16S rRNA gene profiling and genome reconstruction reveal community metabolic interactions and prebiotic potential of medicinal herbs used in neurodegenerative disease and as nootropics.

The prebiotic potential of nervine herbal medicines has been scarcely studied. We therefore used anaerobic human fecal cultivation to investigate whether medicinal herbs commonly used as treatment in neurological health and disease in Ayurveda and other traditional systems of medicine modulate gut microbiota. Profiling of fecal cultures supplemented with either Kapikacchu, Gotu Kola, Bacopa/Brahmi, Shankhapushpi, Boswellia/Frankincense, Jatamansi, Bhringaraj, Guduchi, Ashwagandha or Shatavari by 16S rRNA sequencing revealed profound changes in diverse taxa. Principal coordinate analysis highlights that each herb drives the formation of unique microbial communities predicted to display unique metabolic potential. The relative abundance of approximately one-third of the 243 enumerated species was altered by all herbs. Additional species were impacted in an herb-specific manner. In this study, we combine genome reconstruction of sugar utilization and short chain fatty acid (SCFA) pathways encoded in the genomes of 216 profiled taxa with monosaccharide composition analysis of each medicinal herb by quantitative mass spectrometry to enhance the interpretation of resulting microbial communities and discern potential drivers of microbiota restructuring. Collectively, our results indicate that gut microbiota engage in both protein and glycan catabolism, providing amino acid and sugar substrates that are consumed by fermentative species. We identified taxa that are efficient amino acid fermenters and those capable of both amino acid and sugar fermentation. Herb-induced microbial communities are predicted to alter the relative abundance of taxa encoding SCFA (butyrate and propionate) pathways. Co-occurrence network analyses identified a large number of taxa pairs in medicinal herb cultures. Some of these pairs displayed related culture growth relationships in replicate cultures highlighting potential functional interactions among medicinal herb-induced taxa

Synthetic genomics in an era of personalized medicine

System requirements: Windows Media Player version 9 or above.Synthetic genomics is a new field that engages in the design and assembly of genes and chromosomes from chemically synthesized oligonucleotides to create cells with properties unobtainable by conventional molecular biology methods

High quality protein microarray using in situ protein purification

<p>Abstract</p> <p>Background</p> <p>In the postgenomic era, high throughput protein expression and protein microarray technologies have progressed markedly permitting screening of therapeutic reagents and discovery of novel protein functions. Hexa-histidine is one of the most commonly used fusion tags for protein expression due to its small size and convenient purification via immobilized metal ion affinity chromatography (IMAC). This purification process has been adapted to the protein microarray format, but the quality of <it>in situ </it>His-tagged protein purification on slides has not been systematically evaluated. We established methods to determine the level of purification of such proteins on metal chelate-modified slide surfaces. Optimized <it>in situ </it>purification of His-tagged recombinant proteins has the potential to become the new gold standard for cost-effective generation of high-quality and high-density protein microarrays.</p> <p>Results</p> <p>Two slide surfaces were examined, chelated Cu²⁺slides suspended on a polyethylene glycol (PEG) coating and chelated Ni²⁺slides immobilized on a support without PEG coating. Using PEG-coated chelated Cu²⁺slides, consistently higher purities of recombinant proteins were measured. An optimized wash buffer (PBST) composed of 10 mM phosphate buffer, 2.7 mM KCl, 140 mM NaCl and 0.05% Tween 20, pH 7.4, further improved protein purity levels. Using <it>Escherichia coli </it>cell lysates expressing 90 recombinant <it>Streptococcus pneumoniae </it>proteins, 73 proteins were successfully immobilized, and 66 proteins were <it>in situ </it>purified with greater than 90% purity. We identified several antigens among the <it>in situ</it>-purified proteins via assays with anti-<it>S. pneumoniae </it>rabbit antibodies and a human patient antiserum, as a demonstration project of large scale microarray-based immunoproteomics profiling. The methodology is compatible with higher throughput formats of <it>in vivo </it>protein expression, eliminates the need for resin-based purification and circumvents protein solubility and denaturation problems caused by buffer exchange steps and freeze-thaw cycles, which are associated with resin-based purification, intermittent protein storage and deposition on microarrays.</p> <p>Conclusion</p> <p>An optimized platform for <it>in situ </it>protein purification on microarray slides using His-tagged recombinant proteins is a desirable tool for the screening of novel protein functions and protein-protein interactions. In the context of immunoproteomics, such protein microarrays are complimentary to approaches using non-recombinant methods to discover and characterize bacterial antigens.</p

LUD, a new protein domain associated with lactate utilization.

BackgroundA novel highly conserved protein domain, DUF162 [Pfam: PF02589], can be mapped to two proteins: LutB and LutC. Both proteins are encoded by a highly conserved LutABC operon, which has been implicated in lactate utilization in bacteria. Based on our analysis of its sequence, structure, and recent experimental evidence reported by other groups, we hereby redefine DUF162 as the LUD domain family.ResultsJCSG solved the first crystal structure [PDB:2G40] from the LUD domain family: LutC protein, encoded by ORF DR_1909, of Deinococcus radiodurans. LutC shares features with domains in the functionally diverse ISOCOT superfamily. We have observed that the LUD domain has an increased abundance in the human gut microbiome.ConclusionsWe propose a model for the substrate and cofactor binding and regulation in LUD domain. The significance of LUD-containing proteins in the human gut microbiome, and the implication of lactate metabolism in the radiation-resistance of Deinococcus radiodurans are discussed

Dry-Milling and Fractionation of Transgenic Maize Seed Tissues with Green Fluorescent Protein as a Tissue Marker

The efficiency of fractionating cereal grains (e.g., dry corn milling) can be evaluated and monitored by quantifying the proportions of seed tissues in each of the recovered fractions. The quantities of individual tissues are typically estimated using indirect methods such as quantifying fiber or ash to indicate pericarp and tip cap contents, and oil to indicate germ content. More direct and reliable methods are possible with tissue-specific markers. We used two transgenic maize lines, one containing the fluorescent protein green fluorescent protein (GFP) variant S65T expressed in endosperm, and the other containing GFP expressed in germ to determine the fate of each tissue in the dry-milling fractionation process. The two lines were dry-milled to produce three fractions (bran-, endosperm-, and germ-rich fractions) and GFP fluorescence was quantified in each fraction to estimate the tissue composition. Using a simplified laboratory dry-milling procedure and our GFP-containing grain, we determined that the endosperm-rich fraction contained 4% germ tissue, the germ-rich fraction contained 28% germ, 20% endosperm, and 52% nonendosperm and nonembryo tissues, and the bran-rich fraction contained 44% endosperm, 13% germ, and 43% nonendosperm and nonembryo tissues. GFP-containing grain can be used to optimize existing fractionation methods and to develop improved processing strategies

Recombinant expression and functional analysis of proteases from Streptococcus pneumoniae, Bacillus anthracis, and Yersinia pestis

<p>Abstract</p> <p>Background</p> <p>Uncharacterized proteases naturally expressed by bacterial pathogens represents important topic in infectious disease research, because these enzymes may have critical roles in pathogenicity and cell physiology. It has been observed that cloning, expression and purification of proteases often fail due to their catalytic functions which, in turn, cause toxicity in the <it>E. coli </it>heterologous host.</p> <p>Results</p> <p>In order to address this problem systematically, a modified pipeline of our high-throughput protein expression and purification platform was developed. This included the use of a specific <it>E. coli </it>strain, BL21(DE3) pLysS to tightly control the expression of recombinant proteins and various expression vectors encoding fusion proteins to enhance recombinant protein solubility. Proteases fused to large fusion protein domains, maltosebinding protein (MBP), SP-MBP which contains signal peptide at the N-terminus of MBP, disulfide oxidoreductase (DsbA) and Glutathione S-transferase (GST) improved expression and solubility of proteases. Overall, 86.1% of selected protease genes including hypothetical proteins were expressed and purified using a combination of five different expression vectors. To detect novel proteolytic activities, zymography and fluorescence-based assays were performed and the protease activities of more than 46% of purified proteases and 40% of hypothetical proteins that were predicted to be proteases were confirmed.</p> <p>Conclusions</p> <p>Multiple expression vectors, employing distinct fusion tags in a high throughput pipeline increased overall success rates in expression, solubility and purification of proteases. The combinatorial functional analysis of the purified proteases using fluorescence assays and zymography confirmed their function.</p

Intermediate resolution H-beta spectroscopy and photometric monitoring of 3C 390.3 I. Further evidence of a nuclear accretion disk

We have monitored the AGN 3C390.3 between 1995 and 2000.Two large amplitude outbursts, of different duration, in continuum and H beta light were observed ie.: in October 1994 a brighter flare that lasted about 1000 days and in July 1997 another one that lasted about 700 days were detected. The flux in the H beta wings and line core vary simultaneously, a behavior indicative of predominantly circular motions in the BLR.Important changes of the Hbeta emission profiles were detected: at times, we found profiles with prominent asymmetric wings, as those normaly seen in Sy1s, while at other times, we observe profiles with weak almost symmetrical wings, similar to those seen in Sy1.8s. We found that the radial velocity difference between the red and blue bumps is anticorrelated with the light curves of H beta and continuum radiation.e found that the radial velocity difference between the red and blue bumps is anticorrelated with the light curves of H-beta and continuum radiation. Theoretical H-beta profiles were computed for an accretion disk, the observed profiles are best reproduced by an inclined disk (25 deg) whose region of maximum emission is located roughly at 200 Rg. The mass of the black hole in 3C 390.3, estimated from the reverberation analysis is Mrev = 2.1 x 10^9 Msun, ie. 5 times larger than previous estimatesComment: 18 pages, 13 figures, 4 tables. to appear in Astronomy and Astrophysic