Draft Genome Sequence of the Tomato Stem Endophyte Bacillus safensis TS3.

Tomato stem endophyte Bacillus safensis TS3 was isolated from surface-sterilized stems of greenhouse tomato plants. Here, we sequenced the complete genome of this strain to understand the molecular mechanisms underlying its beneficial activities.

Characterization of Astaxanthin Nanoemulsions Produced by Intense Fluid Shear through a Self-Throttling Nanometer Range Annular Orifice Valve-Based High-Pressure Homogenizer.

Stable, oil-in-water nanoemulsions containing astaxanthin (AsX) were produced by intense fluid shear forces resulting from pumping a coarse reagent emulsion through a self-throttling annular gap valve at 300 MPa. Compared to crude emulsions prepared by conventional homogenization, a size reduction of over two orders of magnitude was observed for AsX-encapsulated oil droplets following just one pass through the annular valve. In krill oil formulations, the mean hydrodynamic diameter of lipid particles was reduced to 60 nm after only two passes through the valve and reached a minimal size of 24 nm after eight passes. Repeated processing of samples through the valve progressively decreased lipid particle size, with an inflection in the rate of particle size reduction generally observed after 2-4 passes. Krill- and argan oil-based nanoemulsions were produced using an Ultra Shear Technology™ (UST™) approach and characterized in terms of their small particle size, low polydispersity, and stability.

High-pressure small-angle X-ray scattering cell for biological solutions and soft materials.

Pressure is a fundamental thermodynamic parameter controlling the behavior of biological macromolecules. Pressure affects protein denaturation, kinetic parameters of enzymes, ligand binding, membrane permeability, ion trans-duction, expression of genetic information, viral infectivity, protein association and aggregation, and chemical processes. In many cases pressure alters the molecular shape. Small-angle X-ray scattering (SAXS) is a primary method to determine the shape and size of macromolecules. However, relatively few SAXS cells described in the literature are suitable for use at high pressures and with biological materials. Described here is a novel high-pressure SAXS sample cell that is suitable for general facility use by prioritization of ease of sample loading, temperature control, mechanical stability and X-ray background minimization. Cell operation at 14 keV is described, providing a q range of 0.01 < q < 0.7 Å-1, pressures of 0-400 MPa and an achievable temperature range of 0-80°C. The high-pressure SAXS cell has recently been commissioned on the ID7A beamline at the Cornell High Energy Synchrotron Source and is available to users on a peer-reviewed proposal basis.

Draft Genome Sequence of Plant Growth-Promoting Bacillus velezensis BS89.

The plant growth-promoting bacterium Bacillus velezensis BS89 was isolated from the rhizosphere of winter wheat. Strain BS89 has the ability to promote plant growth and produce a mix of auxins and vitamins. Here, we sequenced the complete genome of this strain to understand the molecular mechanisms underlying its beneficial activities.

Optical Diagnostics of Supercritical CO2 and CO2-Ethanol Mixture in the Widom Delta.

The supercritical CO2 (scCO2) is widely used as solvent and transport media in different technologies. The technological aspects of scCO2 fluid applications strongly depend on spatial-temporal fluctuations of its thermodynamic parameters. The region of these parameters' maximal fluctuations on the p-T (pressure-temperature) diagram is called Widom delta. It has significant practical and fundamental interest. We offer an approach that combines optical measurements and molecular dynamics simulation in a wide range of pressures and temperatures. We studied the microstructure of supercritical CO2 fluid and its binary mixture with ethanol in a wide range of temperatures and pressures using molecular dynamics (MD) simulation. MD is used to retrieve a set of optical characteristics such as Raman spectra, refractive indexes and molecular refraction and was verified by appropriate experimental measurements. We demonstrated that in the Widom delta the monotonic dependence of the optical properties on the CO2 density is violated. It is caused by the rapid increase of density fluctuations and medium-sized (20-30 molecules) cluster formation. We identified the correlation between cluster parameters and optical properties of the media; in particular, it is established that the clusters in the Widom delta acts as a seed for clustering in molecular jets. MD demonstrates that the cluster formation is stronger in the supercritical CO2-ethanol mixture, where the extended binary clusters are formed; that is, the nonlinear refractive index significantly increased. The influence of the supercritical state in the cell on the formation of supersonic cluster jets is studied using the Mie scattering technique.

Quantitative High-Resolution Imaging of Live Microbial Cells at High Hydrostatic Pressure.

The majority of the Earth's microbial biomass exists in the deep biosphere, in the deep ocean, and within the Earth's crust. Although other physical parameters in these environments, such as temperature or pH, can differ substantially, they are all under high pressures. Beyond emerging genomic information, little is known about the molecular mechanisms underlying the ability of these organisms to survive and grow at pressures that can reach over 1000-fold the pressure on the Earth's surface. The mechanisms of pressure adaptation are also important in food safety, with the increasing use of high-pressure food processing. Advanced imaging represents an important tool for exploring microbial adaptation and response to environmental changes. Here, we describe implementation of a high-pressure sample chamber with a two-photon scanning microscope system, allowing for the first time, to our knowledge, quantitative high-resolution two-photon imaging at 100 MPa of living microbes from all three kingdoms of life. We adapted this setup for fluorescence lifetime imaging microscopy with phasor analysis (FLIM/Phasor) and investigated metabolic responses to pressure of live cells from mesophilic yeast and bacterial strains, as well as the piezophilic archaeon Archaeoglobus fulgidus. We also monitored by fluorescence intensity fluctuation-based methods (scanning number and brightness and raster scanning imaging correlation spectroscopy) the effect of pressure on the chromosome-associated protein HU and on the ParB partition protein in Escherichia coli, revealing partially reversible dissociation of ParB foci and concomitant nucleoid condensation. These results provide a proof of principle that quantitative, high-resolution imaging of live microbial cells can be carried out at pressures equivalent to those in the deepest ocean trenches.

Molecular Epitope Determination of Aptamer Complexes of the Multidomain Protein C-Met by Proteolytic Affinity-Mass Spectrometry.

C-Met protein is a glycosylated receptor tyrosine kinase of the hepatocyte growth factor (HGF), composed of an α and a ß chain. Upon ligand binding, C-Met transmits intracellular signals by a unique multi-substrate docking site. C-Met can be aberrantly activated leading to tumorigenesis and other diseases, and has been recognized as a biomarker in cancer diagnosis. C-Met aptamers have been recently considered a useful tool for detection of cancer biomarkers. Herein we report a molecular interaction study of human C-Met expressed in kidney cells with two DNA aptamers of 60 and 64 bases (CLN0003 and CLN0004), obtained using the SELEX (Systematic Evolution of Ligands by Exponential Enrichment) procedure. Epitope peptides of aptamer-C-Met complexes were identified by proteolytic affinity-mass spectrometry in combination with SPR biosensor analysis (PROTEX-SPR-MS), using high-pressure proteolysis for efficient digestion. High affinities (KD , 80-510 nM) were determined for aptamer-C-Met complexes, with two-step binding suggested by kinetic analysis. A linear epitope, C-Met (381-393) was identified for CLN0004, while the CLN0003 aptamer revealed an assembled epitope comprised of two peptide sequences, C-Met (524-543) and C-Met (557-568). Structure modeling of C-Met-aptamers were consistent with the identified epitopes. Specificities and affinities were ascertained by SPR analysis of the synthetic epitope peptides. The high affinities of aptamers to C-Met, and the specific epitopes revealed render them of high interest for cellular diagnostic studies.

Complete Genome Sequences of Endophytic Bacilli Isolated from Grapevine Plants.

The endophytic strains Bacillus amyloliquefaciens V417 and V167 were isolated from cultured grape plants. We sequenced the complete genomes of these strains to reveal their potential beneficial properties for plant growth promotion and control of fungal pathogens. Genes responsible for the synthesis of antimicrobial compounds and siderophores were identified.

Long-range dispersion C6 coefficient for SF6 dimer: Experimental and theoretical study.

The long-range dispersion C6 coefficient for the SF6 dimer is experimentally measured using a technique that uses the expansion of a supersonic pulse jet into a vacuum. A dynamic model of the jet enables us to correlate the position of the maximal peak in the time-of-flight spectrum with the initial conditions of the experiment and the parameters of the intermolecular interaction potential. Due to the low temperature of the jet target, the C6 coefficient can be extracted directly from the experimental results. Theoretical calculation of the C6 dispersion coefficient is also performed by using linearly approximated explicitly correlated coupled-cluster singles and doubles (CCSD(F12)) method with the subsequent utilization of the Casimir-Polder formula. Good agreement of experimental and theoretical results confirms the reliability of results.

Epitope Ligand Binding Sites of Blood Group Oligosaccharides in Lectins Revealed by Pressure-Assisted Proteolytic Excision Affinity Mass Spectrometry.

Affinity mass spectrometry using selective proteolytic excision and extraction combined with MALDI and ESI mass spectrometry has been applied to the identification of epitope binding sites of lactose, GalNac, and blood group oligosaccharides in two blood group-specific lectins, human galectin-3 and glycine max lectin. The epitope peptides identified comprise all essential amino acids involved in carbohydrate recognition, in complete agreement with available X-ray structures. Tryptic and chymotryptic digestion of lectins for proteolytic extraction/excision-MS was substantially improved by pressure-enhanced digestion using an automated Barocycler procedure (40 kpsi). Both previously established immobilization on affinity microcolumns using divinyl sulfone and coupling of a specific peptide glycoprobe to the gold surface of a biosensor chip were successfully employed for proteolytic excision and extraction of carbohydrate epitopes and affinity measurements. The identified epitope peptides could be differentiated according to the carbohydrate employed, thus demonstrating the specificity of the mass spectrometric approach. The specificities of the epitope ligands for individual carbohydrates were further ascertained by affinity studies using synthetic peptide ligands with immobilized carbohydrates. Binding affinities of the synthetic ligand peptides to lactose, in comparison to the intact full-length lectins, were determined by surface acoustic wave (SAW) biosensor analysis and provided micromolar KD values for the intact lectins, in agreement with results of previous ITC and SPR studies. Binding affinities of the epitope peptides were approximately two orders of magnitude lower, consistent with their smaller size and assembled arrangement in the carbohydrate recognition domains. Graphical Abstract ᅟ.

Reproducible Tissue Homogenization and Protein Extraction for Quantitative Proteomics Using MicroPestle-Assisted Pressure-Cycling Technology.

The reproducible and efficient extraction of proteins from biopsy samples for quantitative analysis is a critical step in biomarker and translational research. Recently, we described a method consisting of pressure-cycling technology (PCT) and sequential windowed acquisition of all theoretical fragment ions-mass spectrometry (SWATH-MS) for the rapid quantification of thousands of proteins from biopsy-size tissue samples. As an improvement of the method, we have incorporated the PCT-MicroPestle into the PCT-SWATH workflow. The PCT-MicroPestle is a novel, miniaturized, disposable mechanical tissue homogenizer that fits directly into the microTube sample container. We optimized the pressure-cycling conditions for tissue lysis with the PCT-MicroPestle and benchmarked the performance of the system against the conventional PCT-MicroCap method using mouse liver, heart, brain, and human kidney tissues as test samples. The data indicate that the digestion of the PCT-MicroPestle-extracted proteins yielded 20-40% more MS-ready peptide mass from all tissues tested with a comparable reproducibility when compared to the conventional PCT method. Subsequent SWATH-MS analysis identified a higher number of biologically informative proteins from a given sample. In conclusion, we have developed a new device that can be seamlessly integrated into the PCT-SWATH workflow, leading to increased sample throughput and improved reproducibility at both the protein extraction and proteomic analysis levels when applied to the quantitative proteomic analysis of biopsy-level samples.

Immunosignature: Serum Antibody Profiling for Cancer Diagnostics.

Biomarkers for preclinical diagnosis of cancer are valuable tools for detection of malignant tumors at early stages in groups at risk and screening healthy people, as well as monitoring disease recurrence after treatment of cancer. However the complexity of the body's response to the pathological processes makes it virtually impossible to evaluate this response to the development of the disease using a single biomarker that is present in the serum at low concentrations. An alternative approach to standard biomarker analysis is called immunosignature. Instead of going after biomarkers themselves this approach rely on the analysis of the humoral immune response to molecular changes associated with the development of pathological processes. It is known that antibodies are produced in response to proteins expressed during cancer development. Accordingly, the changes in antibody repertoire associated with tumor growth can serve as biomarkers of cancer. Immunosignature is a highly sensitive method for antibody repertoire analysis utilizing high density peptide microarrays. In the present review we discuss modern methods for antibody detection, as well as describe the principles and applications of immunosignature in research and clinical practice.

Alcohol and mortality in Russia: prospective observational study of 151,000 adults.

BACKGROUND: Russian adults have extraordinarily high rates of premature death. Retrospective enquiries to the families of about 50,000 deceased Russians had found excess vodka use among those dying from external causes (accident, suicide, violence) and eight particular disease groupings. We now seek prospective evidence of these associations. METHODS: In three Russian cities (Barnaul, Byisk, and Tomsk), we interviewed 200,000 adults during 1999-2008 (with 12,000 re-interviewed some years later) and followed them until 2010 for cause-specific mortality. In 151,000 with no previous disease and some follow-up at ages 35-74 years, Poisson regression (adjusted for age at risk, amount smoked, education, and city) was used to calculate the relative risks associating vodka consumption with mortality. We have combined these relative risks with age-specific death rates to get 20-year absolute risks. FINDINGS: Among 57,361 male smokers with no previous disease, the estimated 20-year risks of death at ages 35-54 years were 16% (95% CI 15-17) for those who reported consuming less than a bottle of vodka per week at baseline, 20% (18-22) for those consuming 1-2·9 bottles per week, and 35% (31-39) for those consuming three or more bottles per week; trend p<0·0001. The corresponding risks of death at ages 55-74 years were 50% (48-52) for those who reported consuming less than a bottle of vodka per week at baseline, 54% (51-57) for those consuming 1-2·9 bottles per week, and 64% (59-69) for those consuming three or more bottles per week; trend p<0·0001. In both age ranges most of the excess mortality in heavier drinkers was from external causes or the eight disease groupings strongly associated with alcohol in the retrospective enquiries. Self-reported drinking fluctuated; of the men who reported drinking three or more bottles of vodka per week who were reinterviewed a few years later, about half (185 of 321) then reported drinking less than one bottle per week. Such fluctuations must have substantially attenuated the apparent hazards of heavy drinking in this study, yet self-reported vodka use at baseline still strongly predicted risk. Among male non-smokers and among females, self-reported heavy drinking was uncommon, but seemed to involve similar absolute excess risks. INTERPRETATION: This large prospective study strongly reinforces other evidence that vodka is a major cause of the high risk of premature death in Russian adults. FUNDING: UK Medical Research Council, British Heart Foundation, Cancer Research UK, European Union, WHO International Agency for Research on Cancer.

Pressure cycling technology (PCT) reduces effects of inhibitors of the PCR.

A common problem in the analysis of forensic human DNA evidence, or for that matter any nucleic acid analysis, is the presence of contaminants or inhibitors. Contaminants may copurify with the DNA, inhibiting downstream PCR or they may present samples effectively as containing fewer templates than exist in the PCR, even when the actual amount of DNA is adequate. Typically, these challenged samples exhibit allele imbalance, allele dropout, and sequence-specific inhibition, leading to interpretational difficulties. Lessening the effects of inhibitors may increase the effective yield of challenged low template copy samples. High pressure may alter some inhibitors and render them less effective at reducing the yield of PCR products. In an attempt to enhance the amplicon yield of inhibited DNA samples, pressure cycling technology was applied to DNA exposed to various concentrations of hematin (0, 1.25, 2.5, 5, and 7 µM) and humic acid (0, 1.25, 2.5, 5, and 7 ng/µL). The effect of high pressure on the inhibitors, and subsequently the PCR process, was assessed by measuring DNA quantity by quantitative PCR and evaluating short tandem repeat typing results. The results support that pressure cycling technology reduces inhibitory effects and thus, in effect, enhances yield of contaminated amplified products of both hematin and humic acid contaminate samples. Based on the results obtained in this study, this method can improve the ability to type challenged or inhibited DNA samples.

Method development for fecal lipidomics profiling.

Robust methodologies for the analysis of fecal material will facilitate the understanding of gut (patho)physiology and its role in health and disease and will help improve care for individual patients, especially high-risk populations, such as premature infants. Because lipidomics offers a biologically and analytically attractive approach, we developed a simple, sensitive, and quantitatively precise method for profiling intact lipids in fecal material. The method utilizes two separate, complementary extraction chemistries, dichloromethane (DCM) and a methyl tert-butyl ether/hexafluoroisopropanol (MTBE) mixture, alone or with high pressure cycling. Extracts were assessed by liquid chromatography-high-resolution mass spectrometry-based profiling with all ion higher energy collisional dissociation fragmentation in both positive and negative ionization modes. This approach provides both class-specific and lipid-specific fragments, enhancing lipid characterization. Solvents preferentially extracted lipids based on hydrophobicity. More polar species preferred MTBE; more hydrophobic compounds preferred DCM. Pressure cycling differentially increased the yield of some lipids. The platform enabled analysis of >500 intact lipophilic species with over 300 lipids spanning 6 LIPID MAPS categories identified in the fecal matter from premature infants. No previous report exists that provides these data; thus, this study represents a new paradigm for assessing nutritional health, inflammation, and infectious disease in vulnerable populations.

Selective reduction of ketones using water as a hydrogen source under high hydrostatic pressure.

A selective reduction of a broad variety of ketones is described. The method is based on the combination of a Ni-Al alloy and high hydrostatic pressure (HHP, 2.8 kbar) in an aqueous medium. The reaction of the Ni-Al alloy with water provides in situ hydrogen generation and the high pressure ensures that the H(2) formed remains in the solution, thus the C=O reduction readily occurs. The application of the HHP resulted in selective formation of the desired products and the common problem of non-selective overhydrogenation could be avoided. In most cases the reductions resulted in high yields and excellent selectivities without the use of any base.

Prognostic Value of Microvessel Density in Tumor and Peritumoral Area as Evaluated by CD31 Protein Expression and Argyrophilic Nucleolar Organizer Region Count in Endothelial Cells in Uterine Leiomyosarcoma.

The objective of this study was to investigate the prognostic value of microvessel density (MVD) in uterine leiomyosarcoma (LMS) and peritumoral area (PA) as evaluated by CD31 expression and argyrophilic nucleolar organizer region (AgNOR) count in endothelial cells. Tissue specimens from 66 patients with uterine LMS were examined. There were no significant differences in the mean MVD between tumor itself and the PA (P = 0.9); moreover, the MVD in the PA often exceeded that in the tumor. No correlation or significant differences were also found in the MVD between different grades of malignancy of LMS (r = 0.1; P = 0.07). The number of AgNORs in tumor endothelial cells was significantly higher in tumor vessels than in the peritumoral area (P < 0.005) and increased with the tumor grade. Analysis of the prognostic value of MVD in uterine LMS and PA showed that the density of tumor vessels was not an independent criterion, while the MVD in the PA affected 10-year survival to a significantly greater extent (χ(2) = 27.5; P = 0.0003). The number of AgNORs also had an important effect on survival of LMS patients: when the threshold of 11.6 granules was exceeded, prognosis was significantly more unfavorable than that prior to exceeding the threshold.

Pressure cycling technology in systems biology.

Systems biologists frequently seek to integrate complex data sets of diverse analytes into a comprehensive picture of an organism's biological state under defined environmental conditions. Although one would prefer to collect these data from the same sample, technical limitations with traditional sample preparation methods often commit the investigator to extracting one type of analyte at the expense of losing all others. Often, volume further constrains the range of experiments that can be collected from a single sample. The practical solution employed to date has been to rely on information collected from multiple replicate experiments and similar historical or reported data. While this approach has been popular, the integration of information collected from disparate single-analyte sample preparation streams increases uncertainty due to nonalignment during comparative analysis, and such gaps accumulate quickly when combining multiple data sets. Regrettably, discontinuities between separate data streams can confound a whole understanding of the biological system being investigated. This difficulty is further compounded for researchers handling highly pathogenic samples, in which it is often necessary to use harsh chemicals or high-energy sterilization procedures that damage the target analytes. Ultra-high pressure cycling technology (PCT), also known as barocycling, is an emerging sample preparation strategy that has distinct advantages for systems biology studies because it neither commits the researcher to pursuing a specific analyte nor leads to the degradation of target material. In fact, samples prepared under pressure cycling conditions have been shown to yield a more complete set of analytes due to uniform disruption of the sample matrix coupled with an advantageous high pressure solvent environment. Fortunately, PCT safely sterilizes and extracts complex or pathogenic viral, bacterial, and spore samples without adversely affecting the constituent biomolecules valued as informative and meaningful analytes. This chapter provides procedures and findings associated with incorporating PCT into systems biology as a new and enabling approach to preanalytical sample treatment.

Isolation of functional mitochondria from rat kidney and skeletal muscle without manual homogenization.

Isolation of functional and intact mitochondria from solid tissue is crucial for studies that focus on the elucidation of normal mitochondrial physiology and/or mitochondrial dysfunction in conditions such as aging, diabetes, and cancer. There is growing recognition of the importance of mitochondria both as targets for drug development and as off-target mediators of drug side effects. Unfortunately, mitochondrial isolation from tissue is generally carried out using homogenizer-based methods that require extensive operator experience to obtain reproducible high-quality preparations. These methods limit dissemination, impede scale-up, and contribute to difficulties in reproducing experimental results over time and across laboratories. Here we describe semiautomated methods to disrupt tissue using kidney and muscle mitochondria preparations as exemplars. These methods use the Barocycler, the PCT Shredder, or both. The PCT Shredder is a mechanical grinder that quickly breaks up tissue without significant risk of overhomogenization. Mitochondria isolated using the PCT Shredder are shown to be comparable to controls. The Barocycler generates controlled pressure pulses that can be adjusted to lyse cells and release organelles. The mitochondria subjected to pressure cycling-mediated tissue disruption are shown to retain functionality, enabling combinations of the PCT Shredder and the Barocycler to be used to purify mitochondrial preparations.

Thermal stabilization of tissues and the preservation of protein phosphorylation states for two-dimensional gel electrophoresis.

2-DE is typically capable of discriminating proteins differing by a single phosphorylation or dephosphorylation event. However, a reliable representation of protein phosphorylation states as they occur in vivo requires that both phosphatases and kinases are rapidly and completely inactivated. Thermal stabilization of mouse cerebral cortex homogenates effectively inactivated these enzymes, as evidenced by comparison with unstabilized tissues where abscissal pI shifts were a common feature in 2-D gels. Of the 588 matched proteins separated on 2-D gels comparing stabilized and unstabilized tissues, 53 proteins exhibited greater than twofold differences in spot volume (ANOVA, p<0.05). Phosphoprotein-specific staining was corroborated by the identification of 16 phosphoproteins by nano-LC MS/MS and phosphotyrosine kinase activity assay.