Removal of phospholipid contaminants through precipitation of glycosylphosphatidylinositols

Parasitic glycosylphosphatidylinositols (GPIs) are thought to be involved in induced cell signaling that leads to proinflammatory responses. Increasing interest in elucidation of the mechanisms involved in signaling pathways drives the finding of rapid and reliable methods to purify GPIs. GPIs are usually extracted using mixtures of chloroform/methanol/water, followed by a phase partition between water and water-saturated n-butanol. GPIs recovered in the butanol phase are separated by thin-layer chromatography, scraped, eluted from the silica, and used for studying the structure-function relationship. The presence of phospholipid contaminants or other hydrophobic components in the samples cannot be excluded. Furthermore, the standard procedures to purify GPIs harbor several drawbacks, including the need to handle large amounts of culture, poor yields, time-consuming, and interfering contaminants. Here we report on the development of a simple and reliable method to isolate and purify both free and bound GPIs from one cell pellet. We exploited the low solubility of GPIs in water-saturated n-butanol to remove the phospholipid contaminants completely. After delipidation, GPI proteins were solubilized from the pellet using a mixture of organic solvent containing ethanol and water.     

Molecular Analysis of Deep-Sea Hydrothermal Vent Aerobic Methanotrophs by Targeting Genes of 16S rRNA and Particulate Methane Monooxygenase

Molecular diversity of deep-sea hydrothermal vent aerobic methanotrophs was studied using both 16S ribosomalDNA and pmoA encoding the subunit A of particulate methane monooxygenase (pMOA). Hydrothermal vent plume and chimney samples were collected from back-arc vent at Mid-Okinawa Trough (MOT), Japan, and the Trans-Atlantic Geotraverse (TAG) site along Mid-Atlantic Ridge, respectively. The target genes were amplified by polymerase chain reaction from the bulk DNA using specific primers and cloned. Fifty clones from each clone library were directly sequenced. The 16S rDNA sequences were grouped into 3 operational taxonomic units (OTUs), 2 from MOT and 1 from TAG. Two OTUs (1 MOT and 1 TAG) were located within the branch of type I methanotrophic ?-Proteobacteria. Another MOT OTU formed a unique phylogenetic lineage related to type I methanotrophs. Direct sequencing of 50 clones each from the MOT and TAG samples yielded 17 and 4 operational pmoA units (OPUs), respectively. The phylogenetic tree based on the pMOA amino acid sequences deduced from OPUs formed diverse phylogenetic lineages within the branch of type I methanotrophs, except for the OPU MOT-pmoA-8 related to type X methanotrophs. The deduced pMOA topologies were similar to those of all known pMOA, which may suggest that the pmoA gene is conserved through evolution. Neither the 16S rDNA nor pmoA molecular analysis could detect type II methanotrophs, which suggests the absence of type II methanotrophs in the collected vent samples.     

Translational coupling in Bacillus subtilis of a heterologous Bacillus subtilis-Escherichia coli gene fusion.

Translational coupling was demonstrated in a gene fusion in which the promoter and the N-terminal region of the Bacillus subtilis subtilisin (aprA) gene were fused to a promoterless Tn9-derived chloramphenicol acetyltransferase (CAT; EC 2.3.1.28) gene. Expression of this gene fusion results in the production of a native-sized CAT product, whereas the Tn9-derived CAT gene is usually not translated from its own ribosome binding site in B. subtilis (D. S. Goldfarb, R. L. Rodriguez, and R. H. Doi, Proc. Natl. Acad. Sci. USA 79:5886-5890, 1982). A 178-base-pair deletion, which removed part of the signal peptide and the propeptide of the aprA gene and created a translational stop codon 230 base pairs upstream of the CAT gene ribosome binding site, reduced expression of the CAT gene. A BamHI 10-mer linker insertion into this deletion site, which restored the reading frame and simultaneously removed the translation stop codon, restored CAT gene expression. The data indicate that expression of the CAT gene was dependent on translation of the truncated aprA gene into the ribosome binding site of the CAT gene.     

The distribution of insertion sequences in the genome of Shigella flexneri strain 2457T

Shigella flexneri, which causes shigellosis in humans, evolved from Escherichia coli. The sequencing of Shigella genomes has revealed that a large number of insertion sequence (IS) elements (over 200 elements) reside in the genome. Although the presence of these elements has been noted previously and summarized, more detailed analyses are required to understand their evolutionary significance. Here, the genome of S. flexneri strain 2457T is used to investigate the spatial distribution of IS copies around the chromosome and the location of elements with respect to genes. It is found that most IS isoforms occur essentially randomly around the genome. Two exceptions are IS91 and IS911, which appear to cluster due to local hopping. The location of IS elements with respect to genes is biased, however, revealing the action of natural selection. The non-coding regions of the genome (no more than 21%) carry disproportionally more IS elements (at least 28%) than the coding regions, implying that selection acts against insertion into genes. Of the genes disrupted by ISs, those involved in signal transduction, intracellular trafficking, and cell motility are most commonly targeted, suggesting selection against genes in these categories.     

Psychobiotic Potential of Gamma-Aminobutyric Acid–Producing Marine Enterococcus faecium SH9 from Marine Shrimp

Probiotics and Antimicrobial Proteins - Psychobiotics are a novel class of probiotics with potential to confer mental wellness via production of neuroactive compounds such as gamma-aminobutyric...     

Gene and protein interaction network analysis in the epithelial-mesenchymal transition of Hertwig's Epithelial Root Sheath reveals periodontal regenerative drug targets – An in silico study

Background and aimHertwig’s Εpithelial Root Sheath (HΕRS) has a major function in the developing tooth roots. Earlier research revealed that it undergoes epithelial–mesenchymal transition, a vital process for the morphogenesis and complete development of the tooth and its surrounding periodontium. Few studies have demonstrated the role of HERS in cementogenesis through ΕMΤ. The background of this in-silico system biology approach is to find a hub protein and gene involved in the EMT of HERS that may uncover novel insights in periodontal regenerative drug targets.Materials and methodsThe protein and gene list involved in epithelial–mesenchymal transition were obtained from literature sources. The protein interaction was constructed using STRING software and the protein interaction network was analyzed. Molecular docking simulation checks the binding energy and stability of protein-ligand complex.ResultsResults revealed the hub gene to be DYRK1A(Hepcidin), and the ligand was identified as isoetharine. SΤRIΝG results showed a confidence cutoff of 0.9 in sensitivity analysis with a condensed protein interaction network. Overall, 98 nodes from 163 nodes of expected edges were found with an average node degree of 11.9. Docking results show binding energy of −4.70, and simulation results show an RMSD value of 5.6 Å at 50 ns.ConclusionIsoetharine could be a potential drug for periodontal regeneration.     

Green roof <Emphasis Type="Italic">Petunia</Emphasis>, <Emphasis Type="Italic">Ageratum</Emphasis>, and <Emphasis Type="Italic">Mentha</Emphasis> responses to water stress,seaweeds, and trinexapac-ethyl treatments

The availability of sufficient irrigation water and the development of drought-tolerant species are challenging factors in the design and maintenance of green roofs in modern cities. Green roof plants of Petunia hybrid Headliner^® Red Star, Ageratum hybrid Artist^® blue, and Mentha spicata L. grown in a simulated green roof pot system under controlled greenhouse conditions. The plants were watered every 2 or 6 days (2DWI/6DWI) for 8 weeks accompanied by either a 6-day treatment of seaweed extracts of Ascophyllum nodosum as a soil drench or foliar spray, or two concentrations of trinexapac-ethyl (TE) biweekly sprays. Following treatments, leaf number, leaf area, dry weights, plant height, stomatal conductanse, photosynthetic and transpiration rates and leaf water potential and relative water content were determined in two seasons during 2016 and 2017. The prolonged irrigation intervals reduced plant growth as revealed by morphological and physiological parameters. The application of SWE as drench treatment improved Petunia and Ageratum plant vegetative growth, stomatal conductance, photosynthetic and transpiration rates and leaf water potential and relative water content during prolonged irrigation intervals. TE increased the vegetative growth as well as the physiological performance of Ageratum plants. However, neither SWE nor TE treatments improved the performance of Mentha plants under prolonged irrigation intervals. It was suggested that improved photosynthetic rates were stimulated by enhanced stomatal conductance leading to improved leaf water potential as well as increased relative water content during prolonged irrigation conditions.     

Place Cell Rate Remapping by CA3 Recurrent Collaterals

Episodic-like memory is thought to be supported by attractor dynamics in the hippocampus. A possible neural substrate for this memory mechanism is rate remapping, in which the spatial map of place cells encodes contextual information through firing rate variability. To test whether memories are stored as multimodal attractors in populations of place cells, recent experiments morphed one familiar context into another while observing the responses of CA3 cell ensembles. Average population activity in CA3 was reported to transition gradually rather than abruptly from one familiar context to the next, suggesting a lack of attractive forces associated with the two stored representations. On the other hand, individual CA3 cells showed a mix of gradual and abrupt transitions at different points along the morph sequence, and some displayed hysteresis which is a signature of attractor dynamics. To understand whether these seemingly conflicting results are commensurate with attractor network theory, we developed a neural network model of the CA3 with attractors for both position and discrete contexts. We found that for memories stored in overlapping neural ensembles within a single spatial map, position-dependent context attractors made transitions at different points along the morph sequence. Smooth transition curves arose from averaging across the population, while a heterogeneous set of responses was observed on the single unit level. In contrast, orthogonal memories led to abrupt and coherent transitions on both population and single unit levels as experimentally observed when remapping between two independent spatial maps. Strong recurrent feedback entailed a hysteretic effect on the network which diminished with the amount of overlap in the stored memories. These results suggest that context-dependent memory can be supported by overlapping local attractors within a spatial map of CA3 place cells. Similar mechanisms for context-dependent memory may also be found in other regions of the cerebral cortex.