In vitro assembly and GTP hydrolysis by bacterial tubulins BtubA and BtubB

Arecent study identified genuine tubulin proteins, BtubA and BtubB, in the bacterial genus Prosthecobacter. We have expressed BtubA and BtubB in Escherichia coli and studied their in vitro assembly. BtubB by itself formed rings with an outer diameter of 35–36 nm in the presence of GTP or GDP. Mixtures of BtubB and BtubA formed long protofilament bundles, 4–7 protofilaments wide (20–30 protofilaments in the three-dimensional bundle). Regardless of the starting stoichiometry, the polymers always contained equal concentrations of BtubA and BtubB, suggesting that BtubA and B alternate along the protofilament. BtubA showed negligible GTP hydrolysis, whereas BtubB hydrolyzed 0.40 mol GTP per min per mol BtubB. This GTPase activity increased to 1.37 per min when mixed 1:1 with BtubA. A critical concentration of 0.4–1.0 μM was indicated by light scattering experiments and extrapolation of GTPase versus concentration, thus suggesting a cooperative assembly mechanism

Identifying avian malaria vectors: sampling methods influence outcomes

Background The role of vectors in the transmission of avian malaria parasites is currently understudied. Many studies that investigate parasite-vector relationships use limited trapping techniques and/or identify potential competent vectors in the field in such ways that cannot distinguish between an infected or infectious vector. Without the use of multiple trapping techniques that address the specific biology of diverse mosquito species, and without looking at the infection status of individual mosquitoes, it is not possible to make dependable conclusions on the role of mosquitoes in the transmission of avian malaria parasites. Methods We conducted two years of mosquito collections at a riparian preserve in California where a wide diversity of species were collected with multiple trap types. We hypothesized that competent mosquito species can influence the distribution and diversity of avian malaria parasites by acting as a compatibility filter for specific Plasmodium species. To determine the infection status of all individual mosquitoes for Plasmodium species/lineages, amplification within the cytochrome b gene was carried out on over 3000 individual mosquito thoraxes, and for those that tested positive we then repeated the same process for abdomens and salivary glands. Results Our data show heterogeneity in the transmissibility of Plasmodium among ornithophillic mosquito species. More specifically, Culex stigmatosoma appears to not be a vector of Plasmodium homopolare, a parasite that is prevalent in the avian population, but is a vector of multiple other Plasmodium species/lineages. Conclusions Our results suggest that conclusions made on the role of vectors from studies that do not use different mosquito trapping methods should be re-evaluated with caution, as we documented the potential for trapping biases, which may cause studies to miss important roles of specific mosquito species in the transmission of avian malaria. Moreover, we document heterogeneity in the transmission of Plasmodium spp. by mosquitoes can influence Plasmodium diversity and prevalence in specific locations to Plasmodium-vector incompatibilities

Microbes in beach sands : integrating environment, ecology and public health

Author Posting. © The Author(s), 2014. This is the author's version of the work. It is posted here by permission of Springer for personal use, not for redistribution. The definitive version was published in Reviews in Environmental Science and Bio/Technology 13 (2014): 329-368, doi:10.1007/s11157-014-9340-8.Beach sand is a habitat that supports many microbes, including viruses, bacteria, fungi and protozoa (micropsammon). The apparently inhospitable conditions of beach sand environments belie the thriving communities found there. Physical factors, such as water availability and protection from insolation; biological factors, such as competition, predation, and biofilm formation; and nutrient availability all contribute to the characteristics of the micropsammon. Sand microbial communities include autochthonous species/phylotypes indigenous to the environment. Allochthonous microbes, including fecal indicator bacteria (FIB) and waterborne pathogens, are deposited via waves, runoff, air, or animals. The fate of these microbes ranges from death, to transient persistence and/or replication, to establishment of thriving populations (naturalization) and integration in the autochthonous community. Transport of the micropsammon within the habitat occurs both horizontally across the beach, and vertically from the sand surface and ground water table, as well as at various scales including interstitial flow within sand pores, sediment transport for particle-associated microbes, and the large-scale processes of wave action and terrestrial runoff. The concept of beach sand as a microbial habitat and reservoir of FIB and pathogens has begun to influence our thinking about human health effects associated with sand exposure and recreational water use. A variety of pathogens have been reported from beach sands, and recent epidemiology studies have found some evidence of health risks associated with sand exposure. Persistent or replicating populations of FIB and enteric pathogens have consequences for watershed/beach management strategies and regulatory standards for safe beaches. This review summarizes our understanding of the community structure, ecology, fate, transport, and public health implications of microbes in beach sand. It concludes with recommendations for future work in this vastly under-studied area.2015-05-0

Understanding the errors of SHAPE-directed RNA structure modeling

Single-nucleotide-resolution chemical mapping for structured RNA is being rapidly advanced by new chemistries, faster readouts, and coupling to computational algorithms. Recent tests have shown that selective 2'-hydroxyl acylation by primer extension (SHAPE) can give near-zero error rates (0-2%) in modeling the helices of RNA secondary structure. Here, we benchmark the method using six molecules for which crystallographic data are available: tRNA(phe) and 5S rRNA from Escherichia coli, the P4-P6 domain of the Tetrahymena group I ribozyme, and ligand-bound domains from riboswitches for adenine, cyclic di-GMP, and glycine. SHAPE-directed modeling of these highly structured RNAs gave an overall false negative rate (FNR) of 17% and a false discovery rate (FDR) of 21%, with at least one helix prediction error in five of the six cases. Extensive variations of data processing, normalization, and modeling parameters did not significantly mitigate modeling errors. Only one varation, filtering out data collected with deoxyinosine triphosphate during primer extension, gave a modest improvement (FNR = 12%, and FDR = 14%). The residual structure modeling errors are explained by the insufficient information content of these RNAs' SHAPE data, as evaluated by a nonparametric bootstrapping analysis. Beyond these benchmark cases, bootstrapping suggests a low level of confidence (<50%) in the majority of helices in a previously proposed SHAPE-directed model for the HIV-1 RNA genome. Thus, SHAPE-directed RNA modeling is not always unambiguous, and helix-by-helix confidence estimates, as described herein, may be critical for interpreting results from this powerful methodology.Comment: Biochemistry, Article ASAP (Aug. 15, 2011

Sub region-specific modulation of synchronous neuronal burst firing after a kainic acid insult in organotypic hippocampal cultures

<p>Abstract</p> <p>Background</p> <p>Excitotoxicity occurs in a number of pathogenic states including stroke and epilepsy. The adaptations of neuronal circuits in response to such insults may be expected to play an underlying role in pathogenesis. Synchronous neuronal firing can be induced in isolated hippocampal slices and involves all regions of this structure, thereby providing a measure of circuit activity. The effect of an excitotoxic insult (kainic acid, KA) on Mg²⁺-free-induced synchronized neuronal firing was tested in organotypic hippocampal culture by measuring extracellular field activity in CA1 and CA3.</p> <p>Results</p> <p>Within 24 hrs of the insult regional specific changes in neuronal firing patterns were evident as: (i) a dramatic <it>reduction </it>in the ability of CA3 to generate firing; and (ii) a contrasting <it>increase </it>in the frequency and duration of synchronized neuronal firing events in CA1. Two distinct processes underlie the increased propensity of CA1 to generate synchronized burst firing; a lack of ability of the CA3 region to 'pace' CA1 resulting in an increased frequency of synchronized events; and a change in the 'intrinsic' properties limited to the CA1 region, which is responsible for increased event duration. Neuronal quantification using NeuN immunoflurescent staining and stereological confocal microscopy revealed no significant cell loss in hippocampal sub regions, suggesting that changes in the properties of neurons within this region were responsible for the KA-mediated excitability changes.</p> <p>Conclusion</p> <p>These results provide novel insight into adaptation of hippocampal circuits following excitotoxic injury. KA-mediated disruption of the interplay between CA3 and CA1 clearly increases the propensity to synchronized firing in CA1.</p

Culture Enriched Molecular Profiling of the Cystic Fibrosis Airway Microbiome

The microbiome of the respiratory tract, including the nasopharyngeal and oropharyngeal microbiota, is a dynamic community of microorganisms that is highly diverse. The cystic fibrosis (CF) airway microbiome refers to the polymicrobial communities present in the lower airways of CF patients. It is comprised of chronic opportunistic pathogens (such as Pseudomonas aeruginosa) and a variety of organisms derived mostly from the normal microbiota of the upper respiratory tract. The complexity of these communities has been inferred primarily from culture independent molecular profiling. As with most microbial communities it is generally assumed that most of the organisms present are not readily cultured. Our culture collection generated using more extensive cultivation approaches, reveals a more complex microbial community than that obtained by conventional CF culture methods. To directly evaluate the cultivability of the airway microbiome, we examined six samples in depth using culture-enriched molecular profiling which combines culture-based methods with the molecular profiling methods of terminal restriction fragment length polymorphisms and 16S rRNA gene sequencing. We demonstrate that combining culture-dependent and culture-independent approaches enhances the sensitivity of either approach alone. Our techniques were able to cultivate 43 of the 48 families detected by deep sequencing; the five families recovered solely by culture-independent approaches were all present at very low abundance (<0.002% total reads). 46% of the molecular signatures detected by culture from the six patients were only identified in an anaerobic environment, suggesting that a large proportion of the cultured airway community is composed of obligate anaerobes. Most significantly, using 20 growth conditions per specimen, half of which included anaerobic cultivation and extended incubation times we demonstrate that the majority of bacteria present can be cultured

Search of the Orion spur for continuous gravitational waves using a loosely coherent algorithm on data from LIGO interferometers

We report results of a wideband search for periodic gravitational waves from isolated neutron stars within the Orion spur towards both the inner and outer regions of our Galaxy. As gravitational waves interact very weakly with matter, the search is unimpeded by dust and concentrations of stars. One search disk (A) is 6.87° in diameter and centered on 20h10m54.71s+33°33′25.29′′, and the other (B) is 7.45° in diameter and centered on 8h35m20.61s-46°49′25.151′′. We explored the frequency range of 50-1500 Hz and frequency derivative from 0 to -5×10-9 Hz/s. A multistage, loosely coherent search program allowed probing more deeply than before in these two regions, while increasing coherence length with every stage. Rigorous follow-up parameters have winnowed the initial coincidence set to only 70 candidates, to be examined manually. None of those 70 candidates proved to be consistent with an isolated gravitational-wave emitter, and 95% confidence level upper limits were placed on continuous-wave strain amplitudes. Near 169 Hz we achieve our lowest 95% C.L. upper limit on the worst-case linearly polarized strain amplitude h0 of 6.3×10-25, while at the high end of our frequency range we achieve a worst-case upper limit of 3.4×10-24 for all polarizations and sky locations. © 2016 American Physical Society