Unsupervised discovery of microbial population structure within metagenomes using nucleotide base composition

An approach to infer the unknown microbial population structure within a metagenome is to cluster nucleotide sequences based on common patterns in base composition, otherwise referred to as binning. When functional roles are assigned to the identified populations, a deeper understanding of microbial communities can be attained, more so than gene-centric approaches that explore overall functionality. In this study, we propose an unsupervised, model-based binning method with two clustering tiers, which uses a novel transformation of the oligonucleotide frequency-derived error gradient and GC content to generate coarse groups at the first tier of clustering; and tetranucleotide frequency to refine these groups at the secondary clustering tier. The proposed method has a demonstrated improvement over PhyloPythia, S-GSOM, TACOA and TaxSOM on all three benchmarks that were used for evaluation in this study. The proposed method is then applied to a pyrosequenced metagenomic library of mud volcano sediment sampled in southwestern Taiwan, with the inferred population structure validated against complementary sequencing of 16S ribosomal RNA marker genes. Finally, the proposed method was further validated against four publicly available metagenomes, including a highly complex Antarctic whale-fall bone sample, which was previously assumed to be too complex for binning prior to functional analysis.     

A spatial cluster analysis of tractor overturns in Kentucky from 1960 to 2002

Background

Agricultural tractor overturns without rollover protective structures are the leading cause of farm fatalities in the United States. To our knowledge, no studies have incorporated the spatial scan statistic in identifying high-risk areas for tractor overturns. The aim of this study was to determine whether tractor overturns cluster in certain parts of Kentucky and identify factors associated with tractor overturns.

Methods

A spatial statistical analysis using Kulldorff''s spatial scan statistic was performed to identify county clusters at greatest risk for tractor overturns. A regression analysis was then performed to identify factors associated with tractor overturns.

Results

The spatial analysis revealed a cluster of higher than expected tractor overturns in four counties in northern Kentucky (RR = 2.55) and 10 counties in eastern Kentucky (RR = 1.97). Higher rates of tractor overturns were associated with steeper average percent slope of pasture land by county (p = 0.0002) and a greater percent of total tractors with less than 40 horsepower by county (p<0.0001).

Conclusions

This study reveals that geographic hotspots of tractor overturns exist in Kentucky and identifies factors associated with overturns. This study provides policymakers a guide to targeted county-level interventions (e.g., roll-over protective structures promotion interventions) with the intention of reducing tractor overturns in the highest risk counties in Kentucky.     

Voltage clamp fluorimetry reveals a novel outer pore instability in a mammalian voltage-gated potassium channel

Voltage-gated potassium (Kv) channel gating involves complex structural rearrangements that regulate the ability of channels to conduct K(+) ions. Fluorescence-based approaches provide a powerful technique to directly report structural dynamics underlying these gating processes in Shaker Kv channels. Here, we apply voltage clamp fluorimetry, for the first time, to study voltage sensor motions in mammalian Kv1.5 channels. Despite the homology between Kv1.5 and the Shaker channel, attaching TMRM or PyMPO fluorescent probes to substituted cysteine residues in the S3-S4 linker of Kv1.5 (M394C-V401C) revealed unique and unusual fluorescence signals. Whereas the fluorescence during voltage sensor movement in Shaker channels was monoexponential and occurred with a similar time course to ionic current activation, the fluorescence report of Kv1.5 voltage sensor motions was transient with a prominent rapidly dequenching component that, with TMRM at A397C (equivalent to Shaker A359C), represented 36 +/- 3% of the total signal and occurred with a tau of 3.4 +/- 0.6 ms at +60 mV (n = 4). Using a number of approaches, including 4-AP drug block and the ILT triple mutation, which dissociate channel opening from voltage sensor movement, we demonstrate that the unique dequenching component of fluorescence is associated with channel opening. By regulating the outer pore structure using raised (99 mM) external K(+) to stabilize the conducting configuration of the selectivity filter, or the mutations W472F (equivalent to Shaker W434F) and H463G to stabilize the nonconducting (P-type inactivated) configuration of the selectivity filter, we show that the dequenching of fluorescence reflects rapid structural events at the selectivity filter gate rather than the intracellular pore gate.     

Site-directed mutagenesis of firefly luciferase: implication of conserved residue(s) in bioluminescence emission spectra among firefly luciferases

Eukaryotic-type serine/threonine protein kinases in bacteria have been implicated in controlling a host of cellular activities. PknA is one of eleven such protein kinases from Mycobacterium tuberculosis which regulates morphological changes associated with cell division. In the present study we provide the evidence for the ability of PknA to transphosphorylate mMurD (mycobacterial UDP-N-acetylmuramoyl-L-alanine:D-glutamate-ligase), the enzyme involved in peptidoglycan biosynthesis. Its co-expression in Escherichia coli along with PknA resulted in phosphorylation of mMurD. Consistent with these observations, results of the solid-phase binding assays revealed a high-affinity in vitro binding between the two proteins. Furthermore, overexpression of m-murD in Mycobacterium smegmatis yielded a phosphorylated protein. The results of the present study therefore point towards the possibility of mMurD being a substrate of PknA.     

Novel anti-inflammatory mechanisms of N-Acetyl-Ser-Asp-Lys-Pro in hypertension-induced target organ damage

High blood pressure (HBP) is an important risk factor for cardiac, renal, and vascular dysfunction. Excess inflammation is the major pathogenic mechanism for HBP-induced target organ damage (TOD). N-acetyl-Ser-Asp-Lys-Pro (Ac-SDKP), a tetrapeptide specifically degraded by angiotensin converting enzyme (ACE), reduces inflammation, fibrosis, and TOD induced by HBP. Our hypothesis is that Ac-SDKP exerts its anti-inflammatory effects by inhibiting: 1) differentiation of bone marrow stem cells (BMSC) to macrophages, 2) activation and migration of macrophages, and 3) release of the proinflammatory cytokine TNF-alpha by activated macrophages. BMSC were freshly isolated and cultured in macrophage growth medium. Differentiation of murine BMSC to macrophages was analyzed by flow cytometry using F4/80 as a marker of macrophage maturation. Macrophage migration was measured in a modified Boyden chamber. TNF-alpha release by activated macrophages in culture was measured by ELISA. Myocardial macrophage activation in mice with ANG II-induced hypertension was studied by Western blotting of Mac-2 (galectin-3) protein. Interstitial collagen deposition was measured by picrosirius red staining. We found that Ac-SDKP (10 nM) reduced differentiation of cultured BMSC to mature macrophages by 24.5% [F4/80 positivity: 14.09 +/- 1.06 mean fluorescent intensity for vehicle and 10.63 +/- 0.35 for Ac-SDKP; P < 0.05]. Ac-SDKP also decreased galectin-3 and macrophage colony-stimulating factor-dependent macrophage migration. In addition, Ac-SDKP decreased secretion of TNF-alpha by macrophages stimulated with bacterial LPS. In mice with ANG II-induced hypertension, Ac-SDKP reduced expression of galectin-3, a protein produced by infiltrating macrophages in the myocardium, and interstitial collagen deposition. In conclusion, this study demonstrates that part of the anti-inflammatory effect of Ac-SDKP is due to its direct effect on BMSC and macrophage, inhibiting their differentiation, activation, and cytokine release. These effects explain some of the anti-inflammatory and antifibrotic properties of Ac-SDKP in hypertension.     

DNA organization by the apicoplast-targeted bacterial histone-like protein of Plasmodium falciparum

Apicomplexans, including the pathogens Plasmodium and Toxoplasma, carry a nonphotosynthetic plastid of secondary endosymbiotic origin called the apicoplast. The P. falciparum apicoplast contains a 35 kb, circular DNA genome with limited coding capacity that lacks genes encoding proteins for DNA organization and replication. We report identification of a nuclear-encoded bacterial histone-like protein (PfHU) involved in DNA compaction in the apicoplast. PfHU is associated with apicoplast DNA and is expressed throughout the parasite's intra-erythocytic cycle. The protein binds DNA in a sequence nonspecific manner with a minimum binding site length of ~27 bp and a K_d of ~63 nM and displays a preference for supercoiled DNA. PfHU is capable of condensing Escherichia coli nucleoids in vivo indicating its role in DNA compaction. The unique 42 aa C-terminal extension of PfHU influences its DNA condensation properties. In contrast to bacterial HUs that bend DNA, PfHU promotes concatenation of linear DNA and inhibits DNA circularization. Atomic Force Microscopic study of PfHU–DNA complexes shows protein concentration-dependent DNA stiffening, intermolecular bundling and formation of DNA bridges followed by assembly of condensed DNA networks. Our results provide the first functional characterization of an apicomplexan HU protein and provide additional evidence for red algal ancestry of the apicoplast.     

Polo Kinase Interacts with RacGAP50C and Is Required to Localize the Cytokinesis Initiation Complex

The assembly and constriction of an actomyosin contractile ring in cytokinesis is dependent on the activation of Rho at the equatorial cortex by a complex, here termed the cytokinesis initiation complex, between a microtubule-associated kinesin-like protein (KLP), a member of the RacGAP family, and the RhoGEF Pebble. Recently, the activity of the mammalian Polo kinase ortholog Plk1 has been implicated in the formation of this complex. We show here that Polo kinase interacts directly with the cytokinesis initiation complex by binding RacGAP50C. We find that a new domain of Polo kinase, termed the intermediate domain, interacts directly with RacGAP50C and that Polo kinase is essential for localization of the KLP-RacGAP centralspindlin complex to the cell equator and spindle midzone. In the absence of Polo kinase, RacGAP50C and Pav-KLP fail to localize normally, instead decorating microtubules along their length. Our results indicate that Polo kinase directly binds the conserved cytokinesis initiation complex and is required to trigger centralspindlin localization as a first step in cytokinesis.     

Phosphorylation of Wat1, human Lst8 homolog is critical for the regulation of TORC2 –Gad8 dependent pathway in fission yeast Schizosacchromyces pombe

Mammalian Lst8 interacts with the kinase domain of mTOR and stabilizes its interaction with Raptor regulating cell growth through the mTOR-S6K1 signalling pathway. Fission yeast Wat1, an ortholog of mammalian Lst8 is also an essential component of TOR complex 1 (TORC1) and TOR Complex 2 (TORC2) that control protein kinases essential for metabolic pathways. Here, we show that in response to osmotic stress, the Wat1 protein undergoes hyper-phosphorylation at S116 position. Wat1 interacts with the C-terminal region of Tor1 that also contain kinase domain. Co-immunoprecipitation and molecular modelling studies suggest that Wat1-Tor1 interaction is stabilized by FATC domain of Tor1 protein present at the C-terminal region. We have also demonstrated a physical interaction of Wat1 with Gad8, an AGC family protein kinase that is dependent on phosphorylation of Wat1 at S116 residue. Wat1 phosphorylation is required for the maintenance of vacuolar integrity and sexual differentiation. Collectively, our study reveals Wat1 phosphorylation regulates Gad8 function in a manner dependent on Tor1 interaction.     

HspX protein as a candidate vaccine against <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis>: an overview

Background

Tuberculosis (TB) is a contagious infectious disease caused by Mycobacterium tuberculosis (Mtb). This disease with two million deaths per year has the highest mortality rate among bacterial infections. The only available vaccine against TB is BCG vaccine. BCG is an effective vaccine against TB in childhood, however, due to some limitations, has not proper efficiency in adults. Also, BCG cannot produce an adequately protective response against reactivation of latent infections.

Objective

In the present study we will review the most recent findings about contribution of HspX protein in the vaccines against tuberculosis.

Methods

Therefore, many attempts have been made to improve BCG or to find its replacement. Most of the subunit vaccines for TB in various phases of clinical trials were constructed as prophylactic vaccines using Mtb proteins expressed in the replicating stage. These vaccines might prevent active TB but not reactivation of latent tuberculosis infection (LTBI). A literature search was performed on various online databases (PubMed, Scopus, and Google Scholar) regarding the roles of HspX protein in tuberculosis vaccines.

Results

Ideal subunit post-exposure vaccines should target all forms of TB infection, including active symptomatic and dormant (latent) asymptomatic forms. Among these subunit vaccines, HspX is the most important latent phase antigen of M. tuberculosis with a strong immunological response. There are many studies that have evaluated the immunogenicity of this protein to improve TB vaccine.

Conclusion

According to the studies, HspX protein is a good candidate for development of subunit vaccines against TB infection.