Development of an antibody fragment that stabilizes GPCR/G-protein complexes.

Single-particle cryo-electron microscopy (cryo-EM) has recently enabled high-resolution structure determination of numerous biological macromolecular complexes. Despite this progress, the application of high-resolution cryo-EM to G protein coupled receptors (GPCRs) in complex with heterotrimeric G proteins remains challenging, owning to both the relative small size and the limited stability of these assemblies. Here we describe the development of antibody fragments that bind and stabilize GPCR-G protein complexes for the application of high-resolution cryo-EM. One antibody in particular, mAb16, stabilizes GPCR/G-protein complexes by recognizing an interface between Gα and Gβγ subunits in the heterotrimer, and confers resistance to GTPγS-triggered dissociation. The unique recognition mode of this antibody makes it possible to transfer its binding and stabilizing effect to other G-protein subtypes through minimal protein engineering. This antibody fragment is thus a broadly applicable tool for structural studies of GPCR/G-protein complexes

Minor structural modifications to alchemix influence mechanism of action and pharmacological activity

Alchemix is an exemplar of a class of anthraquinone with efficacy against multidrug resistant tumors. We have explored further the mechanism of action of alchemix and investigated the effect of extending its side arm bearing the alkylating functionality with regard to DNA binding and activity against multidrug resistant cancer cells. Increasing the distance between the intercalating chromophore and the alkylating functionality of ICT2901 (propyl), ICT2902 (butyl) and ICT2903 (pentyl), led to a higher number of DNA alkylation sites, more potent topoisomerase II inhibition and generated more apoptotic and necrotic cells when analysed in p53-proficient HCT116 cells. Intriguingly, alchemix, the compound with the shortest distance between its intercalative chromophore and alkylating functionality (ethyl), did not conform to this SAR. A different toxicity pattern against DNA repair defective CHO cell lines as well as arrest of cells in G1 supports a somewhat distinct mode of action by alchemix compared with its analogues. Importantly, both alchemix and ICT2901 demonstrated greater cytotoxic activity against anthraquinone-resistant MCF-7/adr cells than wild-type MCF-7 cells. Subtle synthetic modification in this anthraquinone series has led to significant changes to the stability of DNA-compound complexes and cellular activity. Given that the failure of chemotherapy in the clinic is often associated with MDR, the results of both alchemix and ICT2901 represent important advances towards improved therapies

Diffuse Gamma Rays: Galactic and Extragalactic Diffuse Emission

"Diffuse" gamma rays consist of several components: truly diffuse emission from the interstellar medium, the extragalactic background, whose origin is not firmly established yet, and the contribution from unresolved and faint Galactic point sources. One approach to unravel these components is to study the diffuse emission from the interstellar medium, which traces the interactions of high energy particles with interstellar gas and radiation fields. Because of its origin such emission is potentially able to reveal much about the sources and propagation of cosmic rays. The extragalactic background, if reliably determined, can be used in cosmological and blazar studies. Studying the derived "average" spectrum of faint Galactic sources may be able to give a clue to the nature of the emitting objects.Comment: 32 pages, 28 figures, kapproc.cls. Chapter to the book "Cosmic Gamma-Ray Sources," to be published by Kluwer ASSL Series, Edited by K. S. Cheng and G. E. Romero. More details can be found at http://www.gamma.mpe-garching.mpg.de/~aws/aws.htm

Garden and landscape-scale correlates of moths of differing conservation status: significant effects of urbanization and habitat diversity

Moths are abundant and ubiquitous in vegetated terrestrial environments and are pollinators, important herbivores of wild plants, and food for birds, bats and rodents. In recent years, many once abundant and widespread species have shown sharp declines that have been cited by some as indicative of a widespread insect biodiversity crisis. Likely causes of these declines include agricultural intensification, light pollution, climate change, and urbanization; however, the real underlying cause(s) is still open to conjecture. We used data collected from the citizen science Garden Moth Scheme (GMS) to explore the spatial association between the abundance of 195 widespread British species of moth, and garden habitat and landscape features, to see if spatial habitat and landscape associations varied for species of differing conservation status. We found that associations with habitat and landscape composition were species-specific, but that there were consistent trends in species richness and total moth abundance. Gardens with more diverse and extensive microhabitats were associated with higher species richness and moth abundance; gardens near to the coast were associated with higher richness and moth abundance; and gardens in more urbanized locations were associated with lower species richness and moth abundance. The same trends were also found for species classified as increasing, declining and vulnerable under IUCN (World Conservation Union) criteria

Caveolin-1 protects B6129 mice against Helicobacter pylori gastritis.

Caveolin-1 (Cav1) is a scaffold protein and pathogen receptor in the mucosa of the gastrointestinal tract. Chronic infection of gastric epithelial cells by Helicobacter pylori (H. pylori) is a major risk factor for human gastric cancer (GC) where Cav1 is frequently down-regulated. However, the function of Cav1 in H. pylori infection and pathogenesis of GC remained unknown. We show here that Cav1-deficient mice, infected for 11 months with the CagA-delivery deficient H. pylori strain SS1, developed more severe gastritis and tissue damage, including loss of parietal cells and foveolar hyperplasia, and displayed lower colonisation of the gastric mucosa than wild-type B6129 littermates. Cav1-null mice showed enhanced infiltration of macrophages and B-cells and secretion of chemokines (RANTES) but had reduced levels of CD25+ regulatory T-cells. Cav1-deficient human GC cells (AGS), infected with the CagA-delivery proficient H. pylori strain G27, were more sensitive to CagA-related cytoskeletal stress morphologies ("humming bird") compared to AGS cells stably transfected with Cav1 (AGS/Cav1). Infection of AGS/Cav1 cells triggered the recruitment of p120 RhoGTPase-activating protein/deleted in liver cancer-1 (p120RhoGAP/DLC1) to Cav1 and counteracted CagA-induced cytoskeletal rearrangements. In human GC cell lines (MKN45, N87) and mouse stomach tissue, H. pylori down-regulated endogenous expression of Cav1 independently of CagA. Mechanistically, H. pylori activated sterol-responsive element-binding protein-1 (SREBP1) to repress transcription of the human Cav1 gene from sterol-responsive elements (SREs) in the proximal Cav1 promoter. These data suggested a protective role of Cav1 against H. pylori-induced inflammation and tissue damage. We propose that H. pylori exploits down-regulation of Cav1 to subvert the host's immune response and to promote signalling of its virulence factors in host cells

Standardisation of rates using logistic regression: a comparison with the direct method

<p>Abstract</p> <p>Background</p> <p>Standardisation of rates in health services research is generally undertaken using the direct and indirect arithmetic methods. These methods can produce unreliable estimates when the calculations are based on small numbers. Regression based methods are available but are rarely applied in practice. This study demonstrates the advantages of using logistic regression to obtain smoothed standardised estimates of the prevalence of rare disease in the presence of covariates.</p> <p>Methods</p> <p>Step by step worked examples of the logistic and direct methods are presented utilising data from BETS, an observational study designed to estimate the prevalence of subclinical thyroid disease in the elderly. Rates calculated by the direct method were standardised by sex and age categories, whereas rates by the logistic method were standardised by sex and age as a continuous variable.</p> <p>Results</p> <p>The two methods produce estimates of similar magnitude when standardising by age and sex. The standard errors produced by the logistic method were lower than the conventional direct method.</p> <p>Conclusion</p> <p>Regression based standardisation is a practical alternative to the direct method. It produces more reliable estimates than the direct or indirect method when the calculations are based on small numbers. It has greater flexibility in factor selection and allows standardisation by both continuous and categorical variables. It therefore allows standardisation to be performed in situations where the direct method would give unreliable results.</p

Splenic peliosis with spontaneous splenic rupture: report of two cases

BACKGROUND: Peliosis is a rare condition characterised by multiple cyst-like, blood-filled cavities within the parenchyma of solid organs. Most commonly affecting the liver, isolated splenic peliosis is an even more unique phenomenon. Patients with the condition are often asymptomatic. However, this potentially lethal condition can present with spontaneous organ rupture. We present two such cases, discuss their management and review what is currently known in the existing literature. CASE PRESENTATION: A previously well twenty-six year old woman presented with abdominal pain following a trivial episode of coughing. A diagnosis of spontaneous splenic rupture was made following clinical and radiological examination. She underwent emergency splenectomy and made a full, uneventful recovery. Histopathological examination confirmed splenic peliosis. The second case describes an eighty six year old lady who sustained a trivial fall and developed pain in her left side. A CT confirmed splenic rupture. She became haemodynamically unstable during her admission and underwent emergency splenectomy. Histopathological examination revealed splenic peliosis. She went on to make an uneventful recovery. CONCLUSION: Splenic peliosis is very rare. It has a number of associations including immunosuppression, drug therapy and infection. Although patients are often asymptomatic, life-threatening spontaneous organ rupture may occur. If the diagnosis of peliosis is confirmed, additional investigations should be considered to detect its presence in other organs. Furthermore, the presence of the condition may be relevant if further medical or surgical intervention is planned

Vision-based real-time position control of a semi-automated system for robot-assisted joint fracture surgery

Purpose: Joint fracture surgery quality can be improved by robotic system with high-accuracy and high-repeatability fracture fragment manipulation. A new real-time vision-based system for fragment manipulation during robot-assisted fracture surgery was developed and tested. Methods: The control strategy was accomplished by merging fast open-loop control with vision-based control. This two-phase process is designed to eliminate the open-loop positioning errors by closing the control loop using visual feedback provided by an optical tracking system. Evaluation of the control system accuracy was performed using robot positioning trials, and fracture reduction accuracy was tested in trials on ex vivo porcine model.Results: The system resulted in high fracture reduction reliability with a reduction accuracy of 0.09mm (translations) and of (Formula presented.) (rotations), maximum observed errors in the order of 0.12mm (translations) and of (Formula presented.) (rotations), and a reduction repeatability of 0.02mm and (Formula presented.). Conclusions: The proposed vision-based system was shown to be effective and suitable for real joint fracture surgical procedures, contributing a potential improvement of their quality

Simple estimators of the intensity of seasonal occurrence

<p>Abstract</p> <p>Background</p> <p>Edwards's method is a widely used approach for fitting a sine curve to a time-series of monthly frequencies. From this fitted curve, estimates of the seasonal intensity of occurrence (i.e., peak-to-low ratio of the fitted curve) can be generated.</p> <p>Methods</p> <p>We discuss various approaches to the estimation of seasonal intensity assuming Edwards's periodic model, including maximum likelihood estimation (MLE), least squares, weighted least squares, and a new closed-form estimator based on a second-order moment statistic and non-transformed data. Through an extensive Monte Carlo simulation study, we compare the finite sample performance characteristics of the estimators discussed in this paper. Finally, all estimators and confidence interval procedures discussed are compared in a re-analysis of data on the seasonality of monocytic leukemia.</p> <p>Results</p> <p>We find that Edwards's estimator is substantially biased, particularly for small numbers of events and very large or small amounts of seasonality. For the common setting of rare events and moderate seasonality, the new estimator proposed in this paper yields less finite sample bias and better mean squared error than either the MLE or weighted least squares. For large studies and strong seasonality, MLE or weighted least squares appears to be the optimal analytic method among those considered.</p> <p>Conclusion</p> <p>Edwards's estimator of the seasonal relative risk can exhibit substantial finite sample bias. The alternative estimators considered in this paper should be preferred.</p

The coding and long noncoding single-cell atlas of the developing human fetal striatum.

Deciphering how the human striatum develops is necessary for understanding the diseases that affect this region. To decode the transcriptional modules that regulate this structure during development, we compiled a catalog of 1116 long intergenic noncoding RNAs (lincRNAs) identified de novo and then profiled 96,789 single cells from the early human fetal striatum. We found that D1 and D2 medium spiny neurons (D1- and D2-MSNs) arise from a common progenitor and that lineage commitment is established during the postmitotic transition, across a pre-MSN phase that exhibits a continuous spectrum of fate determinants. We then uncovered cell type-specific gene regulatory networks that we validated through in silico perturbation. Finally, we identified human-specific lincRNAs that contribute to the phylogenetic divergence of this structure in humans. This work delineates the cellular hierarchies governing MSN lineage commitment