GroEL/S Overexpression Helps to Purge Deleterious Mutations and Reduce Genetic Diversity during Adaptive Protein Evolution

Chaperones are proteins that help other proteins fold. They also affect the adaptive evolution of their client proteins by buffering the effect of deleterious mutations and increasing the genetic diversity of evolving proteins. We study how the bacterial chaperone GroE (GroEL+GroES) affects the evolution of green fluorescent protein (GFP). To this end, we subjected GFP to multiple rounds of mutation and selection for its color phenotype in four replicate Escherichia coli populations, and studied its evolutionary dynamics through high-throughput sequencing and mutant engineering. We evolved GFP both under stabilizing selection for its ancestral (green) phenotype, and to directional selection for a new (cyan) phenotype. We did so both under low and high expression of the chaperone GroE. In contrast to previous work, we observe that GroE does not just buffer but also helps purge deleterious (fluorescence reducing) mutations from evolving populations. In doing so, GroE helps reduce the genetic diversity of evolving populations. In addition, it causes phenotypic heterogeneity in mutants with the same genotype, helping to enhance their fluorescence in some cells, and reducing it in others. Our observations show that chaperones can affect adaptive evolution in more than one way.     

Prokaryotic expression of chicken infectious anemia apoptin protein and characterization of its polyclonal antibodies

In the present study recombinant VP3 (rVP3) was expressed in E. coli BL21 (DE3) (pLysS) and its polyclonal antibodies were characterized. SDS-PAGE analysis revealed that the expression of recombinant protein was maximum when induced with 1.5 mM IPTG for 6 h at 37 degrees C. The 6xHis-tagged fusion protein was purified on Ni-NTA and confirmed by Western blot using CAV specific antiserum. Rabbits were immunized with purified rVP3 to raise anti-VP3 polyclonal antibodies. Polyclonal serum was tested for specificity and used for confirming expression of VP3 in HeLa cells transfected with pcDNA.cav.vp3 by indirect fluorescent antibody test (IFAT), flow cytometry and Western blot. Available purified rVP3 and polyclonal antibodies against VP3 may be useful to understand its functions which may lead to application of VP3 in cancer therapeutics.     

Targeted mapping of ESTs linked to the adult plant resistance gene Lr46 in wheat using synteny with rice

The gene Lr46 has provided slow-rusting resistance to leaf rust caused by Puccinia triticina in wheat (Triticum aestivum), which has remained durable for almost 30 years. Using linked markers and wheat deletion stocks, we located Lr46 in the deletion bin 1BL (0.84–0.89) comprising 5% of the 1BL arm. The distal part of chromosome 1BL of wheat is syntenic to chromosome 5L of rice. Wheat expressed sequence tags (ESTs) mapping in the terminal 15% of chromosome 1BL with significant homology to sequences from the terminal region of chromosome 5L of rice were chosen for sequence-tagged site (STS) primer design and were mapped physically and genetically. In addition, sequences from two rice bacterial artificial chromosome clones covering the targeted syntenic region were used to identify additional linked wheat ESTs. Fourteen new markers potentially linked to Lr46 were developed; eight were mapped in a segregating population. Markers flanking (2.2 cM proximal and 2.2 cM distal) and cosegregating with Lr46 were identified. The physical location of Lr46 was narrowed to a submicroscopic region between the breakpoints of deletion lines 1BL-13 [fraction length (FL)=0.89–1] and 1BL-10 (FL=0.89–3). We are now developing a high-resolution mapping population for the positional cloning of Lr46.     

A Review on Potential Footprints of Ferulic Acid for Treatment of Neurological Disorders

Ferulic acid is being screened in preclinical settings to combat various neurological disorders. It is a naturally occurring dietary flavonoid commonly found in grains, fruits, and vegetables such as rice, wheat, oats, tomatoes, sweet corn etc., which exhibits protective effects against a number of neurological diseases such as epilepsy, depression, ischemia-reperfusion injury, Alzheimer’s disease, and Parkinson’s disease. Ferulic acid prevents and treats different neurological diseases pertaining to its potent anti-oxidative and anti-inflammatory effects, beside modulating unique neuro-signaling pathways. It stays in the bloodstream for longer periods than other dietary polyphenols and antioxidants and easily crosses blood brain barrier. The use of novel drug delivery systems such as solid-lipid nanoparticles (SLNs) or its salt forms (sodium ferulate, ethyl ferulate, and isopentyl ferulate) further enhance its bioavailability and cerebral penetration. Based on reported studies, ferulic acid appears to be a promising molecule for treatment of neurological disorders; however, more preclinical (in vitro and in vivo) mechanism-based studies should be planned and conceived followed by its testing in clinical settings.

     

Temporal oscillations of phosphatases in N-phthaloyl gamma-aminobutyric acid treated rats

N-pathaloyl gamma-aminobutyric acid (P-GABA) was administered to Wistar and 24 hr rhythms of acid and alkaline phosphatases were studied under light-dark conditions. P-GABA administration advanced the peak times of phosphatases. Since GABA is being involved in conveying dark information to the clock, exogenous administration of P-GABA might reduce the photic information received by the clock. The results could be explained by slight daily advances which would bring the peak times to the points 21 days after the start of administration.     

Microalgae: a sustainable feed source for aquaculture

The need for nutritional sources safer than traditional animal products has renewed interest generally in plants and particularly in microalgae. Microalgae have diverse uses in aquaculture, their applications are mainly to provide nutrition and to enhance the colour of the flesh of salmonids. The larvae of molluscs, echinoderms and crustaceans as well as some fish larvae feed on microalgae. Several studies have confirmed that a live multi-specific, low bacterial and microalgal biomass remains essential for shellfish hatcheries. Major advances are expected from new production system, designs and operations from batch run open tanks to more sophisticated continuously-run and closed loop reactors. Currently, studies are underway to examine the cost-effectiveness of the on- and off-site microalgal production systems which can only be achieved by substantial scaling-up and improved quality control. In order to attain sustainability in the usage of microalgae, a systems-based approach is required which integrates different fields such as biotechnology, bioprocess and management procedures.     

Comparative genome analysis of Lactobacillus casei strains isolated from Actimel and Yakult products reveals marked similarities and points to a common origin

The members of the Lactobacillus genus are widely used in the food and feed industry and show a remarkable ecological adaptability. Several Lactobacillus strains have been marketed as probiotics as they possess health‐promoting properties for the host. In the present study, we used two complementary next‐generation sequencing technologies to deduce the genome sequences of two Lactobacillus casei strains LcA and LcY, which were isolated from the products Actimel and Yakult, commercialized as probiotics. The LcA and LcY draft genomes have, respectively, an estimated size of 3067 and 3082 Mb and a G+C content of 46.3%. Both strains are close to identical to each other and differ by no more than minor chromosomal re‐arrangements, substitutions, insertions and deletions, as evident from the verified presence of one insertion‐deletion (InDel) and only 29 single‐nucleotide polymorphisms (SNPs). In terms of coding capacity, LcA and LcY are predicted to encode a comparable exoproteome, indicating that LcA and LcY are likely to establish similar interactions with human intestinal cells. Moreover, both L. casei LcA and LcY harboured a 59.6 kb plasmid that shared high similarities with plasmids found in other L. casei strains, such as W56 and BD‐II. Further analysis revealed that the L. casei plasmids constitute a good evolution marker within the L. casei species. The plasmids of the LcA and LcY strains are almost identical, as testified by the presence of only three verified SNPs, and share a 3.5 kb region encoding a remnant of a lactose PTS system that is absent from the plasmids of W56 and BD‐II but conserved in another smaller L. casei plasmid (pLC2W). Our observations imply that the results obtained in animal and human experiments performed with the Actimel and Yakult strains can be compared with each other as these strains share a very recent common ancestor.