Intestinal stem cells lacking the Math1 tumour suppressor are refractory to Notch inhibitors

Intestinal cells are constantly produced from a stem cell reservoir that gives rise to proliferating transient amplifying cells, which subsequently differentiate into one of the four principal cell types. Signalling pathways, including the Notch signalling pathway, coordinate these differentiation processes and their deregulation may cause cancer. Pharmacological inhibition through γ-secretase inhibitors or genetic inactivation of the Notch signalling pathway results in the complete loss of proliferating crypt progenitors due to their conversion into post-mitotic goblet cells. The basic helix–loop–helix transcription factor Math1 is essential for intestinal secretory cell differentiation. Because of the critical roles of both Math1 and Notch signalling in intestinal homeostasis and neoplastic transformation, we sought to determine the genetic hierarchy regulating the differentiation of intestinal stem cells into secretory cells. In this paper, we demonstrate that the conversion of intestinal stem cells into goblet cells upon inhibition of the Notch signalling pathway requires Math1

A potential new, stable state of the E-cadherin strand-swapped dimer in solution

E-cadherin is a transmembrane glycoprotein that facilitates inter-cellular adhesion in the epithelium. The ectodomain of the native structure is comprised of five repeated immunoglobulin-like domains. All E-cadherin crystal structures show the protein in one of three alternative conformations: a monomer, a strand-swapped trans homodimer and the so-called X-dimer, which is proposed to be a kinetic intermediate to forming the strand-swapped trans homodimer. However, previous studies have indicated that even once the trans strand-swapped dimer is formed, the complex is highly dynamic and the E-cadherin monomers may reorient relative to each other. Here, molecular dynamics simulations have been used to investigate the stability and conformational flexibility of the human E-cadherin trans strand-swapped dimer. In four independent, 100 ns simulations, the dimer moved away from the starting structure and converged to a previously unreported structure, which we call the Y-dimer. The Y-dimer was present for over 90% of the combined simulation time, suggesting that it represents a stable conformation of the E-cadherin dimer in solution. The Y-dimer conformation is stabilised by interactions present in both the trans strand-swapped dimer and X-dimer crystal structures, as well as additional interactions not found in any E-cadherin dimer crystal structures. The Y-dimer represents a previously unreported, stable conformation of the human E-cadherin trans strand-swapped dimer and suggests that the available crystal structures do not fully capture the conformations that the human E-cadherin trans homodimer adopts in solution

recA mediated spontaneous deletions of the icaADBC operon of clinical Staphylococcus epidermidis isolates: a new mechanism of phenotypic variations

Phenotypic variation of Staphylococcus epidermidis involving the slime related ica operon results in heterogeneity in surface characteristics of individual bacteria in axenic cultures. Five clinical S. epidermidis isolates demonstrated phenotypic variation, i.e. both black and red colonies on Congo Red agar. Black colonies displayed bi-modal electrophoretic mobility distributions at pH 2, but such phenotypic variation was absent in red colonies of the same strain as well as in control strains without phenotypic variation. All red colonies had lost ica and the ability to form biofilms, in contrast to black colonies of the same strain. Real time PCR targeting icaA indicated a reduction in gene copy number within cultures exhibiting phenotypic variation, which correlated with phenotypic variations in biofilm formation and electrophoretic mobility distribution of cells within a culture. Loss of ica was irreversible and independent of the mobile element IS256. Instead, in high frequency switching strains, spontaneous mutations in lexA were found which resulted in deregulation of recA expression, as shown by real time PCR. RecA is involved in genetic deletions and rearrangements and we postulate a model representing a new mechanism of phenotypic variation in clinical isolates of S. epidermidis. This is the first report of S. epidermidis strains irreversibly switching from biofilm-positive to biofilm-negative phenotype by spontaneous deletion of icaADBC

Loss of LMOD1 impairs smooth muscle cytocontractility and causes megacystis microcolon intestinal hypoperistalsis syndrome in humans and mice

Megacystis microcolon intestinal hypoperistalsis syndrome (MMIHS) is a congenital visceral myopathy characterized by severe dilation of the urinary bladder and defective intestinal motility. The genetic basis of MMIHS has been ascribed to spontaneous and autosomal dominant mutations in actin gamma 2 (ACTG2), a smooth muscle contractile gene. However, evidence suggesting a recessive origin of the disease also exists. Using combined homozygosity mapping and whole exome sequencing, a genetically isolated family was found to carry a premature termination codon in Leiomodin1 (LMOD1), a gene preferentially expressed in vascular and visceral smooth muscle cells. Parents heterozygous for the mutation exhibited no abnormalities, but a child homozygous for the premature termination codon displayed symptoms consistent with MMIHS. We used CRISPR-Cas9 (CRISPR-associated protein) genome editing of Lmod1 to generate a similar premature termination codon. Mice homozygous for the mutation showed loss of LMOD1 protein and pathology consistent with MMIHS, including late gestation expansion of the bladder, hydronephrosis, and rapid demise after parturition. Loss of LMOD1 resulted in a reduction of filamentous actin, elongated cytoskeletal dense bodies, and impaired intestinal smooth muscle contractility. These results define LMOD1 as a disease gene for MMIHS and suggest its role in establishing normal smooth muscle cytoskeletal-contractile coupling

The rate of beneficial mutations surfing on the wave of a range expansion

Many theoretical and experimental studies suggest that range expansions can have severe consequences for the gene pool of the expanding population. Due to strongly enhanced genetic drift at the advancing frontier, neutral and weakly deleterious mutations can reach large frequencies in the newly colonized regions, as if they were surfing the front of the range expansion. These findings raise the question of how frequently beneficial mutations successfully surf at shifting range margins, thereby promoting adaptation towards a range-expansion phenotype. Here, we use individual-based simulations to study the surfing statistics of recurrent beneficial mutations on wave-like range expansions in linear habitats. We show that the rate of surfing depends on two strongly antagonistic factors, the probability of surfing given the spatial location of a novel mutation and the rate of occurrence of mutations at that location. The surfing probability strongly increases towards the tip of the wave. Novel mutations are unlikely to surf unless they enjoy a spatial head start compared to the bulk of the population. The needed head start is shown to be proportional to the inverse fitness of the mutant type, and only weakly dependent on the carrying capacity. The second factor is the mutation occurrence which strongly decreases towards the tip of the wave. Thus, most successful mutations arise at an intermediate position in the front of the wave. We present an analytic theory for the tradeoff between these factors that allows to predict how frequently substitutions by beneficial mutations occur at invasion fronts. We find that small amounts of genetic drift increase the fixation rate of beneficial mutations at the advancing front, and thus could be important for adaptation during species invasions.Comment: 21 pages, 7 figures; to appear in PLoS Computational Biolog

Investigation of attentional bias in obsessive compulsive disorder with and without depression in visual search

Copyright: © 2013 Morein-Zamir et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are creditedWhether Obsessive Compulsive Disorder (OCD) is associated with an increased attentional bias to emotive stimuli remains controversial. Additionally, it is unclear whether comorbid depression modulates abnormal emotional processing in OCD. This study examined attentional bias to OC-relevant scenes using a visual search task. Controls, non-depressed and depressed OCD patients searched for their personally selected positive images amongst their negative distractors, and vice versa. Whilst the OCD groups were slower than healthy individuals in rating the images, there were no group differences in the magnitude of negative bias to concern-related scenes. A second experiment employing a common set of images replicated the results on an additional sample of OCD patients. Although there was a larger bias to negative OC-related images without pre-exposure overall, no group differences in attentional bias were observed. However, OCD patients subsequently rated the images more slowly and more negatively, again suggesting post-attentional processing abnormalities. The results argue against a robust attentional bias in OCD patients, regardless of their depression status and speak to generalized difficulties disengaging from negative valence stimuli. Rather, post-attentional processing abnormalities may account for differences in emotional processing in OCD.Peer reviewedFinal Published versio

Low Numbers of FOXP3 Positive Regulatory T Cells Are Present in all Developmental Stages of Human Atherosclerotic Lesions

BACKGROUND: T cell mediated inflammation contributes to atherogenesis and the onset of acute cardiovascular disease. Effector T cell functions are under a tight control of a specialized T cell subset, regulatory T cells (Treg). At present, nothing is known about the in situ presence of Treg in human atherosclerotic tissue. In the present study we investigated the frequency of naturally occurring Treg cells in all developmental stages of human atherosclerotic lesions including complicated thrombosed plaques. METHODOLOGY: Normal arteries, early lesions (American Heart Association classification types I, II, and III), fibrosclerotic plaques (types Vb and Vc) and 'high risk' plaques (types IV, Va and VI) were obtained at surgery and autopsy. Serial sections were immunostained for markers specific for regulatory T cells (FOXP3 and GITR) and the frequency of these cells was expressed as a percentage of the total numbers of CD3+ T cells. Results were compared with Treg counts in biopsies of normal and inflammatory skin lesions (psoriasis, spongiotic dermatitis and lichen planus). PRINCIPLE FINDINGS: In normal vessel fragments T cells were virtually absent. Treg were present in the intima during all stages of plaque development (0.5-5%). Also in the adventitia of atherosclerotic vessels Treg were encountered, in similar low amounts. High risk lesions contained significantly increased numbers of Treg compared to early lesions (mean: 3.9 and 1.2%, respectively). The frequency of FOXP3+ cells in high risk lesions was also higher compared to stable lesions (1.7%), but this difference was not significant. The mean numbers of intimal FOXP3 positive cells in atherosclerotic lesions (2.4%) was much lower than those in normal (24.3%) or inflammatory skin lesions (28%). CONCLUSION: Low frequencies of Treg in all developmental stages of human plaque formation could explain the smoldering chronic inflammatory process that takes place throughout the longstanding course of atherosclerosis

Cell-type-specific profiling of protein-DNA interactions without cell isolation using targeted DamID with next-generation sequencing.

This protocol is an extension to: Nat. Protoc. 2, 1467-1478 (2007); doi:10.1038/nprot.2007.148; published online 7 June 2007The ability to profile transcription and chromatin binding in a cell-type-specific manner is a powerful aid to understanding cell-fate specification and cellular function in multicellular organisms. We recently developed targeted DamID (TaDa) to enable genome-wide, cell-type-specific profiling of DNA- and chromatin-binding proteins in vivo without cell isolation. As a protocol extension, this article describes substantial modifications to an existing protocol, and it offers additional applications. TaDa builds upon DamID, a technique for detecting genome-wide DNA-binding profiles of proteins, by coupling it with the GAL4 system in Drosophila to enable both temporal and spatial resolution. TaDa ensures that Dam-fusion proteins are expressed at very low levels, thus avoiding toxicity and potential artifacts from overexpression. The modifications to the core DamID technique presented here also increase the speed of sample processing and throughput, and adapt the method to next-generation sequencing technology. TaDa is robust, reproducible and highly sensitive. Compared with other methods for cell-type-specific profiling, the technique requires no cell-sorting, cross-linking or antisera, and binding profiles can be generated from as few as 10,000 total induced cells. By profiling the genome-wide binding of RNA polymerase II (Pol II), TaDa can also identify transcribed genes in a cell-type-specific manner. Here we describe a detailed protocol for carrying out TaDa experiments and preparing the material for next-generation sequencing. Although we developed TaDa in Drosophila, it should be easily adapted to other organisms with an inducible expression system. Once transgenic animals are obtained, the entire experimental procedure-from collecting tissue samples to generating sequencing libraries-can be accomplished within 5 d.This work was funded by a Wellcome Trust Senior Investigator Award (103792), Wellcome Trust Programme Grant (092545) and BBSRC Project Grant (BB/L00786X/1) to A.H.B. A.H.B acknowledges core funding to the Gurdon Institute from the Wellcome Trust (092096) and CRUK (C6946/A14492).This is the author accepted manuscript. The final version is available from Nature Publishing Group via http://dx.doi.org/10.1038/nprot.2016.08

The effects of applied water at various fractions of measured evapotranspiration on reproductive growth and water productivity of Thompson Seedless grapevines

The reproductive growth and water productivity (WPb) of Thompson Seedless grapevines were measured as a function of applied water amounts at various fractions of measured grapevine ETc for a total of eight irrigation treatments. Shoots were harvested numerous times during the growing season to calculate water productivity. Berry weight was maximized at the 0.6–0.8 applied water treatments across years. As applied water amounts increased soluble solids decreased. Berry weight measured at veraison and harvest was a linear function of the mean midday leaf water potential measured between anthesis and veraison and anthesis and harvest, respectively. As applied water amounts increased up to the 0.6–0.8 irrigation treatments there was a significant linear increase in yield. Yields at greater applied water amounts either leveled off or decreased. The reduction in yield on either side of the yearly maximum was due to fewer numbers of clusters per vine. Maximum yield occurred at an ETc ranging from 550 to 700 mm. Yield per unit applied water and WPb increased as applied water decreased. The results from this study demonstrated that Thompson Seedless grapevines can be deficit irrigated, increasing water use efficiency while maximizing yields

Нові явища у функціонально-стилістичному вживанні протиставних сполучників в українській літературній мові кінця ХХ — початку ХХІ століть

У статті досліджено зміни у функціонуванні найуживаніших протиставних сполучників у мові української преси та художньої літератури кінця ХХ — почат ку ХХІ століть, обґрунтовано слабку семантико-синтаксичну спеціалізацію протиставних сполучників та визначено їхні транспозиційні можливості.In the article the changes in the functioning of the most used adversative conjunctions in the language of Ukrainian press and artistic literature of the end of the XX — the beginning of the XXI centuries have been investigated, weak semantic-syntactic specialization of adversative conjunctions has been explained and their transisting resources have been determined