Osteopontin Deletion Prevents the Development of Obesity and Hepatic Steatosis via Impaired Adipose Tissue Matrix Remodeling and Reduced Inflammation and Fibrosis in Adipose Tissue and Liver in Mice

Osteopontin (OPN) is a multifunctional extracellular matrix (ECM) protein involved in multiple physiological processes. OPN expression is dramatically increased in visceral adipose tissue in obesity and the lack of OPN protects against the development of insulin resistance and inflammation in mice. We sought to unravel the potential mechanisms involved in the beneficial effects of the absence of OPN. We analyzed the effect of the lack of OPN in the development of obesity and hepatic steatosis induced by a high-fat diet (HFD) using OPN-KO mice. OPN expression was upregulated in epididymal white adipose tissue (EWAT) and liver in wild type (WT) mice with HFD. OPN-KO mice had higher insulin sensitivity, lower body weight and fat mass with reduced adipose tissue ECM remodeling and reduced adipocyte size than WT mice under a HFD. Reduced MMP2 and MMP9 activity was involved in the decreased ECM remodeling. Crown-like structure number in EWAT as well as F4/80-positive cells and Emr1 expression in EWAT and liver increased with HFD, while OPN-deficiency blunted the increase. Moreover, our data show for the first time that OPN-KO under a HFD mice display reduced fibrosis in adipose tissue and liver, as well as reduced oxidative stress in adipose tissue. Gene expression of collagens Col1a1, Col6a1 and Col6a3 in EWAT and liver, as well as the profibrotic cytokine Tgfb1 in EWAT were increased with HFD, while OPN-deficiency prevented this increase. OPN deficiency prevented hepatic steatosis via reduction in the expression of molecules involved in the onset of fat accumulation such as Pparg, Srebf1, Fasn, Mogat1, Dgat2 and Cidec. Furthermore, OPN-KO mice exhibited higher body temperature and improved BAT function. The present data reveal novel mechanisms of OPN in the development of obesity, pointing out the inhibition of OPN as a promising target for the treatment of obesity and fatty liver.     

Proteolytic Scanning Calorimetry: A Novel Methodology that Probes the Fundamental Features of Protein Kinetic Stability

We introduce proteolytic scanning calorimetry, a modification of the differential scanning calorimetry approach to the determination of protein stability in which a proteolytic enzyme (thermolysin) is used to mimic a harsh environment. This methodology allows the straightforward calculation of the rate of irreversible denaturation as a function of temperature and concentration of proteolytic enzyme and, as a result, has the potential to probe efficiently the fundamental biophysical features of protein kinetic stability. In the particular case of Escherichia coli thioredoxin (used as an illustrative example in this article), we find that the rate of irreversible denaturation is determined by 1), the global unfolding mechanism at low thermolysin concentrations, indicating that thermodynamic stability may contribute directly to the kinetic stability of thioredoxin under moderately harsh conditions and 2), the rate of unfolding at high thermolysin concentrations, indicating that the free-energy barrier for unfolding may act as a safety mechanism that ensures significant kinetic stability, even in very harsh environments. This thioredoxin picture, however, is by no means expected to be general and different proteins may show different patterns of kinetic stabilization. Proteolytic scanning calorimetry is particularly well-suited to probe this diversity at a fundamental biophysical level.     

Epigenetic control of retrotransposon expression in human embryonic stem cells

Long interspersed element 1s (LINE-1s or L1s) are a family of non-long-terminal-repeat retrotransposons that predominate in the human genome. Active LINE-1 elements encode proteins required for their mobilization. L1-encoded proteins also act in trans to mobilize short interspersed elements (SINEs), such as Alu elements. L1 and Alu insertions have been implicated in many human diseases, and their retrotransposition provides an ongoing source of human genetic diversity. L1/Alu elements are expected to ensure their transmission to subsequent generations by retrotransposing in germ cells or during early embryonic development. Here, we determined that several subfamilies of Alu elements are expressed in undifferentiated human embryonic stem cells (hESCs) and that most expressed Alu elements are active elements. We also exploited expression from the L1 antisense promoter to map expressed elements in hESCs. Remarkably, we found that expressed Alu elements are enriched in the youngest subfamily, Y, and that expressed L1s are mostly located within genes, suggesting an epigenetic control of retrotransposon expression in hESCs. Together, these data suggest that distinct subsets of active L1/Alu elements are expressed in hESCs and that the degree of somatic mosaicism attributable to L1 insertions during early development may be higher than previously anticipated.     

Synapsis and strand exchange in the resolution and DNA inversion reactions catalysed by the beta recombinase

In the presence of a sequence-independent chromatin-associated protein, such as Hbsu or HMGB, the β recombinase catalyses resolution between two directly oriented recombination sites (six sites) and both resolution and DNA inversion between two inversely oriented six sites. Assembly of the synaptic complex requires binding of the β recombinase to the six sites and the presence of Hbsu. Whether resolution or inversion will take place depends on the relative orientation of the two six sites, the level of DNA supercoiling and the amounts of Hbsu. In this work, the topologies of the products of the resolution and inversion reactions were analysed. The resolution reaction generated mainly singly catenated DNA circles, while DNA inversion gave rise to unknotted circles and small amounts of DNA molecules containing 3- or 5-noded knots. In spite of the distinctive features of the β system, the topology of synapsis and strand exchange during the resolution reaction is similar to that of Tn3 and γδ resolvases. The ability of the β recombinase to catalyse both inversion and resolution reactions probably reflects different possible architectures of the synaptic complex, which to a large extent depends on Hbsu.     

Immunogenicity and efficacy of a chimpanzee adenovirus-vectored Rift Valley Fever vaccine in mice

Background

Rift Valley Fever (RVF) is a viral zoonosis that historically affects livestock production and human health in sub-Saharan Africa, though epizootics have also occurred in the Arabian Peninsula. Whilst an effective live-attenuated vaccine is available for livestock, there is currently no licensed human RVF vaccine. Replication-deficient chimpanzee adenovirus (ChAd) vectors are an ideal platform for development of a human RVF vaccine, given the low prevalence of neutralizing antibodies against them in the human population, and their excellent safety and immunogenicity profile in human clinical trials of vaccines against a wide range of pathogens.

Methods

Here, in BALB/c mice, we evaluated the immunogenicity and efficacy of a replication-deficient chimpanzee adenovirus vector, ChAdOx1, encoding the RVF virus envelope glycoproteins, Gn and Gc, which are targets of virus neutralizing antibodies. The ChAdOx1-GnGc vaccine was assessed in comparison to a replication-deficient human adenovirus type 5 vector encoding Gn and Gc (HAdV5-GnGc), a strategy previously shown to confer protective immunity against RVF in mice.

Results

A single immunization with either of the vaccines conferred protection against RVF virus challenge eight weeks post-immunization. Both vaccines elicited RVF virus neutralizing antibody and a robust CD8⁺ T cell response.

Conclusions

Together the results support further development of RVF vaccines based on replication-deficient adenovirus vectors, with ChAdOx1-GnGc being a potential candidate for use in future human clinical trials.

     

Xp38gamma/SAPK3 promotes meiotic G(2)/M transition in Xenopus oocytes and activates Cdc25C

We have studied the role of p38 mitogen-activated protein kinases (MAPKs) in the meiotic maturation of Xenopus oocytes. Overexpression of a constitutively active mutant of the p38 activator MKK6 accelerates progesterone-induced maturation. Immunoprecipit ation experiments indicate that p38gamma/SAPK3 is the major p38 activated by MKK6 in the oocytes. We have cloned Xenopus p38gamma (Xp38gamma) and show that co-expression of active MKK6 with Xp38gamma induces oocyte maturation in the absence of progesterone. The maturation induced by Xp38gamma requires neither protein synthesis nor activation of the p42 MAPK-p90Rsk pathway, but it is blocked by cAMP-dependent protein kinase. A role for the endogenous Xp38gamma in progesterone-induced maturation is supported by the inhibitory effect of kinase-dead mutants of MKK6 and Xp38gamma. Furthermore, MKK6 can rescue the inhibition of oocyte maturation by anthrax lethal factor, a protease that inactivates MAPK kinases. We also show that Xp38gamma can activate the phosphatase XCdc25C, and we identified Ser205 of XCdc25C as a major phosphorylation site for Xp38gamma. Our results indicate that phosphorylation of XCdc25C by Xp38gamma/SAPK3 is important for the meiotic G(2)/M progression of Xenopus oocytes.     

Development and characterization of monoclonal antibodies against Rift Valley fever virus nucleocapsid protein generated by DNA immunization

This paper describes the generation of monoclonal antibodies directed to immunogenic nucleoprotein N epitopes of Rift Valley fever virus (RVFV), and their application in diagnostics, both for antibody detection in competitive eLISA and for antigen capture in a sandwich eLISA. Monoclonal antibodies (mAbs) were generated after DNA immunization of Balb/c mice and characterized by western blot, ELISA and cell immunostaining assays. At least three different immunorelevant epitopes were defined by mAb competition assays. Interestingly, two of the mAbs generated were able to distinguish between RVFV strains from egyptian or South African lineages. these monoclonal antibodies constitute useful tools for diagnosis, especially for the detection of serum anti-RVFV antibodies from a broad range of species by means of competitive ELISA.Key words: RVF, nucleoprotein, competitive ELISA, RVFV lineages     

TSG-6 binds via its CUB_C domain to the cell-binding domain of fibronectin and increases fibronectin matrix assembly.

Human plasma fibronectin binds with high affinity to the inflammation-induced secreted protein TSG-6. Fibronectin binds to the CUB_C domain of TSG-6 but not to its Link module. TSG-6 can thus act as a bridging molecule to facilitate fibronectin association with the TSG-6 Link module ligand thrombospondin-1. Fibronectin binding to TSG-6 is divalent cation-independent and is conserved in cellular fibronectins. Based on competition binding studies using recombinant and proteolytic fragments of fibronectin, TSG-6 binding localizes to type III repeats 9-14 of fibronectin. This region of fibronectin contains the Arg-Gly-Asp sequence recognized by alpha5beta1 integrin, but deletion of that sequence does not prevent TSG-6 binding, and TSG-6 does not inhibit cell adhesion on fibronectin substrates mediated by this integrin. This region of fibronectin is also involved in fibronectin matrix assembly, and addition of TSG-6 enhances exogenous and endogenous fibronectin matrix assembly by human fibroblasts. Therefore, TSG-6 is a high affinity ligand that can mediate fibronectin interactions with other matrix components and modulate some interactions of fibronectin with cells.     

HLA‐DRB1 and HLA–DQB1 genes on susceptibility to and protection from allergic bronchopulmonary aspergillosis in patients with cystic fibrosis

Allergic bronchopulmonary aspergillosis (ABPA) is a hypersensitivity pulmonary disease that affects both patients with cystic fibrosis (CF) and those with asthma. HLA‐DRB1 alleles have previously been associated with ABPA–CF susceptibility; however, HLA‐DQB1 allele associations have not been clearly established. The aim of the present study was to investigate HLA class II associations in patients with ABPA–CF and determine their roles in susceptibility or protection. Patients with ABPA–CF, patients with CF without ABPA, patients with asthma without ABPA (AST), and healthy controls were included in this study. DNA was extracted by automatic extractor. HLA‐DRB1 and ‐DQB1 genotyping was performed by the Luminex PCR‐SSOP method (One Lambda, Canoga Park, CA, USA). Allele specific PCR‐SSP was also performed by high‐resolution analysis (One Lambda). Statistical analysis was performed with SSPS and Arlequin software. Both HLA‐DRB1*5:01 and ‐DRB1*11:04 alleles occurred with greater frequency in patients with ABPA–CF than in those with AST and CF and control subjects, corroborating previously published data. On the other hand, analysis of haplotypes revealed that almost all patients with ABPA–CF lacking DRB1*15:01 or DRB1*11:04 carry either DRB1*04, DRB1*11:01, or DRB1*07:01 alleles. In the HLA‐DQB1 region, the HLA‐DQB1*06:02 allele occurred more frequently in patients with ABPA–CF than in those with AST and CF and healthy controls, whereas HLA‐DQB1*02:01 occurred less frequently in patients with ABPA–CF. These data confirm that there is a correlation between HLA‐DRB1*15:01, –DRB1*11:04, DRB1*11:01, –DRB1*04 and –DRB1 * 07:01 alleles and ABPA–CF susceptibility and suggest that HLA‐DQB1*02:01 is an ABPA–CF resistance allele.