/HK/W312/97 (H6N1) possesses seven gene segments that are highly homologous to those of highly pathogenic human influenza H5N1 viruses, suggesting that a W312-like H6N1 virus might have been involved in the generation of the A/HK/97 H5N1 viruses. The continuous circulation and reassortment of influenza H6 subtype viruses in birds highlight the need to develop an H6 vaccine to prevent potential influenza pandemics caused by the H6 viruses. Based on the serum antibody cross-reactivity data obtained from 14 different H6 viruses from Eurasian and North American lineages, A/duck/HK/182/77, A/teal/HK/W312/97, and A/mallard/Alberta/89/85 were selected to produce live attenuated H6 candidate vaccines. Each of the H6 vaccine strains is a 6:2 reassortant ca virus containing HA and NA gene segments from an H6 virus and the six internal gene segments from cold-adapted A/Ann Arbor/6/60 (AA ca), the master donor virus that is used to make live attenuated influenza virus FluMist (intranasal) vaccine. All three H6 vaccine candidates exhibited phenotypic properties of temperature sensitivity (ts), ca, and attenuation (att) conferred by the internal gene segments from AA ca. Intranasal administration of a single dose of the three H6 ca vaccine viruses induced neutralizing antibodies in mice and ferrets and fully protected mice and ferrets from homologous wild-type (wt) virus challenge. Among the three H6 vaccine candidates, the A/teal/HK/W312/97 ca virus provided the broadest cross-protection against challenge with three antigenically distinct H6 wt viruses. These data support the rationale for further evaluating the A/teal/HK/W312/97 ca vaccine in humans.     

Identification of two proteins that interact with the Erp virulence factor from Mycobacterium tuberculosis by using the bacterial two-hybrid system

Background  

The exported repetitive protein (erp) gene encodes a secreted 36-kDa protein with a central domain containing several proline-glycine-leucine-threonine-serine (PGLTS) repeats. It has been demonstrated that erp is a virulence-associated factor since the disruption of this gene impairs the growth of Mycobacterium bovis and Mycobacterium tuberculosis in mice.     

Sertoli Cells Maintain Leydig Cell Number and Peritubular Myoid Cell Activity in the Adult Mouse Testis

The Sertoli cells are critical regulators of testis differentiation and development. In the adult, however, their known function is restricted largely to maintenance of spermatogenesis. To determine whether the Sertoli cells regulate other aspects of adult testis biology we have used a novel transgenic mouse model in which Amh-Cre induces expression of the receptor for Diphtheria toxin (iDTR) specifically within Sertoli cells. This causes controlled, cell-specific and acute ablation of the Sertoli cell population in the adult animal following Diphtheria toxin injection. Results show that Sertoli cell ablation leads to rapid loss of all germ cell populations. In addition, adult Leydig cell numbers decline by 75% with the remaining cells concentrated around the rete and in the sub-capsular region. In the absence of Sertoli cells, peritubular myoid cell activity is reduced but the cells retain an ability to exclude immune cells from the seminiferous tubules. These data demonstrate that, in addition to support of spermatogenesis, Sertoli cells are required in the adult testis both for retention of the normal adult Leydig cell population and for support of normal peritubular myoid cell function. This has implications for our understanding of male reproductive disorders and wider androgen-related conditions affecting male health.     

Plasma lipidome is independently associated with variability in metabolic syndrome in Mexican American families

Plasma lipidome is now increasingly recognized as a potentially important marker of chronic diseases, but the exact extent of its contribution to the interindividual phenotypic variability in family studies is unknown. Here, we used the rich data from the ongoing San Antonio Family Heart Study (SAFHS) and developed a novel statistical approach to quantify the independent and additive value of the plasma lipidome in explaining metabolic syndrome (MS) variability in Mexican American families recruited in the SAFHS. Our analytical approach included two preprocessing steps: principal components analysis of the high-resolution plasma lipidomics data and construction of a subject-subject lipidomic similarity matrix. We then used the Sequential Oligogenic Linkage Analysis Routines software to model the complex family relationships, lipidomic similarities, and other important covariates in a variance components framework. Our results suggested that even after accounting for the shared genetic influences, indicators of lipemic status (total serum cholesterol, TGs, and HDL cholesterol), and obesity, the plasma lipidome independently explained 22% of variability in the homeostatic model of assessment-insulin resistance trait and 16% to 22% variability in glucose, insulin, and waist circumference. Our results demonstrate that plasma lipidomic studies can additively contribute to an understanding of the interindividual variability in MS.     

Polo-like kinase 1 (PLK1) and protein phosphatase 6 (PP6) regulate DNA-dependent protein kinase catalytic subunit (DNA-PKcs) phosphorylation in mitosis

The protein kinase activity of the DNA-PKcs (DNA-dependent protein kinase catalytic subunit) and its autophosphorylation are critical for DBS (DNA double-strand break) repair via NHEJ (non-homologous end-joining). Recent studies have shown that depletion or inactivation of DNA-PKcs kinase activity also results in mitotic defects. DNA-PKcs is autophosphorylated on Ser²⁰⁵⁶, Thr²⁶⁴⁷ and Thr²⁶⁰⁹ in mitosis and phosphorylated DNA-PKcs localize to centrosomes, mitotic spindles and the midbody. DNA-PKcs also interacts with PP6 (protein phosphatase 6), and PP6 has been shown to dephosphorylate Aurora A kinase in mitosis. Here we report that DNA-PKcs is phosphorylated on Ser³²⁰⁵ and Thr³⁹⁵⁰ in mitosis. Phosphorylation of Thr³⁹⁵⁰ is DNA-PK-dependent, whereas phosphorylation of Ser³²⁰⁵ requires PLK1 (polo-like kinase 1). Moreover, PLK1 phosphorylates DNA-PKcs on Ser³²⁰⁵ in vitro and interacts with DNA-PKcs in mitosis. In addition, PP6 dephosphorylates DNA-PKcs at Ser³²⁰⁵ in mitosis and after IR (ionizing radiation). DNA-PKcs also phosphorylates Chk2 on Thr⁶⁸ in mitosis and both phosphorylation of Chk2 and autophosphorylation of DNA-PKcs in mitosis occur in the apparent absence of Ku and DNA damage. Our findings provide mechanistic insight into the roles of DNA-PKcs and PP6 in mitosis and suggest that DNA-PKcs’ role in mitosis may be mechanistically distinct from its well-established role in NHEJ.     

The role of GABARAPL1/GEC1 in autophagic flux and mitochondrial quality control in MDA-MB-436 breast cancer cells

GABARAPL1/GEC1 is an early estrogen-induced gene which encodes a protein highly conserved from C. elegans to humans. Overexpressed GABARAPL1 interacts with GABA_A or kappa opioid receptors, associates with autophagic vesicles, and inhibits breast cancer cell proliferation. However, the function of endogenous GABARAPL1 has not been extensively studied. We hypothesized that GABARAPL1 is required for maintaining normal autophagic flux, and plays an important role in regulating cellular bioenergetics and metabolism. To test this hypothesis, we knocked down GABARAPL1 expression in the breast cancer MDA-MB-436 cell line by shRNA. Decreased expression of GABARAPL1 activated procancer responses of the MDA-MB-436 cells including increased proliferation, colony formation, and invasion. In addition, cells with decreased expression of GABARAPL1 exhibited attenuated autophagic flux and a decreased number of lysosomes. Moreover, decreased GABARAPL1 expression led to cellular bioenergetic changes including increased basal oxygen consumption rate, increased intracellular ATP, increased total glutathione, and an accumulation of damaged mitochondria. Taken together, our results demonstrate that GABARAPL1 plays an important role in cell proliferation, invasion, and autophagic flux, as well as in mitochondrial homeostasis and cellular metabolic programs.     

Functional characterization of cytochrome P450-derived epoxyeicosatrienoic acids in adipogenesis and obesity

Adipogenesis plays a critical role in the initiation and progression of obesity. Although cytochrome P450 (CYP)-derived epoxyeicosatrienoic acids (EETs) have emerged as a potential therapeutic target for cardiometabolic disease, the functional contribution of EETs to adipogenesis and the pathogenesis of obesity remain poorly understood. Our studies demonstrated that induction of adipogenesis in differentiated 3T3-L1 cells (in vitro) and obesity-associated adipose expansion in high-fat diet (HFD)-fed mice (in vivo) significantly dysregulate the CYP epoxygenase pathway and evoke a marked suppression of adipose-derived EET levels. Subsequent in vitro experiments demonstrated that exogenous EET analog administration elicits potent anti-adipogenic effects via inhibition of the early phase of adipogenesis. Furthermore, EET analog administration to mice significantly mitigated HFD-induced weight gain, adipose tissue expansion, pro-adipogenic gene expression, and glucose intolerance. Collectively, these findings suggest that suppression of EET bioavailability in adipose tissue is a key pathological consequence of obesity, and strategies that promote the protective effects of EETs in adipose tissue offer enormous therapeutic potential for obesity and its downstream pathological consequences.     

Cytokine profiles in the joint depend on pathology,but are different between synovial fluid,cartilage tissue and cultured chondrocytes

Introduction

This study aimed to evaluate whether profiles of several soluble mediators in synovial fluid and cartilage tissue are pathology-dependent and how their production is related to in vitro tissue formation by chondrocytes from diseased and healthy tissue.

Methods

Samples were obtained from donors without joint pathology (n = 39), with focal defects (n = 65) and osteoarthritis (n = 61). A multiplex bead assay (Luminex) was performed measuring up to 21 cytokines: Interleukin (IL)-1α, IL-1β, IL-1RA, IL-4, IL-6, IL-6Rα, IL-7, IL-8, IL-10, IL-13, tumor necrosis factor (TNF)α, Interferon (IFN)γ, oncostatin M (OSM), leukemia inhibitory factor (LIF), adiponectin, leptin, monocyte chemotactic factor (MCP)1, RANTES, basic fibroblast growth factor (bFGF), hepatocyte growth factor (HGF), vascular growth factor (VEGF).

Results

In synovial fluid of patients with cartilage pathology, IL-6, IL-13, IFNγ and OSM levels were higher than in donors without joint pathology (P ≤0.001). IL-13, IFNγ and OSM were also different between donors with cartilage defects and OA (P <0.05). In cartilage tissue from debrided defects, VEGF was higher than in non-pathological or osteoarthritic joints (P ≤0.001). IL-1α, IL-6, TNFα and OSM concentrations (in ng/ml) were markedly higher in cartilage tissue than in synovial fluid (P <0.01). Culture of chondrocytes generally led to a massive induction of most cytokines (P <0.001). Although the release of inflammatory cytokines was also here dependent on the pathological condition (P <0.001) the actual profiles were different from tissue or synovial fluid and between non-expanded and expanded chondrocytes. Cartilage formation was lower by healthy unexpanded chondrocytes than by osteoarthritic or defect chondrocytes.

Conclusions

Several pro-inflammatory, pro-angiogenic and pro-repair cytokines were elevated in joints with symptomatic cartilage defects and/or osteoarthritis, although different cytokines were elevated in synovial fluid compared to tissue or cells. Hence a clear molecular profile was evident dependent on disease status of the joint, which however changed in composition depending on the biological sample analysed. These alterations did not affect in vitro tissue formation with these chondrocytes, as this was at least as effective or even better compared to healthy chondrocytes.     

Extracellular Polymeric Substances (EPS) of Freshwater Biofilms Stabilize and Modify CeO2 and Ag Nanoparticles

Streams are potential receiving compartments for engineered nanoparticles (NP). In streams, NP may remain dispersed or settle to the benthic compartment. Both dispersed and settling NP can accumulate in benthic biofilms called periphyton that are essential to stream ecosystems. Periphytic organisms excrete extracellular polymeric substances (EPS) that interact with any material reaching the biofilms. To understand the interaction of NP with periphyton it is therefore crucial to study the interaction of NP with EPS. We investigated the influence of EPS on the physicochemical properties of selected NP (CeO₂, Ag) under controlled conditions at pH 6, 7.6, 8.6 and light or dark exposure. We extracted EPS from five different periphyton communities, characterized the extracts, and exposed CeO₂ and carbonate-stabilized Ag NP (0.5 and 5 mg/L, both 25 nm primary particle size) and AgNO₃ to EPS (10 mg/L) over two weeks. We measured NP size distribution, shape, primary particle size, surface plasmon resonance, and dissolution. All EPS extracts were composed of biopolymers, building blocks of humic substances, low molecular weight (M_r) acids, and small amphiphilic or neutral compounds in varying concentrations. CeO₂ NP were stabilized by EPS independent of pH and light/dark while dissolution increased over time in the dark at pH 6. EPS induced a size increase in Ag NP in the light with decreasing pH and the formation of metallic Ag NP from AgNO₃ at the same conditions via EPS-enhanced photoreduction. NP transformation and formation were slower in the extract with the lowest biopolymer and low M_r acid concentrations. Periphytic EPS in combination with naturally varying pH and light/dark conditions influence the properties of the Ag and CeO₂ NP tested and thus the exposure conditions within biofilms. Our results indicate that periphytic organisms may be exposed to a constantly changing mixture of engineered and naturally formed Ag NP and Ag⁺.