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.     

Ionization of His 55 at the dimer interface of dynein light-chain LC8 is coupled to dimer dissociation

LC8 is a highly conserved light-chain subunit of cytoplasmic dynein that interacts with a wide variety of cellular proteins and is presumed to play a fundamental role in dynein assembly and cargo recruitment and in the assembly of protein complexes unrelated to dynein. LC8 is a dimer at physiological pH but dissociates to a folded monomer at pH < 4.8. We have suggested that acid-induced dimer dissociation is due to protonation of His 55, which is stacked against His 55' and completely buried in the dimer interface. In this work, we show that the pH-induced dissociation is reversible and indeed governed by the ionization state of His 55. Mutagenesis of His 55 to Lys results in a monomer in the pH range of 3-8, while the mutation to Ala results in a dimer in the same pH range. Mutations that disrupt intermolecular hydrogen bonds between Tyr 65 and Lys 44' and His 55 and Thr 67' do not change the association state of the dimer. Titration curves for His 55 and the two other histidines, His 72 and 68, were determined by (13)C-(1)H NMR for H55K and for WT-LC8 in the monomeric and dimeric states. The pK(a) values of His 72 and His 68 are 6 in the WT dimer and 6.2-6.5 in monomeric H55K, while the pK(a) of His 55 is about 4.5 in the WT dimer. These results indicate that deprotonation of His 55 is linked to dimer formation and that mutation of His 55 to a small neutral residue or to a positively charged residue uncouples the protonation and dissociation processes.     

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.     

The chemo enzymatic functionalization of chitosan zeolite particles provides antioxidant and antimicrobial properties

Silicate‐based microporous materials like zeolites are nano enabled particles and used for various applications including pharmaceutical formulations. This study reports on the chemo‐enzymatic functionalization of chitosan‐zeolite particles (CTS‐zeolites) with caffeic acid (CA) and glucose oxidase (GOX) to impart combined antioxidant and antimicrobial properties. CA was grafted on the chitosan moieties by using laccase generating stable particles (zeta potential –36.7 mV) of high antioxidant activity (44% DPPH inhibition). GOX was immobilized both on CTS‐zeolites and on CA modified CTS‐zeolites and creating a hydrogen peroxide generation system continuously and in‐situ producing this oxidative and antimicrobial agent. The system prevented bacterial growth of E. coli and S. aureus over 24 h whereby a steady‐state concentration of around 60 μM hydrogen peroxide in the culture medium was observed. CA and GOX functionalized CTS‐zeolite particles additionally showed combinatorial antioxidant and antimicrobial properties providing a powerful bioactive system for medical applications. These particles proved their suitability for incorporation in bioactive formulations which could be used, inter alia, for topical wound treatments.     

Withanolides of Datura spp. and hybrids

A new steroidal lactone of the withanolide group has been isolated from two Datura species and three hybrids and characterized as (20R,22R-5α,12α-dihydroxy-1-oxo-6α, 7α-epoxywitha-2,24-dienolide. The distribution of six withanolides throughout section Stramonium of the genus has been investigated.     

Field studies on colour preferences of Ctenarytaina thysanura in Tasmanian boronia farms

Field tests on attraction of Ctenarytaina thysanura (Hemiptera: Psyllidae) adults to different coloured 30×30 cm sticky traps revealed a preference for yellow. Among the enamel colours tested, more psyllids were captured on yellow traps followed by green, then blue and least on red, cyan and magenta. Dilution of yellow enamel with 50% white (1Y: 1W) and 75% white (1Y: 3W) to produce yellow-white hues resulted in a significant decrease in psyllid capture indicating that the psyllids response to yellow was one of positive attraction and could suggest true colour discrimination. Reflectance spectra of painted surfaces of the enamel colours and also yellow to white hues indicated that psyllid capture rates were directly related to the proportion of light reflected in the 500–560 nm region. The biological basis of the observed C. thysanura response may be that yellow is the most intensely reflective colour in the general part of the spectrum for leaves which reflect most light in the 500–600 nm (peak 550 nm) range.     

Aminoalkyl adenylate and aminoacyl sulfamate intermediate analogues differing greatly in affinity for their cognate Staphylococcus aureus aminoacyl tRNA synthetases

Aminoalkyl adenylates and aminoacyl sulfamates derived from arginine, histidine and threonine, have been prepared and tested as inhibitors of their cognate Staphylococcus aureus aminoacyl tRNA synthetases. The arginyl derivatives were both potent nanomolar inhibitors of the Class I arginyl tRNA synthetase whereas for the Class II histidyl and threonyl tRNA synthetases, the acyl sulfamates were potent inhibitors but the adenylates had very little affinity.     

The antimicrobial natural product chuangxinmycin and some synthetic analogues are potent and selective inhibitors of bacterial tryptophanyl tRNA synthetase

The antimicrobial natural product chuangxinmycin has been found to be a potent and selective inhibitor of bacterial tryptophanyl tRNA synthetase (WRS). A number of analogues have been synthesised. The interaction with WRS appears to be highly constrained, as only sterically smaller analogues afforded significant inhibition. The only analogue to show inhibition comparable to chuangxinmycin also had antibacterial activity. WRS inhibition may contribute to the antibacterial action of chuangxinmycin.     

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.