Differences in hydration coupled to specific and nonspecific competitive binding and to specific DNA Binding of the restriction endonuclease BamHI

Using the osmotic stress technique together with a self-cleavage assay we measure directly differences in sequestered water between specific and nonspecific DNA-BamHI complexes as well as the numbers of water molecules released coupled to specific complex formation. The difference between specific and nonspecific binding free energy of the BamHI scales linearly with solute osmolal concentration for seven neutral solutes used to set water activity. The observed osmotic dependence indicates that the nonspecific DNA-BamHI complex sequesters some 120-150 more water molecules than the specific complex. The weak sensitivity of the difference in number of waters to the solute identity suggests that these waters are sterically inaccessible to solutes. This result is in close agreement with differences in the structures determined by x-ray crystallography. We demonstrate additionally that when the same solutes that were used in competition experiments are used to probe changes accompanying the binding of free BamHI to its specific DNA sequence, the measured number of water molecules released in the binding process is strikingly solute-dependent (with up to 10-fold difference between solutes). This result is expected for reactions resulting in a large change in a surface exposed area.     

Functionally diverse rhizobacteria of Saccharum munja (a native wild grass) colonizing abandoned morrum mine in Aravalli hills (Delhi)

Characterization of the rhizobacteria of native grasses naturally colonizing abandoned mine sites may help in identification of microbial inoculants for ecological-restoration programmes. Eighty one strains of Saccharum munja rhizobacteria isolated from an abandoned mine located on Aravalli mountain and 50 from bulk-region were identified using 16S rRNA sequence analyses. Based on chemical- and biological-assays they were categorized into ecologically diverse functional groups (siderophore-, IAA-, ACC-deaminase-, HCN-, polyphosphate-producers; phosphate-solubilizer; antagonistic). Eight genera, 25 species from rhizosphere and 2 genera, 5 species from bulk-region were dominated by Bacillus spp. (B. barbaricus, B. cereus, B. firmus, B. flexus, B. foraminis, B. licheniformis, B. megaterium, B. pumilus, B. subtilis, B. thuringiensis) and Paenibacillus spp. (P. alvei, P. apiarius, P. lautus, P. lentimorbus, P. polymyxa, P. popillae). Siderophore-producers were common in rhizosphere and bulk soil, whereas IAA-producers, N₂-fixers and FePO₄-solubilizers dominated rhizosphere samples. During the reproductive phase (winter) of S. munja, siderophore-, ACC-deaminase- and polyP-producers were predominant; however dominance of HCN-producers in summer might be associated with termite-infestation. In vivo ability of selected rhizobacteria (B. megaterium BOSm201, B. subtilis BGSm253, B. pumilus BGSm157, P. alvei BGSm255, P. putida BOSm217, P. aeruginosa BGSm 306) to enhance seed-germination and seedling-growth of S. munja in mine-spoil suggest their significance in natural colonization and potential for ecological-restoration of Bhatti mine.     

Development of single nucleotide polymorphisms in Phaseolus vulgaris and related Phaseolus spp

In this study, new single nucleotide polymorphism (SNP) markers were developed for common bean (Phaseolus vulgaris L.) and related Phaseolus species. The applied strategy presents new and interesting aspects, such as the choice of accessions used, which was aimed at capturing a large portion of the genetic diversity present in the common bean, with particular focus on wild and domesticated materials from Mesoamerica and the identification of loci for sequencing. Indeed, the primer pairs for 34 loci were designed with the main strategy being to search for single-copy orthologous genes among the legumes (for use in other legume species and comparative analyses). The 10 remaining loci were selected as being near to domestication quantitative trait loci or detected as putatively under selection during domestication in previous studies. To provide an efficient and inexpensive genotyping platform for geneticists and breeders, we used sequence data to develop 60 new SNP markers for KASPar assay genotyping. The same sample was also genotyped with SNP markers developed for common bean in other studies for the same assay. This allowed testing for systematic bias according to the criteria chosen to select the genotypes in which the genetic diversity is surveyed during SNP discovery. Finally, we show that most of the SNP markers worked well in a set of accessions of other species belonging to the Phaseolus genus. The genetic resources developed will be very useful not only for breeding, but also for biodiversity conservation management and evolutionary studies on legumes.     

Identification and characterization of hADSC-derived exosome proteins from different isolation methods

Exosomes are secreted into the extracellular space by most cell types and contain various molecular constituents, which play roles in many biological processes. Adipose-derived mesenchymal stem cells (ADSCs) can differentiate into a variety of cell types and secrete a series of paracrine factors through exosomes. ADSC-derived exosomes have shown diagnostic and therapeutic potential in many clinical diseases. The molecular components are critical for their mechanisms. Several methods have been developed for exosome purification, including ultracentrifugation, ultrafiltration, density gradient purification, size-based isolation, polymer precipitation and immuno-affinity purification. Thus, we employed four methods to isolate exosomes from the hADSC culture medium, including ultracentrifugation, size exclusion chromatography, ExoQuick-TC precipitation and ExoQuick-TC ULTRA isolation. Following exosome isolation, we performed quantitative proteomic analysis of the exosome proteins using isobaric tags for relative and absolute quantification (iTRAQ) labelling, combined with 2D-LC-MS/MS. There were 599 universal and 138 stably expressed proteins in hADSC-derived exosomes. We proved that these proteins were potential hADSC-derived exosomes markers, including CD109, CD166, HSPA4, TRAP1, RAB2A, RAB11B and RAB14. From the quantitative proteomic analysis, we demonstrated that hADSC-derived exosome protein expression varied, with lipopolysaccharide (LPS) treatment, in the different isolation methods. Pathway analysis and proliferation, migration and endothelial tube formation assays showed varying effects in cells stimulated with hADSC-derived exosomes from different isolation methods. Our study revealed that different isolation methods might introduce variations in the protein composition in exosomes, which reflects their effects on biological function. The pros and cons of these methods are important points to consider for downstream research applications.     

Biophysical Response to Pulsed Laser Microbeam‐Induced Cell Lysis and Molecular Delivery

Cell lysis and molecular delivery in confluent monolayers of PtK₂ cells are achieved by the delivery of 6 ns, λ = 532 nm laser pulses via a 40×, 0.8 NA microscope objective. With increasing distance from the point of laser focus we find regions of (a) immediate cell lysis; (b) necrotic cells that detach during the fluorescence assays; (c) permeabilized cells sufficient to facilitate the uptake of small (3 kDa) FITC‐conjugated Dextran molecules in viable cells; and (d) unaffected, viable cells. The spatial extent of cell lysis, cell detachment, and molecular delivery increased with laser pulse energy. Hydrodynamic analysis from time‐resolved imaging studies reveal that the maximum wall shear stress associated with the pulsed laser microbeam‐induced cavitation bubble expansion governs the location and spatial extent of each of these regions independent of laser pulse energy. Specifically, cells exposed to maximum wall shear stresses τ_{w, max} > 190 ± 20 kPa are immediately lysed while cells exposed to τ_{w, max} > 18 ± 2 kPa are necrotic and subsequently detach. Cells exposed to τ_{w, max} in the range 8–18 kPa are viable and successfully optoporated with 3 kDa Dextran molecules. Cells exposed to τ_{w, max} < 8 ± 1 kPa remain viable without molecular delivery. These findings provide the first direct correlation between pulsed laser microbeam‐induced shear stresses and subsequent cellular outcome. (© 2008 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Phylogenetic relationships and phylogeography of the genus Sinocyrtaspis Liu, 2000 (Orthoptera: Tettigoniidae: Meconematinae) reveal speciation processes related to climate change

Species of Sinocyrtaspis Liu inhabit moist montane environments in south China, and occur allopatrically in restricted regions without overlapping areas. This study presents the first phylogeny of Sinocyrtaspis based on one nuclear DNA sequence [partial ITS1-5.8S rDNA-ITS2 (ITS)] as well as two mitochondrial genes [cytochrome oxidase subunit I (COI) and cytochrome b (CYTB)] using Bayesian inference and maximum likelihood. In addition, reconstructing the ancestral geographical range on the basis of the combined genes and distribution information, MaxEnt modelling was used to predict potential distribution areas in the Last Glacial Maximum (LGM), the mid-Holocene, the present-day and in the year 2070, in order to understand speciation processes and responses to climate change in this genus. The phylogenetic analyses supported the monophyly of Sinocyrtaspis. The results of species delimitation confirmed seven Sinocyrtaspis species and that the speciation events mainly occurred in the late Miocene to early Pliocene when the climate became colder and drier. The conclusions of the phylogeographical analysis are as follows: (i) the centres of origin of Sinocyrtaspis were Hunan and north-east Guangxi; (ii) two dispersal routes became obvious with the final destinations of Jiangxi and Zhejiang, respectively; (iii) after the Miocene cooling period, the ancestor species partly remained in Guizhou and partly spread; (iv) as a response to climate change, species altered distribution areas by moving along altitude gradients in mountain regions, whereas the anthropogenic global warming trend has promoted some species moving to high-latitude areas or caused population differentiation.