Amide H/D exchange in the thermal transition of bovine pancreatic ribonuclease A

The H/D exchange behavior of RNase A at pH 2.5 at a number of temperatures spanning the thermal transition region has been examined by NMR spectroscopy. The amide proton of V116 has a slow rate of H/D exchange even at temperatures above the midpoint of the thermal transition. The H/D exchange behavior of the peptide corresponding to residues 105-124 of RNase A and the peptide corresponding to residues 115-117 is compared with that of RNase A, showing that folding/unfolding cannot be described by a two-state model, and that both short- and long-range interactions are responsible for the slow rate of H/D exchange.     

A Synergistic Framework for Evaluating Business Process Improvements

The evaluation, selection, or justification of business process improvements, or business process reengineering efforts, is similar to many strategic initiatives and their justification methodologies. This similarity arises from the fact that there are multiple factors that need to be considered, many of which have long-term and broad implications for an organization. There are many intangible measures and qualitative concerns when evaluating business process improvements. These improvements necessarily have to link to the strategic objectives of the organization. The proposed methodological framework will involve the synthesis of the analytical network process and data envelopment analysis. These two techniques, when used together, can provide subjective and objective evaluations for managerial decision makers. An illustrative example provides some insights into the application of this methodology. Additional issues and research questions are also identified.     

Perspectives of pluripotent stem cells in livestock

The recent progress in derivation of pluripotent stem cells(PSCs)from farm animals opens new approaches not only for reproduction,genetic engineering,treatment and conservation of these species,but also for screening novel drugs for their efficacy and toxicity,and modelling of human diseases.Initial attempts to derive PSCs from the inner cell mass of blastocyst stages in farm animals were largely unsuccessful as either the cells survived for only a few passages,or lost their cellular potency;indicating that the protocols which allowed the derivation of murine or human embryonic stem(ES)cells were not sufficient to support the maintenance of ES cells from farm animals.This scenario changed by the innovation of induced pluripotency and by the development of the 3 inhibitor culture conditions to support na?ve pluripotency in ES cells from livestock species.However,the long-term culture of livestock PSCs while maintaining the full pluripotency is still challenging,and requires further refinements.Here,we review the current achievements in the derivation of PSCs from farm animals,and discuss the potential application areas.     

Mutation of the LXCXE Binding Cleft of pRb Facilitates Transformation by ras In Vitro but Does Not Promote Tumorigenesis In Vivo

Background

The Retinoblastoma protein (pRB) is a key tumor suppressor that is functionally inactivated in most cancers. pRB regulates the cell division cycle and cell cycle exit through protein–protein interactions mediated by its multiple binding interfaces. The LXCXE binding cleft region of pRB mediates interactions with cellular proteins that have chromatin regulatory functions. Chromatin regulation mediated by pRB is required for a stress responsive cell cycle arrest, including oncogene induced senescence. The in vivo role of chromatin regulation by pRB during senescence, and its relevance to cancer is not clear.

Methodology/Principal Findings

Using gene-targeted mice, uniquely defective for pRB mediated chromatin regulation, we investigated its role during transformation and tumor progression in response to activation of oncogenic ras. We report that the pRB^∆L mutation confers susceptibility to escape from HrasV12 induced senescence and allows transformation in vitro, although these cells possess high levels of DNA damage. Intriguingly, LSL-Kras, Rb1 ^∆L/∆L mice show delayed lung tumor formation compared to controls. This is likely due to the increased apoptosis seen in the early hyperplastic lesions shortly following ras activation that inhibits tumor progression. Furthermore, DMBA treatment to induce sporadic ras mutations in other tissues also failed to reveal greater susceptibility to cancer in Rb1 ^∆L/∆L mice.

Conclusions/Significance

Our data suggests that chromatin regulation by pRB can function to limit proliferation, but its loss fails to contribute to cancer susceptibility in ras driven tumor models because of elevated levels of DNA damage and apoptosis.     

Production of lactic acid using a new homofermentative Enterococcus faecalis isolate

Lactic acid is an intermediate-volume specialty chemical for a wide range of food and industrial applications such as pharmaceuticals, cosmetics and chemical syntheses. Although lactic acid production has been well documented, improved production parameters that lead to reduced production costs are always of interest in industrial developments. In this study, we describe the production of lactic acid at high concentration, yield and volumetric productivity utilizing a novel homofermentative, facultative anaerobe Enterococcus faecalis CBRD01. The highest concentration of 182 g lactic acid l⁻¹ was achieved after 38 h of fed-batch fermentation on glucose. The bacterial isolate utilized only 2–13% of carbon for its growth and energy metabolism, while 87–98% of carbon was converted to lactic acid at an overall volumetric productivity of 5 g l⁻¹ h⁻¹. At 13 h of fermentation, the volumetric productivity of lactate production reached 10.3 g l⁻¹ h⁻¹, which is the highest ever reported for microbial production of lactic acid. The lactic acid produced was of high purity as formation of other metabolites was less than 0.1%. The present investigation demonstrates a new opportunity for enhanced production of lactic acid with potential for reduced purification costs.     

Identification and re-addressing of a transcriptionally permissive locus in the porcine genome

Recently, we established the Sleeping Beauty transposon system for germ line competent transgenesis in the pig. Here, we extend this approach to re-target a transposon-tagged locus for a site-specific gene knock-in, and generated a syngeneic cohort of piglets carrying either the original transposon or the re-targeted event. A Cre-loxP-mediated cassette exchange of the tagging transposon with a different reporter gene was performed, followed by flow cytometric sorting and somatic cell nuclear transfer of recombined cells. In parallel, the original cells were employed in somatic cell nuclear transfer to generate clone siblings, thereby resulting in a clone cohort of piglets carrying different reporter transposons at an identical chromosomal location. Importantly, this strategy supersedes the need for an antibiotic selection marker. This approach expands the arsenal of genome engineering technologies in domestic animals, and will facilitate the development of large animal models for human diseases. Potentially, the syngeneic cohort of pigs will be instrumental for vital tracking of transplanted cells in pre-clinical assessments of novel cell therapies.     

Gametes with somatic chromosome number and their significance in interspecific hybridization in Fuchsia

Sexual polyploidization has both a theoretical as well as an applied significance. Morphological screening for large pollen grains and shape of pollen produced by the individual, cytological investigation of hybrid progeny, and unbalanced separation of chromosomes at anaphase I in pollen mother cells were used to detect the gametes with somatic chromosome number in Fuchsia. The interspecific hybrids of F. fulgens (sect. Ellobium) × F. magellanica (sect. Quelusia), F. fulgens (sect. Ellobium) × F. splendens (sect. Ellobium), and F. triphylla (sect. Fuchsia) × F. splendens (sect. Ellobium) produced at the University of Auckland, New Zealand, showed both large and normal pollen grains in the same anther indicating the presence of unreduced gametes. Cytological investigation carried out on the hybrid progeny of F. fulgens (diploid, 2n=22, sect. Ellobium) × F. magellanica (tetraploid, 2n=44, sect. Quelusia) and F. triphylla (diploid, sect. Fuchsia) × F. arborescens (diploid, sect. Schufia) revealed unexpected chromosome numbers of 2n=44 and 2n=33, respectively. In general, the hybrids showed low fertility caused by genetically unbalanced gametes resulted from random disjunction of chromosomes at anaphase I. Studies on meiosis together with the presence of different shapes and sizes of pollen grains in Fuchsia proved indirectly that unreduced gametes are the products of first division meiotic nuclear restitution. These unreduced gametes were viable irrespective of pollen shape, their predominance in the hybrids, nuclear DNA amount and species phylogenetic position.     

A Linkage Disequilibrium–Based Approach to Selecting Disease-Associated Rare Variants

Rare variants have increasingly been cited as major contributors in the disease etiology of several complex disorders. Recently, several approaches have been proposed for analyzing the association of rare variants with disease. These approaches include collapsing rare variants, summing rare variant test statistics within a particular locus to improve power, and selecting a subset of rare variants for association testing, e.g., the step-up approach. We found that (a) if the variants being pooled are in linkage disequilibrium, the standard step-up method of selecting the best subset of variants results in loss of power compared to a model that pools all rare variants and (b) if the variants are in linkage equilibrium, performing a subset selection using step-based selection methods results in a gain of power of association compared to a model that pools all rare variants. Therefore, we propose an approach to selecting the best subset of variants to include in the model that is based on the linkage disequilibrium pattern among the rare variants. The proposed linkage disequilibrium–based variant selection model is flexible and borrows strength from the model that pools all rare variants when the rare variants are in linkage disequilibrium and from step-based selection methods when the variants are in linkage equilibrium. We performed simulations under three different realistic scenarios based on: (1) the HapMap3 dataset of the DRD2 gene, and CHRNA3/A5/B4 gene cluster (2) the block structure of linkage disequilibrium, and (3) linkage equilibrium. We proposed a permutation-based approach to control the type 1 error rate. The power comparisons after controlling the type 1 error show that the proposed linkage disequilibrium–based subset selection approach is an attractive alternative method for subset selection of rare variants.