Entropic Tension in Crowded Membranes

Unlike their model membrane counterparts, biological membranes are richly decorated with a heterogeneous assembly of membrane proteins. These proteins are so tightly packed that their excluded area interactions can alter the free energy landscape controlling the conformational transitions suffered by such proteins. For membrane channels, this effect can alter the critical membrane tension at which they undergo a transition from a closed to an open state, and therefore influence protein function \emph{in vivo}. Despite their obvious importance, crowding phenomena in membranes are much less well studied than in the cytoplasm. Using statistical mechanics results for hard disk liquids, we show that crowding induces an entropic tension in the membrane, which influences transitions that alter the projected area and circumference of a membrane protein. As a specific case study in this effect, we consider the impact of crowding on the gating properties of bacterial mechanosensitive membrane channels, which are thought to confer osmoprotection when these cells are subjected to osmotic shock. We find that crowding can alter the gating energies by more than $2\;k_BT$ in physiological conditions, a substantial fraction of the total gating energies in some cases. Given the ubiquity of membrane crowding, the nonspecific nature of excluded volume interactions, and the fact that the function of many membrane proteins involve significant conformational changes, this specific case study highlights a general aspect in the function of membrane proteins.Comment: 20 pages (inclduing supporting information), 4 figures, to appear in PLoS Comp. Bio

Elective amputation and bionic substitution restore functional hand use after critical soft tissue injuries

Critical soft tissue injuries may lead to a non-functional and insensate limb. In these cases standard reconstructive techniques will not suffice to provide a useful outcome, and solutions outside the biological arena must be considered and offered to these patients. We propose a concept which, after all reconstructive options have been exhausted, involves an elective amputation along with a bionic substitution, implementing an actuated prosthetic hand via a structured tech-neuro-rehabilitation program. Here, three patients are presented in whom this concept has been successfully applied after mutilating hand injuries. Clinical tests conducted before, during and after the procedure, evaluating both functional and psychometric parameters, document the benefits of this approach. Additionally, in one of the patients, we show the possibility of implementing a highly functional and natural control of an advanced prosthesis providing both proportional and simultaneous movements of the wrist and hand for completing tasks of daily living with substantially less compensatory movements compared to the traditional systems. It is concluded that the proposed procedure is a viable solution for re-gaining highly functional hand use following critical soft tissue injuries when existing surgical measures fail. Our results are clinically applicable and can be extended to institutions with similar resources

Comparison of embedded and added motor imagery training in patients after stroke: Study protocol of a randomised controlled pilot trial using a mixed methods approach

Copyright @ 2009 Schuster et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Background: Two different approaches have been adopted when applying motor imagery (MI) to stroke patients. MI can be conducted either added to conventional physiotherapy or integrated within therapy sessions. The proposed study aims to compare the efficacy of embedded MI to an added MI intervention. Evidence from pilot studies reported in the literature suggests that both approaches can improve performance of a complex motor skill involving whole body movements, however, it remains to be demonstrated, which is the more effective one.Methods/Design: A single blinded, randomised controlled trial (RCT) with a pre-post intervention design will be carried out. The study design includes two experimental groups and a control group (CG). Both experimental groups (EG1, EG2) will receive physical practice of a clinical relevant motor task ('Going down, laying on the floor, and getting up again') over a two week intervention period: EG1 with embedded MI training, EG2 with MI training added after physiotherapy. The CG will receive standard physiotherapy intervention and an additional control intervention not related to MI.The primary study outcome is the time difference to perform the task from pre to post-intervention. Secondary outcomes include level of help needed, stages of motor task completion, degree of motor impairment, balance ability, fear of falling measure, motivation score, and motor imagery ability score. Four data collection points are proposed: twice during baseline phase, once following the intervention period, and once after a two week follow up. A nested qualitative part should add an important insight into patients' experience and attitudes towards MI. Semi-structured interviews of six to ten patients, who participate in the RCT, will be conducted to investigate patients' previous experience with MI and their expectations towards the MI intervention in the study. Patients will be interviewed prior and after the intervention period.Discussion: Results will determine whether embedded MI is superior to added MI. Findings of the semi-structured interviews will help to integrate patient's expectations of MI interventions in the design of research studies to improve practical applicability using MI as an adjunct therapy technique

Inactivation of a Single Copy of Crebbp Selectively Alters Pre-mRNA Processing in Mouse Hematopoietic Stem Cells

Global expression analysis of fetal liver hematopoietic stem cells (FL HSCs) revealed the presence of unspliced pre-mRNA for a number of genes in normal FL HSCs. In a subset of these genes, Crebbp+/− FL HSCs had less unprocessed pre-mRNA without a corresponding reduction in total mRNA levels. Among the genes thus identified were the key regulators of HSC function Itga4, Msi2 and Tcf4. A similar but much weaker effect was apparent in Ep300+/− FL HSCs, indicating that, in this context as in others, the two paralogs are not interchangeable. As a group, the down-regulated intronic probe sets could discriminate adult HSCs from more mature cell types, suggesting that the underlying mechanism is regulated with differentiation stage and is active in both fetal and adult hematopoiesis. Consistent with increased myelopoiesis in Crebbp hemizygous mice, targeted reduction of CREBBP abundance by shRNA in the multipotent EML cell line triggered spontaneous myeloid differentiation in the absence of the normally required inductive signals. In addition, differences in protein levels between phenotypically distinct EML subpopulations were better predicted by taking into account not only the total mRNA signal but also the amount of unspliced message present. CREBBP thus appears to selectively influence the timing and degree of pre-mRNA processing of genes essential for HSC regulation and thereby has the potential to alter subsequent cell fate decisions in HSCs

Pioglitazone is as effective as dexamethasone in a cockroach allergen-induced murine model of asthma

<p>Abstract</p> <p>Background</p> <p>While glucocorticoids are currently the most effective therapy for asthma, associated side effects limit enthusiasm for their use. Peroxisome proliferator-activated receptor-γ (PPAR-γ) activators include the synthetic thiazolidinediones (TZDs) which exhibit anti-inflammatory effects that suggest usefulness in diseases such as asthma. How the ability of TZDs to modulate the asthmatic response compares to that of glucocorticoids remains unclear, however, because these two nuclear receptor agonists have never been studied concurrently. Additionally, effects of PPAR-γ agonists have never been examined in a model involving an allergen commonly associated with human asthma.</p> <p>Methods</p> <p>We compared the effectiveness of the PPAR-γ agonist pioglitazone (PIO) to the established effectiveness of a glucocorticoid receptor agonist, dexamethasone (DEX), in a murine model of asthma induced by cockroach allergen (CRA). After sensitization to CRA and airway localization by intranasal instillation of the allergen, Balb/c mice were challenged twice at 48-h intervals with intratracheal CRA. Either PIO (25 mg/kg/d), DEX (1 mg/kg/d), or vehicle was administered throughout the period of airway CRA exposure.</p> <p>Results</p> <p>PIO and DEX demonstrated similar abilities to reduce airway hyperresponsiveness, pulmonary recruitment of inflammatory cells, serum IgE, and lung levels of IL-4, IL-5, TNF-α, TGF-β, RANTES, eotaxin, MIP3-α, Gob-5, and Muc5-ac. Likewise, intratracheal administration of an adenovirus containing a constitutively active PPAR-γ expression construct blocked CRA induction of Gob-5 and Muc5-ac.</p> <p>Conclusion</p> <p>Given the potent effectiveness shown by PIO, we conclude that PPAR-γ agonists deserve investigation as potential therapies for human asthma.</p

Highly Precise and Developmentally Programmed Genome Assembly in Paramecium Requires Ligase IV–Dependent End Joining

During the sexual cycle of the ciliate Paramecium, assembly of the somatic genome includes the precise excision of tens of thousands of short, non-coding germline sequences (Internal Eliminated Sequences or IESs), each one flanked by two TA dinucleotides. It has been reported previously that these genome rearrangements are initiated by the introduction of developmentally programmed DNA double-strand breaks (DSBs), which depend on the domesticated transposase PiggyMac. These DSBs all exhibit a characteristic geometry, with 4-base 5′ overhangs centered on the conserved TA, and may readily align and undergo ligation with minimal processing. However, the molecular steps and actors involved in the final and precise assembly of somatic genes have remained unknown. We demonstrate here that Ligase IV and Xrcc4p, core components of the non-homologous end-joining pathway (NHEJ), are required both for the repair of IES excision sites and for the circularization of excised IESs. The transcription of LIG4 and XRCC4 is induced early during the sexual cycle and a Lig4p-GFP fusion protein accumulates in the developing somatic nucleus by the time IES excision takes place. RNAi–mediated silencing of either gene results in the persistence of free broken DNA ends, apparently protected against extensive resection. At the nucleotide level, controlled removal of the 5′-terminal nucleotide occurs normally in LIG4-silenced cells, while nucleotide addition to the 3′ ends of the breaks is blocked, together with the final joining step, indicative of a coupling between NHEJ polymerase and ligase activities. Taken together, our data indicate that IES excision is a “cut-and-close” mechanism, which involves the introduction of initiating double-strand cleavages at both ends of each IES, followed by DSB repair via highly precise end joining. This work broadens our current view on how the cellular NHEJ pathway has cooperated with domesticated transposases for the emergence of new mechanisms involved in genome dynamics

The effectiveness of a low-intensity problem-solving intervention for common adolescent mental health problems in New Delhi, India: protocol for a school-based, individually randomized controlled trial with an embedded stepped-wedge cluster randomized controlled recruitment trial

Background Conduct, anxiety and depressive disorders account for over 75% of the adolescent mental health burden globally. The current protocol will test a low-intensity problem-solving intervention for school-going adolescents with common mental health problems in India. The protocol also tests the effects of a classroom-based sensitization intervention on the demand for counselling services in an embedded recruitment trial. Methods We will conduct a two-arm individually randomized controlled trial in six Government-run secondary schools in New Delhi. The targeted sample is 240 adolescents in grades 9-12 with persistent, elevated mental health symptoms and associated impact. Participants will receive either a brief problem-solving intervention delivered over 3 weeks by lay counsellors (intervention), or enhanced usual care comprised of problem-solving booklets (control). Self-reported adolescent mental health symptoms and idiographic problems will be assessed at 6 weeks (co-primary outcomes) and again at 12 weeks post-randomization. In addition, adolescent-reported impact of mental health difficulties, perceived stress, mental wellbeing and clinical remission, as well as parent-reported adolescent mental health symptoms and impact scores, will be assessed at 6 and 12 weeks post-randomization. We will also complete a parallel process evaluation, including estimations of the costs of delivering the interventions. An embedded recruitment trial will apply a stepped-wedge, cluster (class)-randomized controlled design in 70 classes across the six schools. This will evaluate the added impact of a classroom-based sensitization intervention over school-level recruitment sensitization activities on the primary outcome of referral rate into the host trial (i.e. the proportion of adolescents referred as a function of the total sampling frame in each condition of the embedded recruitment trial). Other outcomes will be the proportion of referrals eligible to participate in the host trial, proportion of self-generated referrals, and severity and pattern of symptoms among referred adolescents in each condition. Power calculations were undertaken separately for each trial. A detailed statistical analysis plan will be developed separately for each trial prior to unblinding. Discussion Both trials were initiated on 20 August 2018. A single research protocol for both trials offers a resource-efficient methodology for testing the effectiveness of linked procedures to enhance uptake and outcomes of a school-based psychological intervention for common adolescent mental health problems

The Effects of NMDA Subunit Composition on Calcium Influx and Spike Timing-Dependent Plasticity in Striatal Medium Spiny Neurons

Calcium through NMDA receptors (NMDARs) is necessary for the long-term potentiation (LTP) of synaptic strength; however, NMDARs differ in several properties that can influence the amount of calcium influx into the spine. These properties, such as sensitivity to magnesium block and conductance decay kinetics, change the receptor's response to spike timing dependent plasticity (STDP) protocols, and thereby shape synaptic integration and information processing. This study investigates the role of GluN2 subunit differences on spine calcium concentration during several STDP protocols in a model of a striatal medium spiny projection neuron (MSPN). The multi-compartment, multi-channel model exhibits firing frequency, spike width, and latency to first spike similar to current clamp data from mouse dorsal striatum MSPN. We find that NMDAR-mediated calcium is dependent on GluN2 subunit type, action potential timing, duration of somatic depolarization, and number of action potentials. Furthermore, the model demonstrates that in MSPNs, GluN2A and GluN2B control which STDP intervals allow for substantial calcium elevation in spines. The model predicts that blocking GluN2B subunits would modulate the range of intervals that cause long term potentiation. We confirmed this prediction experimentally, demonstrating that blocking GluN2B in the striatum, narrows the range of STDP intervals that cause long term potentiation. This ability of the GluN2 subunit to modulate the shape of the STDP curve could underlie the role that GluN2 subunits play in learning and development

Early loss of Crebbp confers malignant stem cell properties on lymphoid progenitors.

Loss-of-function mutations of cyclic-AMP response element binding protein, binding protein (CREBBP) are prevalent in lymphoid malignancies. However, the tumour suppressor functions of CREBBP remain unclear. We demonstrate that loss of Crebbp in murine haematopoietic stem and progenitor cells (HSPCs) leads to increased development of B-cell lymphomas. This is preceded by accumulation of hyperproliferative lymphoid progenitors with a defective DNA damage response (DDR) due to a failure to acetylate p53. We identify a premalignant lymphoma stem cell population with decreased H3K27ac, which undergoes transcriptional and genetic evolution due to the altered DDR, resulting in lymphomagenesis. Importantly, when Crebbp is lost later in lymphopoiesis, cellular abnormalities are lost and tumour generation is attenuated. We also document that CREBBP mutations may occur in HSPCs from patients with CREBBP-mutated lymphoma. These data suggest that earlier loss of Crebbp is advantageous for lymphoid transformation and inform the cellular origins and subsequent evolution of lymphoid malignancies