Site-directed mutagenesis of UbiA to promote menaquinone biosynthesis in Elizabethkingia meningoseptica

To increase the menaquinone (MK) content of an Elizabethkingia meningoseptica, site-directed mutagenesis was generated to suppress 4-hydroxybenzoate octaprenyl transferase (UbiA) activity and subsequently blocked the ubiquinone (UQ) biosynthesis pathway. Fourteen conserved residues except L174 and G211 were mutated to analyze the effect of site-directed mutagenesis. The expression of UbiA in twelve mutants was decreased in both mRNA and protein levels, which resulted in the decrease of UQ concentration. Based on MenA expression level, 12 mutants were divided into two groups. Second group such as N72A, D76A, K81A, L139A, and D198A enhanced the expression of MenA, which increased MK production by 127.1%, 87.9%, 96.2%, 109.7% and 130.0% in wt-EmUbiA, respectively. In general, blocking UQ synthesis pathway for by site-directed mutagenesis of the active site of UbiA in E. meningoseptica was a promising strategy to increase MK production in E. meningoseptica.     

Insights into the discrepant luminescence for BaSiO3:Eu2+ phosphors prepared by solid‐state reaction and precipitation reaction methods

Two synthesis routes, solid‐state reaction and precipitation reaction, were employed to prepare BaSiO₃:Eu²⁺ phosphors in this study. Discrepancies in the luminescence green emission at 505 nm for the solid‐state reaction method sample and in the yellow emission at 570 nm for the sample prepared by the precipitation reaction method, were observed respectively. A detail investigation about the discrepant luminescence of BaSiO₃:Eu²⁺ phosphors was performed by evaluation of X‐ray diffraction (XRD), photoluminescence (PL)/photoluminescence excitation (PLE), decay time and thermal quenching properties. The results showed that the yellow emission was generated from the BaSiO₃:Eu²⁺ phosphor, while the green emission was ascribed to a small amount of Ba₂SiO₄:Eu²⁺ compound that was present in the solid‐state reaction sample. This work clarifies the luminescence properties of Eu²⁺ ions in BaSiO₃ and Ba₂SiO₄ hosts.     

Side chain flexibility and coupling between the S4‐S5 linker and the TRP domain in thermo‐sensitive TRP channels: Insights from protein modeling

The transient receptor potential (TRP) superfamily is subdivided into several subfamilies on the basis of sequence similarity, which is highly heterogeneous but shows a molecular architecture that resembles the one present in members of the Kv channel superfamily. Because of this diversity, they produce a large variety of channels with different gating and permeability properties. Elucidation of these particular features necessarily requires comparative studies based on structural and functional data. The present study aims to compilate, analyze, and determine, in a coherent way, the relationship between intrinsic side‐chain flexibility and the allosteric coupling in members of the TRPV, TRPM, and TRPC families. Based on the recently determined structures of TRPV1 and TRPV2, we have generated protein models for single subunits of TRPV5, TRPM8, and TRPC5 channels. With these models, we focused our attention on the apparently crucial role of the GP dipeptide at the center of the S4‐S5 linker and discussed its role in the interaction with the TRP domain, specifically with the highly‐conserved Trp during this coupling. Our analysis suggests an important role of the S4‐S5L flexibility in the thermosensitivity, where heat‐activated channels possess rigid S4‐S5 linkers, whereas cold‐activated channels have flexible ones. Finally, we also present evidence of the key interaction between the conserved Trp residue of the TRP box and of several residues in the S4‐S5L, importantly the central Pro. Proteins 2017; 85:630–646. © 2016 Wiley Periodicals, Inc.     

Rhodobacteraceae on the marine brown alga Fucus spiralis are abundant and show physiological adaptation to an epiphytic lifestyle

Macroalgae harbour specific microbial communities on their surface that have functions related to host health and defence. In this study, the bacterial biofilm of the marine brown alga Fucus spiralis was investigated using 16S rRNA gene amplicon-based analysis and isolation of bacteria. Rhodobacteraceae (Alphaproteobacteria) were the predominant family constituting 23% of the epibacterial community. At the genus level, Sulfitobacter, Loktanella, Octadecabacter and a previously undescribed cluster were most abundant, and together they comprised 89% of the Rhodobacteraceae. Supported by a specific PCR approach, 23 different Rhodobacteraceae-affiliated strains were isolated from the surface of F. spiralis, which belonged to 12 established and three new genera. For seven strains, closely related sequences were detected in the 16S rRNA gene dataset. Growth experiments with substrates known to be produced by Fucus spp. showed that all of them were consumed by at least three strains, and vitamin B₁₂ was produced by 70% of the isolates. Since growth of F. spiralis depends on B₁₂ supplementation, bacteria may provide the alga with this vitamin. Most strains produced siderophores, which can enhance algal growth under iron-deficient conditions. Inhibiting properties against other bacteria were only observed when F. spiralis material was present in the medium. Thus, the physiological properties of the isolates indicated adaption to an epiphytic lifestyle.     

Multiple molecular dynamics simulations of human LOX‐1 and Trp150Ala mutant reveal the structural determinants causing the full deactivation of the receptor

Multiple classical molecular dynamics simulations have been applied to the human LOX‐1 receptor to clarify the role of the Trp150Ala mutation in the loss of binding activity. Results indicate that the substitution of this crucial residue, located at the dimer interface, markedly disrupts the wild‐type receptor dynamics. The mutation causes an irreversible rearrangement of the subunits interaction pattern that in the wild‐type protein allows the maintaining of a specific symmetrical motion of the monomers. The subunits dislocation determines a loss of linearity of the arginines residues composing the basic spine and a consequent alteration of the long‐range electrostatic attraction of the substrate. Moreover, the anomalous subunits arrangement observed in the mutated receptor also affects the integrity of the hydrophobic tunnel, actively involved in the short‐range hydrophobic recognition of the substrate. The combined effect of these structural rearrangements generates the impairing of the receptor function.     

Isolation,Culture and Long-Term Maintenance of Primary Mesencephalic Dopaminergic Neurons From Embryonic Rodent Brains

Degeneration of mesencephalic dopaminergic (mesDA) neurons is the pathological hallmark of Parkinson’s diseae. Study of the biological processes involved in physiological functions and vulnerability and death of these neurons is imparative to understanding the underlying causes and unraveling the cure for this common neurodegenerative disorder. Primary cultures of mesDA neurons provide a tool for investigation of the molecular, biochemical and electrophysiological properties, in order to understand the development, long-term survival and degeneration of these neurons during the course of disease. Here we present a detailed method for the isolation, culturing and maintenance of midbrain dopaminergic neurons from E12.5 mouse (or E14.5 rat) embryos. Optimized cell culture conditions in this protocol result in presence of axonal and dendritic projections, synaptic connections and other neuronal morphological properties, which make the cultures suitable for study of the physiological, cell biological and molecular characteristics of this neuronal population.