First Evidence of Simultaneous Circulation of Three Different Dengue Virus Serotypes in Africa

Gabon, in Central Africa, was affected for the first time in 2007 and then in 2010 by simultaneous outbreaks of chikungunya and Dengue serotype 2 (DENV-2) viruses. Through the national surveillance of dengue-like syndromes between 2007 and 2010, we observed continuous circulation of DENV-2 in a southward movement. This rapid spread of DENV-2 was associated with the emergence of DENV-1 in 2007 and DENV-3 in 2010. Interestingly, we detected six DENV-2 infected patients with hemorrhagic signs during the second outbreak in 2010. Although these cases do not meet all standard WHO criteria for severe Dengue with hemorrhage (formerly DHF), this is the first report of several dengue fever cases associated with hemorrhagic signs during a simultaneous circulation of different DENV serotypes in Africa. Together, these findings suggest that DENV is becoming more widely established on this continent and that DHF will likely become a serious public-health problem in the near future.     

Thermostability enhancement of polyethylene terephthalate degrading PETase using self- and nonself-ligating protein scaffolding approaches

Polyethylene terephthalate (PET) hydrolase enzymes show promise for enzymatic PET degradation and green recycling of single-use PET vessels representing a major source of global pollution. Their full potential can be unlocked with enzyme engineering to render activities on recalcitrant PET substrates commensurate with cost-effective recycling at scale. Thermostability is a highly desirable property in industrial enzymes, often imparting increased robustness and significantly reducing quantities required. To date, most engineered PET hydrolases show improved thermostability over their parental enzymes. Here, we report engineered thermostable variants of Ideonella sakaiensis PET hydrolase enzyme (IsPETase) developed using two scaffolding strategies. The first employed SpyCatcher-SpyTag technology to covalently cyclize IsPETase, resulting in increased thermostability that was concomitant with reduced turnover of PET substrates compared to native IsPETase. The second approach using a GFP-nanobody fusion protein (vGFP) as a scaffold yielded a construct with a melting temperature of 80°C. This was further increased to 85°C when a thermostable PETase variant (FAST PETase) was scaffolded into vGFP, the highest reported so far for an engineered PET hydrolase derived from IsPETase. Thermostability enhancement using the vGFP scaffold did not compromise activity on PET compared to IsPETase. These contrasting results highlight potential topological and dynamic constraints imposed by scaffold choice as determinants of enzyme activity.     

Transactivation of EGFR by LPS induces COX-2 expression in enterocytes

Necrotizing enterocolitis (NEC) is the leading cause of gastrointestinal morbidity and mortality in preterm infants. NEC is characterized by an exaggerated inflammatory response to bacterial flora leading to bowel necrosis. Bacterial lipopolysaccharide (LPS) mediates inflammation through TLR4 activation and is a key molecule in the pathogenesis of NEC. However, LPS also induces cyclooxygenase-2 (COX-2), which promotes intestinal barrier restitution through stimulation of intestinal cell survival, proliferation, and migration. Epidermal growth factor receptor (EGFR) activation prevents experimental NEC and may play a critical role in LPS-stimulated COX-2 production. We hypothesized that EGFR is required for LPS induction of COX-2 expression. Our data show that inhibiting EGFR kinase activity blocks LPS-induced COX-2 expression in small intestinal epithelial cells. LPS induction of COX-2 requires Src-family kinase signaling while LPS transactivation of EGFR requires matrix metalloprotease (MMP) activity. EGFR tyrosine kinase inhibitors block LPS stimulation of mitogen-activated protein kinase ERK, suggesting an important role of the MAPK/ERK pathway in EGFR-mediated COX-2 expression. LPS stimulates proliferation of IEC-6 cells, but this stimulation is inhibited with either the EGFR kinase inhibitor AG1478, or the selective COX-2 inhibitor Celecoxib. Taken together, these data show that EGFR plays an important role in LPS-induction of COX-2 expression in enterocytes, which may be one mechanism for EGF in inhibition of NEC.     

Phosphorylation sites on two domains of the beta 2-adrenergic receptor are involved in distinct pathways of receptor desensitization

Continuous exposure of cells to neurotransmitter or hormone agonists often results in a rapid desensitization of the cellular response. For example, pretreatment of Chinese hamster fibroblasts (CHW cells) expressing beta 2-adrenergic receptors (beta 2AR) with low (nanomolar) concentrations of isoproterenol, a beta-adrenergic agonist, causes decreases in the sensitivity of the cellular adenylyl cyclase response to the agonist, without changing the maximal responsiveness. In contrast, exposure of CHW cells to high (micromolar) concentrations of isoproterenol results in decreases in both sensitivity and the maximal responsiveness to agonist. To explore the role(s) of receptor phosphorylation in these processes, we expressed in CHW cells three mutant beta 2AR genes encoding receptors lacking putative phosphorylation sites for the cAMP-dependent protein kinase A and/or the cAMP-independent beta 2AR kinase. Using these mutants we found that exposure of cells to low concentrations of agonist appears to preferentially induce phosphorylation at protein kinase A sites. This phosphorylation correlates with the decreased sensitivity to agonist stimulation of the adenylyl cyclase response. At higher agonist concentrations phosphorylation on both the beta 2AR kinase and protein kinase A sites occurs, and only then is the maximal cyclase responsiveness elicited by agonist reduced. We conclude that low or high concentrations of agonist elicit phosphorylation of beta 2AR on distinct domains, with different implications for the functional coupling of the receptors with effector molecules.     

Toward a phylogeny and biogeography of Diaphanosoma (Crustacea: Cladocera)

Aquatic Ecology - Diaphanosoma s.l., with 40+? described species, is the largest genus of the Sididae and the Ctenopoda, similar in many ways to the anomopod genus Daphnia. Here, we offer a c...     

Chemical reactivity of the carbon-centered free radicals and ferrous iron in coals: Role of bioavailable Fe2+ in coal workers' pneumoconiosis

Striking differences in the prevalence of coal workers' pneumoconiosis (CWP) exist between different coal mine regions. The major factors responsible for the observed regional differences in CWP have not yet been identified. In the present study, chemical reactivity of the carbon-centered free radicals in coals and lung tissues, as well as ferrous iron in the coals, were studied by ESR techniques. The ESR spectra clearly demonstrated the presence of at least two types of carbon-centered free radical species, which might respectively attribute to the macromolecular phase and the molecular phase of coal. Grinding produced free radicals in coals. Exposure of freshly ground coal to air for 28 h induced a slight increase of free radicals for most of the coals, and a slight decrease after 4 months' exposure. The lung tissue samples of coal workers deceased of CWP showed similar ESR spectra as coal samples, and these radicals were highly stable in the lung. After incubation of coals with glutathione, hydrogen peroxide, sodium formate or oxygen, the coal sample from the Gardanne mine which has never induced CWP, and thus is the least hazardous coal, showed the most significant change in the carbon-centered free radical concentration. No significant changes were observed among other coals reported to induce CWP. On the other hand, we found that the coals released different amounts of Fe²⁺ in an acidic medium. Interestingly, the prevalence of CWP correlates positively with the released Fe²⁺ content in these coals and with the amount of oxygen radicals produced by the interaction of Fe²⁺ with O₂ in the acidified coal filtrates. Our studies indicate that the carbon-centered free radicals may not be biologically relevant to coal dust-induced pneumoconiosis, whereas the acid soluble Fe²⁺, which may be dissolved in the phagolysosomes of macrophages, can then lead to Fe²⁺-induced oxidative stress and eventual CWP development.