WDR5, BRCA1, and BARD1 Co-regulate the DNA Damage Response and Modulate the Mesenchymal-to-Epithelial Transition during Early Reprogramming.

Differentiated cells are epigenetically stable, but can be reprogrammed to pluripotency by expression of the OSKM transcription factors. Despite significant effort, relatively little is known about the cellular requirements for reprogramming and how they affect the properties of induced pluripotent stem cells. We have performed high-content screening with small interfering RNAs targeting 300 chromatin-associated factors and extracted colony-level quantitative features. This revealed five morphological phenotypes in early reprogramming, including one displaying large round colonies exhibiting an early block of reprogramming. Using RNA sequencing, we identified transcriptional changes associated with these phenotypes. Furthermore, double knockdown epistasis experiments revealed that BRCA1, BARD1, and WDR5 functionally interact and are required for the DNA damage response. In addition, the mesenchymal-to-epithelial transition is affected in Brca1, Bard1, and Wdr5 knockdowns. Our data provide a resource of chromatin-associated factors in early reprogramming and underline colony morphology as an important high-dimensional readout for reprogramming quality

A Small Conductance Calcium-Activated K⁺ Channel in C. elegans, KCNL-2, Plays a Role in the Regulation of the Rate of Egg-Laying

In the nervous system of mice, small conductance calcium-activated potassium (SK) channels function to regulate neuronal excitability through the generation of a component of the medium afterhyperpolarization that follows action potentials. In humans, irregular action potential firing frequency underlies diseases such as ataxia, epilepsy, schizophrenia and Parkinson's disease. Due to the complexity of studying protein function in the mammalian nervous system, we sought to characterize an SK channel homologue, KCNL-2, in C. elegans, a genetically tractable system in which the lineage of individual neurons was mapped from their early developmental stages. Sequence analysis of the KCNL-2 protein reveals that the six transmembrane domains, the potassium-selective pore and the calmodulin binding domain are highly conserved with the mammalian homologues. We used widefield and confocal fluorescent imaging to show that a fusion construct of KCNL-2 with GFP in transgenic lines is expressed in the nervous system of C. elegans. We also show that a KCNL-2 null strain, kcnl-2(tm1885), demonstrates a mild egg-laying defective phenotype, a phenotype that is rescued in a KCNL-2-dependent manner. Conversely, we show that transgenic lines that overexpress KCNL-2 demonstrate a hyperactive egg-laying phenotype. In this study, we show that the vulva of transgenic hermaphrodites is highly innervated by neuronal processes and by the VC4 and VC5 neurons that express GFP-tagged KCNL-2. We propose that KCNL-2 functions in the nervous system of C. elegans to regulate the rate of egg-laying. © 2013 Chotoo et al

Holocentric Chromosomes of Luzula elegans Are Characterized by a Longitudinal Centromere Groove, Chromosome Bending, and a Terminal Nucleolus Organizer Region

The structure of holocentric chromosomes was analyzed in mitotic cells of Luzula elegans. Light and scanning electron microscopy observations provided evidence for the existence of a longitudinal groove along each sister chromatid. The centromere-specific histone H3 variant, CENH3, colocalized with this groove and with microtubule attachment sites. The terminal chromosomal regions were CENH3-negative. During metaphase to anaphase transition, L. elegans chromosomes typically curved to a sickle-like shape, a process that is likely to be influenced by the pulling forces of microtubules along the holocentric axis towards the corresponding microtubule organizing regions. A single pair of 45S rDNA sites, situated distal to Arabidopsis-telomere repeats, was observed at the terminal region of one chromosome pair. We suggest that the 45S rDNA position in distal centromere-free regions could be required to ensure chromosome stability. Copyright (C) 2011 S. Karger AG, Base

Small Polarons in Transition Metal Oxides

The formation of polarons is a pervasive phenomenon in transition metal oxide compounds, with a strong impact on the physical properties and functionalities of the hosting materials. In its original formulation the polaron problem considers a single charge carrier in a polar crystal interacting with its surrounding lattice. Depending on the spatial extension of the polaron quasiparticle, originating from the coupling between the excess charge and the phonon field, one speaks of small or large polarons. This chapter discusses the modeling of small polarons in real materials, with a particular focus on the archetypal polaron material TiO2. After an introductory part, surveying the fundamental theoretical and experimental aspects of the physics of polarons, the chapter examines how to model small polarons using first principles schemes in order to predict, understand and interpret a variety of polaron properties in bulk phases and surfaces. Following the spirit of this handbook, different types of computational procedures and prescriptions are presented with specific instructions on the setup required to model polaron effects.Comment: 36 pages, 12 figure

High throughput method for analysis of repeat number for 28 phase variable loci of C. jejuni strain NCTC11168

Mutations in simple sequence repeat tracts are a major mechanism of phase variation in several bacterial species including Campylobacter jejuni. Changes in repeat number of tracts located within the reading frame can produce a high frequency of reversible switches in gene expression between ON and OFF states. The genome of C. jejuni strain NCTC11168 contains 29 loci with polyG/polyC tracts of seven or more repeats. This protocol outlines a method for rapidly determining ON/OFF states of these 28 phase-variable loci in a large number of individual colonies. The method combines a series of multiplex PCR assays with a GeneScan assay and automated extraction of tract length, repeat number and expression state. This high throughput, multiplex assay has utility for detecting shifts in phase variation states within and between populations over time and for exploring the effects of phase variation on adaptation to differing selective pressures. An important output of this assay is combinatorial expression states that cannot be determined by other methods. This method can be adapted to analysis of phase variation in other C. jejuni strains and in a diverse range of bacterial species

Seasonal influenza risk in hospital healthcare workers is more strongly associated with household than occupational exposures: results from a prospective cohort study in Berlin, Germany, 2006/07

Background: Influenza immunisation for healthcare workers is encouraged to protect their often vulnerable patients but also due to a perceived higher risk for influenza. We aimed to compare the risk of influenza infection in healthcare workers in acute hospital care with that in non-healthcare workers over the same season. Methods: We conducted a prospective, multicentre cohort study during the 2006/07 influenza season in Berlin, Germany. Recruited participants gave serum samples before and after the season, and completed questionnaires to determine their relevant exposures and possible confounding factors. The main outcome measure was serologically confirmed influenza infection (SCII), defined as a fourfold or greater rise in haemagglutination inhibition antibody titres to a circulating strain of influenza (with post-season titre at least 1:40). Weekly mobile phone text messages were used to prompt participants to report respiratory illnesses during the influenza season. A logistic regression model was used to assess the influence of potential risk factors. Results: We recruited 250 hospital healthcare workers (mean age 35.7 years) and 486 non-healthcare workers (mean age 39.2 years) from administrative centres, blood donors and colleges. Overall SCII attack rate was 10.6%. Being a healthcare worker was not a risk factor for SCII (relative risk 1.1, p=0.70). The final multivariate model had three significant factors: living with children (odds ratio [OR] 3.7, p=0.005), immunization (OR 0.50, p=0.02), and - among persons living in households without children - ownership of a car (OR 3.0, p=0.02). Living with three or more children (OR 13.8,

The effect of dopamine agonists on adaptive and aberrant salience in Parkinson's disease

Clinical evidence suggests that after initiation of dopaminergic medications some patients with Parkinson's disease (PD) develop psychotic symptoms, such as hallucinations and delusions. Here, we tested the hypothesis that the neurocognitive basis of this phenomenon can be defined as the formation of arbitrary and illusory associations between conditioned stimuli and reward signals, called aberrant salience. Young, never-medicated PD patients and matched controls were assessed on a speeded reaction time task in which the probe stimulus was preceded by conditioned stimuli that could signal monetary reward by color or shape. The patients and controls were re-evaluated after 12 weeks during which the patients received a dopamine agonist (pramipexole or ropinirole). Results indicated that dopamine agonists increased both adaptive and aberrant salience in PD patients, that is, formation of real and illusory associations between conditioned stimuli and reward, respectively. This effect was present when associations were assessed by means of faster responding after conditioned stimuli signaling reward (implicit salience) and overt rating of stimulus-reward links (explicit salience). However, unusual feelings and experiences, which are subclinical manifestations of psychotic-like symptoms, were specifically related to irrelevant and illusory stimulus-reward associations (aberrant salience) in PD patients receiving dopamine agonists. The learning of relevant and real stimulus-reward associations (adaptive salience) was not related to unusual experiences. These results suggest that dopamine agonists may increase psychotic-like experiences in young patients with PD, possibly by facilitating dopaminergic transmission in the ventral striatum, which results in aberrant associations between conditioned stimuli and reward

Specific Immunoassays Confirm Association of Mycobacterium avium Subsp. paratuberculosis with Type-1 but Not Type-2 Diabetes Mellitus

Mycobacterium avium subspecies paratuberculosis (MAP) is a versatile pathogen with a broad host range. Its association with type-1 diabetes mellitus (T1DM) has been recently proposed. Rapid identification of infectious agents such as MAP in diabetic patients at the level of clinics might be helpful in deciphering the role of chronic bacterial infection in the development of autoimmune diseases such as T1DM.We describe use of an ELISA method to identify live circulating MAP through the detection of a cell envelope protein, MptD by a specific M13 phage--fMptD. We also used another ELISA format to detect immune response to MptD peptide. Both the methods were tested with blood plasma obtained from T1DM, type-2 diabetes (T2DM) patients and non-diabetic controls. Our results demonstrate MptD and fMptD ELISA assays to be accurate and sensitive to detect MAP bacilli in a large fraction (47.3%) of T1DM patients as compared to non-diabetic controls (12.6%) and those with confirmed T2DM (7.7%). Comparative analysis of ELISA assays performed here with 3 other MAP antigen preparations, namely HbHA, Gsd and whole cell MAP lysates confirmed comparable sensitivity of the MptD peptide and the fMptD based ELISA assays. Moreover, we were successful in demonstrating positive bacterial culture in two of the clinical specimen derived from T1DM patients.The MptD peptide/fMptD based ELISA or similar tests could be suggested as rapid and specific field level diagnostic tests for the identification of MAP in diabetic patients and for finding the explanations towards the occurrence of type-1 or type-2 diabetes in the light of an active infectious trigger

Inhaled nitric oxide alleviates hyperoxia suppressed phosphatidylcholine synthesis in endotoxin-induced injury in mature rat lungs

BACKGROUND: We investigated efficacy of inhaled nitric oxide (NO) in modulation of metabolism of phosphatidylcholine (PC) of pulmonary surfactant and in anti-inflammatory mechanism of mature lungs with inflammatory injury. METHODS: Healthy adult rats were divided into a group of lung inflammation induced by i.v. lipopolysaccharides (LPS) or a normal control (C) for 24 h, and then exposed to: room air (Air), 95% oxygen (O), NO (20 parts per million, NO), both O and NO (ONO) as subgroups, whereas [(3)H]-choline was injected i.v. for incorporation into PC of the lungs which were processed subsequently at 10 min, 4, 8, 12 and 24 h, respectively, for measurement of PC synthesis and proinflammatory cytokine production. RESULTS: LPS-NO subgroup had the lowest level of labeled PC in total phospholipids and disaturated PC in bronchoalveolar lavage fluid and lung tissue (decreased by 46–59%), along with the lowest activity of cytidine triphosphate: phosphocholine cytidylyltransferase (-14–18%) in the lungs, compared to all other subgroups at 4 h (p < 0.01), but not at 8 and 12 h. After 24-h, all LPS-subgroups had lower labeled PC than the corresponding C-subgroups (p < 0.05). LPS-ONO had higher labeled PC in total phospholipids and disaturated PC, activity of cytidylyltransferase, and lower activity of nuclear transcription factor-κB and expression of proinflammatory cytokine mRNA, than that in the LPS-O subgroup (p < 0.05). CONCLUSION: In LPS-induced lung inflammation in association with hyperoxia, depressed PC synthesis and enhanced proinflammatory cytokine production may be alleviated by iNO. NO alone only transiently suppressed the PC synthesis as a result of lower activity of cytidylyltransferase

Jet energy measurement with the ATLAS detector in proton-proton collisions at root s=7 TeV

The jet energy scale and its systematic uncertainty are determined for jets measured with the ATLAS detector at the LHC in proton-proton collision data at a centre-of-mass energy of √s = 7TeV corresponding to an integrated luminosity of 38 pb-1. Jets are reconstructed with the anti-kt algorithm with distance parameters R=0. 4 or R=0. 6. Jet energy and angle corrections are determined from Monte Carlo simulations to calibrate jets with transverse momenta pT≥20 GeV and pseudorapidities {pipe}η{pipe}<4. 5. The jet energy systematic uncertainty is estimated using the single isolated hadron response measured in situ and in test-beams, exploiting the transverse momentum balance between central and forward jets in events with dijet topologies and studying systematic variations in Monte Carlo simulations. The jet energy uncertainty is less than 2. 5 % in the central calorimeter region ({pipe}η{pipe}<0. 8) for jets with 60≤pT<800 GeV, and is maximally 14 % for pT<30 GeV in the most forward region 3. 2≤{pipe}η{pipe}<4. 5. The jet energy is validated for jet transverse momenta up to 1 TeV to the level of a few percent using several in situ techniques by comparing a well-known reference such as the recoiling photon pT, the sum of the transverse momenta of tracks associated to the jet, or a system of low-pT jets recoiling against a high-pT jet. More sophisticated jet calibration schemes are presented based on calorimeter cell energy density weighting or hadronic properties of jets, aiming for an improved jet energy resolution and a reduced flavour dependence of the jet response. The systematic uncertainty of the jet energy determined from a combination of in situ techniques is consistent with the one derived from single hadron response measurements over a wide kinematic range. The nominal corrections and uncertainties are derived for isolated jets in an inclusive sample of high-pT jets. Special cases such as event topologies with close-by jets, or selections of samples with an enhanced content of jets originating from light quarks, heavy quarks or gluons are also discussed and the corresponding uncertainties are determined. © 2013 CERN for the benefit of the ATLAS collaboration