Additive and Transcript-Specific Effects of KPAP1 and TbRND Activities on 3′ Non-Encoded Tail Characteristics and mRNA Stability in Trypanosoma brucei

Short, non-encoded oligo(A), oligo(U), or A/U tails can impact mRNA stability in kinetoplastid mitochondria. However, a comprehensive picture of the relative effects of these modifications in RNA stability is lacking. Furthermore, while the U-preferring exoribonuclease TbRND acts on U-tailed gRNAs, its role in decay of uridylated mRNAs has only been cursorily investigated. Here, we analyzed the roles of mRNA 3′ tail composition and TbRND in RNA decay using cells harbouring single or double knockdown of TbRND and the KPAP1 poly(A) polymerase. Analysis of mRNA abundance and tail composition reveals dramatic and transcript-specific effects of adenylation and uridylation on mitochondrial RNAs. Oligo(A) and A-rich tails can stabilize a proportion of edited and never-edited RNAs. However, non-tailed RNAs are not inherently unstable, implicating additional stability determinants and/or spatial segregation of sub-populations of a given RNA in regulation of RNA decay. Oligo(U) tails, which have been shown to contribute to decay of some never-edited RNAs, are not universally destabilizing. We also show that RNAs display very different susceptibility to uridylation in the absence of KPAP1, a factor that may contribute to regulation of decay. Finally, 3′ tail composition apparently impacts the ability of an RNA to be edited

Reintegrating Biology through the Nexus of Energy, Information, and Matter

Information, energy, and matter are fundamental properties of all levels of biological organization, and life emerges from the continuous flux of matter, energy, and information. This perspective piece defines and explains each of the three pillars of this nexus. We propose that a quantitative characterization of the complex interconversions between matter, energy, and information that compose this nexus will help us derive biological insights that connect phenomena across different levels of biological organization. We articulate examples from multiple biological scales that highlight how this nexus approach leads to a more complete understanding of the biological system. Metrics of energy, information, and matter can provide a common currency that helps link phenomena across levels of biological organization. The propagation of energy and information through levels of biological organization can result in emergent properties and system-wide changes that impact other hierarchical levels. Deeper consideration of measured imbalances in energy, information, and matter can help researchers identify key factors that influence system function at one scale, highlighting avenues to link phenomena across levels of biological organization and develop predictive models of biological systems

Mitochondrial RNA editing in Trypanoplasma borreli: new tools, new revelations

The kinetoplastids are unicellular flagellates that derive their name from their ‘kinetoplast’, a region within each flagellate’s single mitochondrion harboring its organellar genome of high DNA content. Some protein products of this mitochondrial genome are encoded as cryptogenes; their transcripts require editing to generate an open reading frame. This happens through an RNA editing process, whereby small regulatory guide RNAs direct the proper insertion and deletion of one or more uridines at each editing site within specific transcript regions. An accurate perspective of the mitochondrial DNA expansion of kinetoplastids and the evolution of their unique uridine insertion/deletion editing across the entire group has been difficult to achieve. Here, we resolved outstanding questions about the organization of the mitochondrial genome and its editing in the kinetoplastid Trypanoplasma borreli that is evolutionarily distant from the frequently-studied trypanosomatids. We find that its mitochondrial DNA consists of circular molecules of 42 kb that harbor the rRNA and mRNAs, and 17 different contigs of approximately 70 kb carrying an average of 23 putative guide RNA loci per contig. These contigs may be linear molecules; they contain repetitive termini. Our analysis uncovered a putative gRNA population with unique length and sequence parameters that is massive relative to the editing needs of this parasite. We validated or determined the sequence identity of each of the four edited mRNA species – including one coding for ATP synthase 6 that was previously thought to be missing. We utilized our computational methods to show that the T. borreli transcriptome includes a substantial number of transcripts with editing patterns not consistent with the identified product, a result of non-canonical editing. We also discovered that this species is more likely than other kinetoplastids to utilize uridine deletion to enforce amino acid conservation of cryptogene products, although deletion is still less common than insertion. Finally, in three tested kinetoplastid mitochondrial transcriptomes, uridine deletion is more common in the raw mitochondrial read population than it appears when the fully edited translationally competent mRNAs only are considered. We conclude that the organization of mitochondrial DNA across all kinetoplastids can be described as variations on several central themes. These themes include partitioned coding and repetitive regions of a circular molecule encoding mRNA and rRNA, and guide RNA loci positioned on a malleable population of multiple molecules that differ in relative abundance in different strains. Likewise, while all kinetoplastids possess the central mechanism of uridine insertion/deletion RNA editing, its output parameters are species-specific

In-reach specialist nursing teams for residential care homes : uptake of services, impact on care provision and cost-effectiveness

Background: A joint NHS-Local Authority initiative in England designed to provide a dedicated nursing and physiotherapy in-reach team (IRT) to four residential care homes has been evaluated.The IRT supported 131 residents and maintained 15 'virtual' beds for specialist nursing in these care homes. Methods: Data captured prospectively (July 2005 to June 2007) included: numbers of referrals; reason for referral; outcome (e.g. admission to IRT bed, short-term IRT support); length of stay in IRT; prevented hospital admissions; early hospital discharges; avoided nursing home transfers; and detection of unrecognised illnesses. An economic analysis was undertaken. Results: 733 referrals were made during the 2 years (range 0.5 to 13.0 per resident per annum)resulting in a total of 6,528 visits. Two thirds of referrals aimed at maintaining the resident's independence in the care home. According to expert panel assessment, 197 hospital admissions were averted over the period; 20 early discharges facilitated; and 28 resident transfers to a nursing home prevented. Detection of previously unrecognised illnesses accounted for a high number of visits. Investment in IRT equalled £44.38 per resident per week. Savings through reduced hospital admissions, early discharges, delayed transfers to nursing homes, and identification of previously unrecognised illnesses are conservatively estimated to produce a final reduction in care cost of £6.33 per resident per week. A sensitivity analysis indicates this figure might range from a weekly overall saving of £36.90 per resident to a 'worst case' estimate of £2.70 extra expenditure per resident per week. Evaluation early in implementation may underestimate some cost-saving activities and greater savings may emerge over a longer time period. Similarly, IRT costs may reduce over time due to the potential for refinement of team without major loss in effectiveness. Conclusion: Introduction of a specialist nursing in-reach team for residential homes is at least cost neutral and, in all probability, cost saving. Further benefits include development of new skills in the care home workforce and enhanced quality of care. Residents are enabled to stay in familiar surroundings rather than unnecessarily spending time in hospital or being transferred to a higher dependency nursing home setting

Gliadin Peptide P31-43 Localises to Endocytic Vesicles and Interferes with Their Maturation

BACKGROUND: Celiac Disease (CD) is both a frequent disease (1:100) and an interesting model of a disease induced by food. It consists in an immunogenic reaction to wheat gluten and glutenins that has been found to arise in a specific genetic background; however, this reaction is still only partially understood. Activation of innate immunity by gliadin peptides is an important component of the early events of the disease. In particular the so-called "toxic" A-gliadin peptide P31-43 induces several pleiotropic effects including Epidermal Growth Factor Receptor (EGFR)-dependent actin remodelling and proliferation in cultured cell lines and in enterocytes from CD patients. These effects are mediated by delayed EGFR degradation and prolonged EGFR activation in endocytic vesicles. In the present study we investigated the effects of gliadin peptides on the trafficking and maturation of endocytic vesicles. METHODS/PRINCIPAL FINDINGS: Both P31-43 and the control P57-68 peptide labelled with fluorochromes were found to enter CaCo-2 cells and interact with the endocytic compartment in pulse and chase, time-lapse, experiments. P31-43 was localised to vesicles carrying early endocytic markers at time points when P57-68-carrying vesicles mature into late endosomes. In time-lapse experiments the trafficking of P31-43-labelled vesicles was delayed, regardless of the cargo they were carrying. Furthermore in celiac enterocytes, from cultured duodenal biopsies, P31-43 trafficking is delayed in early endocytic vesicles. A sequence similarity search revealed that P31-43 is strikingly similar to Hrs, a key molecule regulating endocytic maturation. A-gliadin peptide P31-43 interfered with Hrs correct localisation to early endosomes as revealed by western blot and immunofluorescence microscopy. CONCLUSIONS: P31-43 and P57-68 enter cells by endocytosis. Only P31-43 localises at the endocytic membranes and delays vesicle trafficking by interfering with Hrs-mediated maturation to late endosomes in cells and intestinal biopsies. Consequently, in P31-43-treated cells, Receptor Tyrosine Kinase (RTK) activation is extended. This finding may explain the role played by gliadin peptides in inducing proliferation and other effects in enterocytes from CD biopsies

The \u3cem\u3eChlamydomonas\u3c/em\u3e Genome Reveals the Evolution of Key Animal and Plant Functions

Chlamydomonas reinhardtii is a unicellular green alga whose lineage diverged from land plants over 1 billion years ago. It is a model system for studying chloroplast-based photosynthesis, as well as the structure, assembly, and function of eukaryotic flagella (cilia), which were inherited from the common ancestor of plants and animals, but lost in land plants. We sequenced the ∼120-megabase nuclear genome of Chlamydomonas and performed comparative phylogenomic analyses, identifying genes encoding uncharacterized proteins that are likely associated with the function and biogenesis of chloroplasts or eukaryotic flagella. Analyses of the Chlamydomonas genome advance our understanding of the ancestral eukaryotic cell, reveal previously unknown genes associated with photosynthetic and flagellar functions, and establish links between ciliopathy and the composition and function of flagella

A large scale hearing loss screen reveals an extensive unexplored genetic landscape for auditory dysfunction

The developmental and physiological complexity of the auditory system is likely reflected in the underlying set of genes involved in auditory function. In humans, over 150 non-syndromic loci have been identified, and there are more than 400 human genetic syndromes with a hearing loss component. Over 100 non-syndromic hearing loss genes have been identified in mouse and human, but we remain ignorant of the full extent of the genetic landscape involved in auditory dysfunction. As part of the International Mouse Phenotyping Consortium, we undertook a hearing loss screen in a cohort of 3006 mouse knockout strains. In total, we identify 67 candidate hearing loss genes. We detect known hearing loss genes, but the vast majority, 52, of the candidate genes were novel. Our analysis reveals a large and unexplored genetic landscape involved with auditory function

Primary B-Cell Deficiencies Reveal a Link between Human IL-17-Producing CD4 T-Cell Homeostasis and B-Cell Differentiation

IL-17 is a pro-inflammatory cytokine implicated in autoimmune and inflammatory conditions. The development/survival of IL-17-producing CD4 T cells (Th17) share critical cues with B-cell differentiation and the circulating follicular T helper subset was recently shown to be enriched in Th17 cells able to help B-cell differentiation. We investigated a putative link between Th17-cell homeostasis and B cells by studying the Th17-cell compartment in primary B-cell immunodeficiencies. Common Variable Immunodeficiency Disorders (CVID), defined by defects in B-cell differentiation into plasma and memory B cells, are frequently associated with autoimmune and inflammatory manifestations but we found no relationship between these and Th17-cell frequency. In fact, CVID patients showed a decrease in Th17-cell frequency in parallel with the expansion of activated non-differentiated B cells (CD21lowCD38low). Moreover, Congenital Agammaglobulinemia patients, lacking B cells due to impaired early B-cell development, had a severe reduction of circulating Th17 cells. Finally, we found a direct correlation in healthy individuals between circulating Th17-cell frequency and both switched-memory B cells and serum BAFF levels, a crucial cytokine for B-cell survival. Overall, our data support a relationship between Th17-cell homeostasis and B-cell maturation, with implications for the understanding of the pathogenesis of inflammatory/autoimmune diseases and the physiology of B-cell depleting therapies

Genetic Applications in Avian Conservation

A fundamental need in conserving species and their habitats is defining distinct entities that range from individuals to species to ecosystems and beyond (Table 1; Ryder 1986, Moritz 1994, Mayden and Wood 1995, Haig and Avise 1996, Hazevoet 1996, Palumbi and Cipriano 1998, Hebert et al. 2004, Mace 2004, Wheeler et al. 2004, Armstrong and Ball 2005, Baker 2008, Ellis et al. 2010, Winker and Haig 2010). Rapid progression in this interdisciplinary field continues at an exponential rate; thus, periodic updates on theory, techniques, and applications are important for informing practitioners and consumers of genetic information. Here, we outline conservation topics for which genetic information can be helpful, provide examples of where genetic techniques have been used best in avian conservation, and point to current technical bottlenecks that prevent better use of genomics to resolve conservation issues related to birds. We hope this review will provide geneticists and avian ecologists with a mutually beneficial dialogue on how this integrated field can solve current and future problems

Power estimation for non-standardized multisite studies

AbstractA concern for researchers planning multisite studies is that scanner and T1-weighted sequence-related biases on regional volumes could overshadow true effects, especially for studies with a heterogeneous set of scanners and sequences. Current approaches attempt to harmonize data by standardizing hardware, pulse sequences, and protocols, or by calibrating across sites using phantom-based corrections to ensure the same raw image intensities. We propose to avoid harmonization and phantom-based correction entirely. We hypothesized that the bias of estimated regional volumes is scaled between sites due to the contrast and gradient distortion differences between scanners and sequences. Given this assumption, we provide a new statistical framework and derive a power equation to define inclusion criteria for a set of sites based on the variability of their scaling factors. We estimated the scaling factors of 20 scanners with heterogeneous hardware and sequence parameters by scanning a single set of 12 subjects at sites across the United States and Europe. Regional volumes and their scaling factors were estimated for each site using Freesurfer's segmentation algorithm and ordinary least squares, respectively. The scaling factors were validated by comparing the theoretical and simulated power curves, performing a leave-one-out calibration of regional volumes, and evaluating the absolute agreement of all regional volumes between sites before and after calibration. Using our derived power equation, we were able to define the conditions under which harmonization is not necessary to achieve 80% power. This approach can inform choice of processing pipelines and outcome metrics for multisite studies based on scaling factor variability across sites, enabling collaboration between clinical and research institutions