Transcriptome sequencing and microsatellite marker discovery in Ailanthus altissima (Mill.) Swingle (Simaroubaceae)

Molecular Biology Reports - Ailanthus altissima Swingle, is a tree species native to East Asia and has a great potential in decorative, bioenergy and industrial applications in many countries. To...     

Computational investigation of the key factors affecting the second stage activation mechanisms of domain II m-calpain

The unique conformation of the active site in calpains along with the implication of their role in several diseases has prompted widespread research interest in the scientific community. Structural studies devoted to m- and μ-calpains have proposed a two-stage calcium-dependent activation mechanism for calpains. In this work, we performed conventional and targeted molecular dynamics simulations to investigate global and local changes in the structure of the protease core of m-calpain upon calcium binding. Simulations were performed on the protease core of calcium free (pdbid: 1kfu) and calcium bound (pdbid: 3df0) m-calpain with and without the presence of calcium ions. Our results indicate that the inactive, open conformation of the protease core does not transform into the active, closed conformation simply upon removal of constraints from the neighbor domains. The role of other factors, including calcium binding and the subsequent formation of an Arg94–Glu305 inter-domain salt bridge and the change in the orientation of Trp288 side chain, in the activation of the protease core is elicited.     

A new clade of Dickeya spp. plays a major role in potato blackleg outbreaks in North Finland

Until recently Dickeya was regarded as a pathogen not established in Finland. As a result the blackleg symptom observed on potato was often associated with Pectobacterium atrosepticum. The occurrence of Dickeya spp. on potato in Finland was first reported in 2004. Since then the prevalence of Dickeya has been monitored through surveys and routine test of seed lots produced in the country. The results of monitoring of Dickeya spp. in seed lots produced in Finland between the years 2004 and 2008 indicated a steady increase in the incidence of Dickeya spp. The highest incidence was observed in samples from the 2006 growing season where about 37% were positive for Dickeya spp. The summer in 2006 was one of the warmest summers recorded in 100 years in Finland. The majority of infected lots were imported varieties. Since recently heavy blackleg outbreaks have occurred in production fields in the High Grade (HG) zone. A detailed study of these incidents of blackleg outbreaks in North Finland during the years 2008 and 2010 indicated that Dickeya spp. was the major component in the observed blackleg complex. It was detected and isolated from almost all symptomatic plants investigated. Repetitive sequences PCR (REP‐PCR) and Pulsed Field Gel Electrophoresis (PFGE) analysis of strains isolated in Finland showed identical pattern with those isolated recently in other European countries with a proposed name ‘Dickeya solani’. Moreover, the dnaX gene sequence of the representative strains isolated in Finland indicated 100% similarity to the dnaX sequences of ‘D. solani’. The study presents the first report of a detailed analysis of bacteria involved in potato blackleg complex from natural field outbreaks in North Finland HG zone and characterisation of the ‘D. solani’ strains playing the major role in the disease complex.     

Pre-PCR Processes in the Molecular Detection of Blackleg and Soft Rot Erwiniae in Seed Potatoes

The polymerase chain reaction (PCR) based detection of blackleg and soft rot erwiniae involves pre‐PCR processing steps which may compromise the sensitivity of detection. The aim of this study was to standardize these various steps to develop reproducible diagnostic PCR protocol for the detection of the three known soft rot erwiniae as they occur in the tuber, singly or in combination. Comparison of tuber peel and stolon end tissue as a starting material for enrichment of the bacteria indicated that tuber peel samples resulted in more representative and sensitive detection of the strains than extract from stolon end tissues. Substances of potato origin in the peel extract were found to be highly inhibitory to the PCR. Addition of the antioxidant Dethiotreitol to the samples before enrichment did not have any significant effect on detection during the 24 h period incubation of the peel extract at room temperature. Bulk washing of tubers with one rotten tuber included with the working sample caused surface contamination on 67–91% of the healthy tubers. Washing tubers individually circumvents the problem. The optimum temperature for enrichment of all the three strains was 27°C. At 37°C, Pectobacterium carotovorum failed to be detected while PCR on Pectobacterium atrosepticum and isolates of Dickeya spp. always produced amplification of the specific DNA fragments. Viability test on Nutrient Agar showed that only Dickeya isolates were viable after 48 h of incubation at 37°C suggesting that the detection of P. atrosepticum at 37°C was from dead or non‐viable cells. Post cell death detection experiment further confirmed that DNA was amplified from dead cells of all the strains at 27°C and 33°C whereas at 37°C, only DNA from dead cells of isolates of Dickeya and P. atrosepticum were amplified. There was no amplification from the dead cells of all isolates of P. carotovorum following the 48 h post death incubation at 37°C. The reason for this difference in post death longevity is not clear at this stage.     

Conformational properties of interacting neurofilaments: Monte Carlo simulations of cylindrically grafted apposing neurofilament brushes

Neurofilaments are essential cytoskeletal filaments that impart mechanical stability to axons. They are mostly assembled from three neurofilament proteins that form the core of the filament and its sidearms. Adjacent neurofilaments interact with each other through their apposing sidearms and attain unique conformations depending on the ionic condition, phosphorylation state, and interfilament separations. To understand the conformational properties of apposing sidearms under various conditions and gain insight into interfilament interactions, we performed Monte Carlo simulations of neurofilament pairs. We employed a sequence-based coarse-grained model of apposing NF sidearms that are end-tethered to cylindrical geometries according to the stoichiometry of the three neurofilament subunits. Monte Carlo simulations were conducted under different conditions such as phosphorylation state, ionic condition, and interfilament separations. Under salt-free conditions, apposing sidearms are found to adopt mutually excluding stretched but bent away conformations that are reminiscent of a repulsive type of interaction. Under physiological conditions, apposing sidearms are found to be in a coiled conformation, suggesting a short-range steric repulsive type of interaction. Increased sidearm mutual interpenetration and a simultaneous decrease in the individual brush heights were observed as the interfilament separation was reduced from 60 to 40 nm. The observed conformations suggest entropic interaction as a likely mechanism for sidearm-mediated interfilament interactions under physiological conditions.     

Lipid production from Yarrowia lipolytica Po1g grown in sugarcane bagasse hydrolysate

This study investigated the possibility of utilizing detoxified sugarcane bagasse hydrolysate (DSCBH) as an alternative carbon source to culture Yarrowia lipolytica Po1g for microbial oil and biodiesel production. Sugarcane bagasse hydrolysis with 2.5% HCl resulted in maximum total sugar concentration (21.38 g/L) in which 13.59 g/L is xylose, 3.98 g/L is glucose, and 2.78 g/L is arabinose. Detoxification of SCBH by Ca(OH)₂ neutralization reduced the concentration of 5-hydroxymethylfurfural and furfural by 21.31% and 24.84%, respectively. Growth of Y. lipolytica Po1g in DSCBH with peptone as the nitrogen source gave maximum biomass concentration (11.42 g/L) compared to NH₄NO₃ (6.49 g/L). With peptone as the nitrogen source, DSCBH resulted in better biomass concentration than d-glucose (10.19 g/L), d-xylose (9.89 g/L) and NDSCBH (5.88 g/L). The maximum lipid content, lipid yield and lipid productivity of Y. lipolytica Po1g grown in DSCBH and peptone was 58.5%, 6.68 g/L and 1.76 g/L-day, respectively.     

Phosphorylation-mediated conformational changes in the mouse neurofilament architecture: insight from a neurofilament brush model

Neurofilaments (NFs) are important cytoskeletal filaments that consist of long flexible C-terminal tails that are abundant with charges. The tails attain additional negative charges through serine phosphorylation of Lys-Ser-Pro (KSP) repeat motifs that are particularly found in neurofilament heavy (NF-H) and neurofilament medium (NF-M) proteins. These side-arm protrusions mediate the interaction between neighboring filaments and maintain axonal diameter. However, the precise role of NF proteins and their phosphorylation in regulating interfilament distances and axonal diameter still remains unclear. In this regard, a recent gene replacement study revealed that the phosphorylation of mouse NF-M KSP repeats does not affect axonal cytoarchitecture, challenging the conventional viewpoint on the role of NF phosphorylation. To better understand the effect of phosphorylation, particularly NF-M phosphorylation, we applied a computational method to reveal phosphorylation-mediated conformational changes in mouse NF architecture. We employed a three-dimensional sequence-based coarse-grained NF brush model to perform Monte Carlo simulations of mouse NF by using the sequence and stoichiometry of mouse NF proteins. Our result shows that the phosphorylation of mouse NF-M does not change the radial extension of NF-M side arms under a salt-free condition and in ionic solution, highlighting a structural factor that supports the notion that NF-M KSP phosphorylation has no effect on the axonal diameter of mouse. On the other hand, significant phosphorylation-mediated conformational changes were found in NF-H side arms under the salt-free condition, while the changes in ionic solution are not significant. However, NF-H side arms are found at the periphery of mouse NF architecture, implying a role in linking neighboring filaments.     

Mesoscopic modeling of bacterial flagellar microhydrodynamics

A particle-based hybrid method of elastic network model and smooth-particle hydrodynamics has been employed to describe the propulsion of bacterial flagella in a viscous hydrodynamic environment. The method explicitly models the two aspects of bacterial propulsion that involve flagellar flexibility and long-range hydrodynamic interaction of low-Reynolds-number flow. The model further incorporates the molecular organization of the flagellar filament at a coarse-grained level in terms of the 11 protofilaments. Each of these protofilaments is represented by a collection of material points that represent the flagellin proteins. A computational model of a single flexible helical segment representing the filament of a bacterial flagellum is presented. The propulsive dynamics and the flow fields generated by the motion of the model filament are examined. The nature of flagellar deformation and the influence of hydrodynamics in determining the shape of deformations are examined based on the helical filament.