Using the RISCI Genetic Screening Platform for Elucidating Apoptosis Signalling Network

Considerable development in the field of nanotechnology is increasingly yielding novel applications of nanoparticles. The unique properties of nanoparticles in particular their high aspect ratio (length : width ratio), however could pose potential risks to the user. A high throughput genetic screening platform, RISCI (robotic single cDNA investigation), was previously established for the systematic evaluation of single gene activities. Here, RISCI was utilised to identify pro-apoptotic genes as well as genes involved in the positive and negative regulation of silica nanoparticle-induced cell death. This project describes the further development of the screening platform by harnessing its capability to screen a cDNA library comprising approximately 30,000 full length, completely annotated, and sequenced human genes for novel regulators of apoptosis. It integrates an extensive skill sets and is broadly organised into three major phases: Setup, Screen and Analysis. The integration of a pro-apoptosis treatment to screen for inhibitors and sensitizers is a novel aspect of the current experimental setup, along with the low redundancy library. The extensive setup phase focused on technical aspects. The cDNA library, acquired as plasmid DNA, was transformed into a bacterial host for replication and subsequent DNA isolation. A new high-throughput process was developed encompassing the production of competent bacteria and a heat shock transformation protocol, which was subsequently transferred onto the robotic platform. In parallel, the software controlling the robots was redeveloped to allow for execution of user-defined protocols while novel transfection protocols were adapted for automation. The screen identified 699 apoptosis inducers, 1,141 inhibitors and 626 sensitizers. Bioinformatics analysis revealed that the inducers were highly enriched for cell death associated terms, while the inhibitors were strongly associated with cancer profiles. Both inducers and sensitizers were predominantly achieving the functional effect on the protein level, but inhibitors were mainly transcription based. Enriched metal response genes also suggest that the silica nanoparticles were causing their toxicity through reactive oxygen species generation. Intriguingly, the screen identified many noncoding sequences as being functionally capable of regulating apoptosis. These noncoding candidates are capable of regulating the protein coding counterparts identified from the screen. The truly interesting part of the project outcome remains those unknown candidates that were implicated in apoptosis regulation for the first time. Dissemination of the consolidated candidate list would help accelerate the experimental validation of these candidates and aid other researchers in deriving novel hypotheses when the candidates are placed in their research context. [For supplementary files please contact author]

Supplementary material to "Replacement of GroEL in Escherichia coli by the group II chaperonin from the archaeon Methanococcus maripaludis"

Supplementary material to "Replacement of GroEL in Escherichia coli by the group II chaperonin from the archaeon Methanococcus maripaludis

Statistical Modelling of Wear and Damage Trajectories of Railway Wheelsets

This paper discusses the use of Linear Mixed Models (LMM) and Generalized Linear Mixed Models (GLMM) to predict the wear and damage trajectories of railway wheelsets for a fleet of modern multiple unit trains. The wear trajectory is described by the evolution of the wheel flange thickness, the flange height and the tread diameter; whereas the damage trajectory is assessed through the probabilities of various types of wheel tread damage such as rolling contact fatigue, wheel flats and cavities occurring. Different model specifications are compared based on an information criterion

Prenatal diagnosis of proximal focal femoral deficiency: Literature review of prenatal sonographic findings

Proximal focal femoral deficiency (PFFD) is a rare musculoskeletal malformation that occurs in 0.11-0.2 per 10,000 live births. This congenital anomaly involves the pelvis and proximal femur with widely variable manifestations, from mild femoral shortening and hypoplasia to the absence of any functional femur and acetabular aplasia. Prenatal diagnosis of PFFD is still a challenge, but early recognition of this malformation could provide useful information to both parents and physicians concerning management and therapeutic planning. For this review, we analyzed all the cases of prenatally diagnosed PFFD that were reported in the literature from 1990 to 2014 and provide a description of the most common prenatal sonographic findings

Optimization of inhomogeneous electron correlation factors in periodic solids

A method is presented for the optimization of one-body and inhomogeneous two-body terms in correlated electronic wave functions of Jastrow-Slater type. The most general form of inhomogeneous correlation term which is compatible with crystal symmetry is used and the energy is minimized with respect to all parameters using a rapidly convergent iterative approach, based on Monte Carlo sampling of the energy and fitting energy fluctuations. The energy minimization is performed exactly within statistical sampling error for the energy derivatives and the resulting one- and two-body terms of the wave function are found to be well-determined. The largest calculations performed require the optimization of over 3000 parameters. The inhomogeneous two-electron correlation terms are calculated for diamond and rhombohedral graphite. The optimal terms in diamond are found to be approximately homogeneous and isotropic over all ranges of electron separation, but exhibit some inhomogeneity at short- and intermediate-range, whereas those in graphite are found to be homogeneous at short-range, but inhomogeneous and anisotropic at intermediate- and long-range electron separation.Comment: 23 pages, 15 figures, 1 table, REVTeX4, submitted to PR

Replacement of GroEL in Escherichia coli by the group II chaperonin from the archaeon Methanococcus maripaludis

Chaperonins are required for correct folding of many proteins. They exist in two phylogenetic groups: group I, found in bacteria and eukaryotic organelles, and group II, found in archaea and eukaryotic cytoplasm. The two groups, while homologous, differ significantly in structure and mechanism. The evolution of group II chaperonins has been proposed to have been crucial in enabling the expansion of the proteome required for eukaryotic evolution. In an archaeal species that expresses both groups of chaperonins, client selection is determined by structural and biochemical properties rather than phylogenetic origin. It is thus predicted that group II chaperonins will be poor at replacing group I chaperonins. We have tested this hypothesis and report here that the group II chaperonin from Methanococcus maripaludis (Mm-cpn) can partially functionally replace GroEL, the group I chaperonin of Escherichia coli. Furthermore, we identify and characterize two single point mutations in Mm-cpn that have an enhanced ability to replace GroEL function, including one that allows E. coli growth after deletion of the groEL gene. The biochemical properties of the wild-type and mutant Mm-cpn proteins are reported. These data show that the two groups are not as functionally diverse as has been thought and provide a novel platform for genetic dissection of group II chaperonins. IMPORTANCE The two phylogenetic groups of the essential and ubiquitous chaperonins diverged approximately 3.7 billion years ago. They have similar structures, with two rings of multiple subunits, and their major role is to assist protein folding. However, they differ with regard to the details of their structure, their cofactor requirements, and their reaction cycles. Despite this, we show here that a group II chaperonin from a methanogenic archaeon can partially substitute for the essential group I chaperonin GroEL in E. coli and that we can easily isolate mutant forms of this chaperonin with further improved functionality. This is the first demonstration that these two groups, despite the long time since they diverged, still overlap significantly in their functional properties

Academics' Responses to Encountered Information: Context Matters

An increasing number of tools are being developed to help academics interact with information, but little is known about the benefits of those tools for their users. This study evaluated academics' receptiveness to information proposed by a mobile app, the SerenA Notebook: information that is based in their inferred interests but does not relate directly to a prior recognized need. The evaluated app aimed at creating the experience of serendipitous encounters: generating ideas and inspiring thoughts, and potentially triggering follow-up actions, by providing users with suggestions related to their work and leisure interests. We studied how 20 academics interacted with messages sent by the mobile app (3 per day over 10 consecutive days). Collected data sets were analyzed using thematic analysis. We found that contextual factors (location, activity, and focus) strongly influenced their responses to messages. Academics described some unsolicited information as interesting but irrelevant when they could not make immediate use of it. They highlighted filtering information as their major struggle rather than finding information. Some messages that were positively received acted as reminders of activities participants were meant to be doing but were postponing, or were relevant to ongoing activities at the time the information was received

220th ENMC workshop: Dystroglycan and the dystroglycanopathies Naarden, The Netherlands, 27–29 May 2016

Highlights • Review of clinical phenotypes associated with the dystroglycanopathies. • Discussion of current animal models and their contribution to understanding the disease process. • New insight into the glycosylation of alpha dystroglycan and the role of LARGE. • Structural information on the LARGE glycan

An Integrated Physical, Genetic and Cytogenetic Map of Brachypodium distachyon, a Model System for Grass Research

The pooid subfamily of grasses includes some of the most important crop, forage and turf species, such as wheat, barley and Lolium. Developing genomic resources, such as whole-genome physical maps, for analysing the large and complex genomes of these crops and for facilitating biological research in grasses is an important goal in plant biology. We describe a bacterial artificial chromosome (BAC)-based physical map of the wild pooid grass Brachypodium distachyon and integrate this with whole genome shotgun sequence (WGS) assemblies using BAC end sequences (BES). The resulting physical map contains 26 contigs spanning the 272 Mb genome. BES from the physical map were also used to integrate a genetic map. This provides an independent vaildation and confirmation of the published WGS assembly. Mapped BACs were used in Fluorescence In Situ Hybridisation (FISH) experiments to align the integrated physical map and sequence assemblies to chromosomes with high resolution. The physical, genetic and cytogenetic maps, integrated with whole genome shotgun sequence assemblies, enhance the accuracy and durability of this important genome sequence and will directly facilitate gene isolation