RANKL induces heterogeneous DC‐STAMPlo and DC‐STAMPhi osteoclast precursors of which the DC‐STAMPlo precursors are the master fusogens

Osteoclasts (OC) are multinucleated bone resorbing cells that form via RANKL‐induced fusion of heterogeneous mononuclear OC precursors (OCP). Currently, there are no unique surface markers to distinguish these OCP populations, which are diagnostic for erosive and metabolic bone diseases using culture assays. Thus, we investigated expression of DC‐STAMP, a surface receptor required for OCP fusion, during osteoclastogenesis in vitro using a novel monoclonal antibody (1A2). Immunoprecipitation‐Western blot analysis of OCP membrane proteins detected 106 kDa dimeric and 53 kDa monomeric DC‐STAMP in non‐denaturing and denaturing conditions, respectively, with greater sensitivity versus rabbit anti‐sera (KR104). 1A2 also detected 99.9% of undifferentiated monocytes as a single population by flow cytometry with a MFI 100‐fold over background, while KR104 was not useful in this assay. Functionally, 1A2 inhibited OCP fusion in vitro. RANKL stimulation of OCP induced DC‐STAMP^lo and DC‐STAMP^hi cells, which mature into OC and mononuclear cells respectively as determined by fluorescent microscopy and TRAP assays. Addition of DC‐STAMP^hi cells to purified DC‐STAMP^lo cultures produced larger, more nucleated OC vs. pure DC‐STAMP^lo cultures. RT‐qPCR analysis of these two populations showed that OC markers (Trap and Oc‐stamp) and fusogenic gene expression (Cd9 and Cd47), were significantly increased in DC‐STAMP^lo vs. DC‐STAMP^hi cells. Collectively, these results demonstrate that DC‐STAMP is expressed on OCP as a dimer, which is efficiently detected by 1A2 via flow cytometry. RANKL induces osteoclastogenesis by stimulating DC‐STAMP internalization in some OCP, and these DC‐STAMP^lo cells display the “master fusogen” phenotype. In contrast, DC‐STAMP^hi OCP can only act as mononuclear donors. J. Cell. Physiol. 223: 76–83, 2010. © 2009 Wiley‐Liss, Inc.     

Field and laboratory studies on water conditions affecting the potency of VectoBac (Bacillus thuringiensis serotype H-14) against larvae of the blackfly, Simulium damnosum

River water conditions that might influence the efficacy of VectoBac, a formulation of the microbial insecticide Bacillus thuringiensis H-14 Berliner against Simulium damnosum sensu lato Theobald (Diptera: Simuliidae) larvae were investigated. A standard formulation was assayed 130 times over 15 months using a mini-gutter system at a field station beside the River Pra in Ghana. The lethal concentration (LC) values, river temperature, conductivity, turbidity and pH were analysed using univariate and multivariate statistics to identify which of these parameters influenced its performance. River temperature, conductivity and turbidity (in that order) were identified as having direct effects on the potency of VectoBac. Water temperature and conductivity were negatively correlated, whereas turbidity and pH were positively correlated with LC values. Analyses of river water samples revealed that despite observed differences in total solids, sodium and potassium cations and chloride concentrations, all the parameters measured did not differ significantly between wet and dry seasons. A simple method for rearing S. damnosum s.l. in the laboratory was then adopted to study the effect of conductivity on potency of VectoBac under controlled conditions. Increasing the conductivity of the water medium up to 3,000 microS enhanced potency by about 42%, whereas increasing that of the insecticide alone raised it by 37%. The results obtained suggest that for effective use of VectoBac for blackfly control in West Africa, river temperature, conductivity and turbidity should be taken into consideration, perhaps by only selecting rivers with optimal conditions for treatment. The laboratory-based system developed for assaying the product overcomes the vagaries associated with field conditions and also the demand for huge logistic requirements of the mini-gutter system, which has to be sited near rivers.     

Les dysfonctions érectiles chez le diabétique: Rôle des facteurs neurologiques

Objective

To assess the etiological factors of erectile dysfunction in male diabetics.

Material and methods

We have performed a prospective evaluation including 69 diabetic patients suffering from erectile dysfunction. Studied parameters including age, type and duration of diabetes, complications, treatments and associated risk factors were analysed. Comparison was done with a control group of 138 diabetic patients without erectile dysfunction.

Results

There was a significant difference between the diabetic with neurologic complications and the others without neuropathy (p=0.0004). The duration of the diabete was was another risk factor of erectile dysfunction (p=0.049)

Conclusion

We confirm various authors who demonstrated that diabetic impotence seems to be mainly neuropathic in etiology even though it was a multifactorial discomfort.     

Adsorptive control of water in esterification with immobilized enzymes. Continuous operation in a periodic counter-current reactor

A periodic counter-current adsorptive-reactor system is developed to carry out continuous esterifications in organic solvents with immobilized enzymes. The system comprises a number of fixed-beds distributed between a reaction-adsorption zone and a regeneration zone and operated in a "merry-go-round" sequence. Water formed in the reaction is adsorbed preventing the formation of a free-water phase and deactivation of the biocatalyst. The adsorbed water is, in turn, recovered by desorption in the regeneration zone. The concept is tested experimentally on a laboratory-scale using, as a model, the esterification of isoamyl alcohol and propionic acid in hexane catalyzed by an immobilized lipase. Pure isoamyl alcohol is used as a regenerant to remove excess water from the biocatalyst. In the periodic steady-state, improvements in ester productivity greater than 50% over that achievable with a conventional fixed-bed reactor are demonstrated experimentally with just two beds in a series arrangement. Use of a water-selective adsorbent in conjunction with the biocatalyst provides further improvements by reducing accumulation of water on the enzyme. A mathematical model is also developed to predict the thermodynamic activity of water along the reactor and describe the dynamic behavior of the system. The model, based on independently developed rate and equilibrium parameters, successfully predicts the experimental behavior and provides an effective tool for scale-up and optimization.     

The mycobiota of the sand fly Phlebotomus perniciosus: Involvement of yeast symbionts in uric acid metabolism

The knowledge of the fungal mycobiota of arthropods, including the vectors of human and animal diseases, is still limited. Here, the mycobiota associated with the sand fly Phlebotomus perniciosus, the main vector of leishmaniasis in the western Mediterranean area, by a culture‐dependent approach (microbiological analyses and sequencing of the 26S rRNA gene), internal transcribed spacer (ITS) rRNA amplicon‐based next‐generation sequencing, fluorescence in situ hybridisation (FISH), and genome sequencing of the dominant yeast species was investigated. The dominant species was Meyerozyma guilliermondii, known for its biotechnological applications. The focus was on this yeast and its prevalence in adults, pupae and larvae of reared sand flies (overall prevalence: 57.5%) and of field‐collected individuals (overall prevalence: 9%) was investigated. Using whole‐mount FISH and microscopic examination, it was further showed that M. guilliermondii colonizes the midgut of females, males and larvae and the distal part of Malpighian tubules of female sand flies, suggesting a possible role in urate degradation. Finally, the sequencing and analysis of the genome of M. guilliermondii allowed predicting the complete uric acid degradation pathway, suggesting that the yeast could contribute to the removal of the excess of nitrogenous wastes after the blood meal of the insect host.     

TPR-Mediated self-association of plant SGT1

The tetratricopeptide repeat (TPR) domain mediates inter-protein associations in a number of systems. The domain is also thought to mediate oligomerization of some proteins, but this has remained controversial, with conflicting data appearing in the literature. By way of investigating such TPR-mediated self-associations we used a variety of biophysical techniques to characterize purified recombinant Sgt1, a TPR-containing protein found in all eukaryotes that is involved in a broad range of biological processes, including kinetochore assembly in humans and yeast and disease resistance in plants. We show that recombinant Sgt1 from Arabidopsis, barley, and yeast self-associates in vitro while recombinant human Sgt1 does not. Further experiments on barley Sgt1 demonstrate unambiguously a TPR-mediated dimerization, which is concentration- and ionic-strength-dependent and results in a global increase in helical structure and stability of the protein. Dimerization is also redox sensitive, being completely abolished by the formation of an intramolecular disulfide bond where the contributing cysteines are conserved in plant Sgt1s. The dimer interface was mapped through cross-linking and mass spectrometry to the C-terminal region of the TPR domain. Our study, which provides the first biophysical characterization of plant Sgt1, highlights how TPR domains can mediate self-association in solution and that sequence variation in the regions involved in oligomerization affects the propensity of TPR-containing proteins to dimerize.