The C Terminus of the Alb3 Membrane Insertase Recruits cpSRP43 to the Thylakoid Membrane

The YidC/Oxa1/Alb3 family of membrane proteins controls the insertion and assembly of membrane proteins in bacteria, mitochondria, and chloroplasts. Here we describe the molecular mechanisms underlying the interaction of Alb3 with the chloroplast signal recognition particle (cpSRP). The Alb3 C-terminal domain (A3CT) is intrinsically disordered and recruits cpSRP to the thylakoid membrane by a coupled binding and folding mechanism. Two conserved, positively charged motifs reminiscent of chromodomain interaction motifs in histone tails are identified in A3CT that are essential for the Alb3-cpSRP43 interaction. They are absent in the C-terminal domain of Alb4, which therefore does not interact with cpSRP43. Chromodomain 2 in cpSRP43 appears as a central binding platform that can interact simultaneously with A3CT and cpSRP54. The observed negative cooperativity of the two binding events provides the first insights into cargo release at the thylakoid membrane. Taken together, our data show how Alb3 participates in cpSRP-dependent membrane targeting, and our data provide a molecular explanation why Alb4 cannot compensate for the loss of Alb3. Oxa1 and YidC utilize their positively charged, C-terminal domains for ribosome interaction in co-translational targeting. Alb3 is adapted for the chloroplast-specific Alb3-cpSRP43 interaction in post-translational targeting by extending the spectrum of chromodomain interactions.     

Working-Poor Mothers and Middle-Class Others: Psychosocial Considerations in Home-School Relations and Research

This article draws from a three-year ethnographic study of girls and their mothers in a high-poverty, predominantly white community. Informed by critical and feminist theories of social class, I present four cases that highlight psychosocial tensions within the mother-daughterteacher-researcher triangle and argue that white, middle-class female teachers and ethnographers need to be particularly reflexive when working with children across the social class divide.     

The acute impact of continuous positive airway pressure on nasal resistance: a randomized controlled comparison.

Subjective nasal obstruction is common among users of continuous positive airway pressure (CPAP). The aim of this study was to measure the acute effect of CPAP on nasal resistance and nasal symptoms in awake normal subjects. Twenty-four healthy CPAP-naive adults [8 men, 16 women; mean age 30 yr (SD 14)] underwent a randomized controlled crossover study comparing nasal CPAP (8 cmH(2)O) for 6 h on one occasion and the control condition (nasal mask without CPAP) on the other. Nasal resistance measurements (posterior active rhinometry) before and after the test exposure were similar on both test days. Nasal resistance during CPAP exposure [2.04 cmH(2)O.l(-1).s (SD 0.72)] was significantly lower than that of the control [2.67 cmH(2)O.l(-1).s (SD 1.07)]: mean difference 0.66 cmH(2)O.l(-1).s, 95% confidence interval 0.19-1.13 cmH(2)O.l(-1).s. The gradient in pressure from CPAP mask to posterior naris during CPAP exposure varied from 1.6 to 2 cmH(2)O but was not significantly different between time points. Subjective nasal symptom scores and peak nasal inspiratory flow rates did not change significantly on either test day. We conclude that in awake CPAP-naive normal subjects, acute CPAP exposure is associated with a reduction in nasal resistance compared with the control condition, but it is not associated with an immediate post-CPAP change in subjective or objective nasal resistance.     

Natural Killer Cells Promote Long-Term Hepatobiliary Inflammation in a Low-Dose Rotavirus Model of Experimental Biliary Atresia

ConclusionsLower inoculation of RRV-induced progressive liver injury and fibrosis via NK cells. These findings point to the potential use of NK cell-depleting strategies to block progression of liver disease in biliary atresia.     

Induction of Neuron-Specific Degradation of Coenzyme A Models Pantothenate Kinase-Associated Neurodegeneration by Reducing Motor Coordination in Mice

Background

Pantothenate kinase-associated neurodegeneration, PKAN, is an inherited disorder characterized by progressive impairment in motor coordination and caused by mutations in PANK2, a human gene that encodes one of four pantothenate kinase (PanK) isoforms. PanK initiates the synthesis of coenzyme A (CoA), an essential cofactor that plays a key role in energy metabolism and lipid synthesis. Most of the mutations in PANK2 reduce or abolish the activity of the enzyme. This evidence has led to the hypothesis that lower CoA might be the underlying cause of the neurodegeneration in PKAN patients; however, no mouse model of the disease is currently available to investigate the connection between neuronal CoA levels and neurodegeneration. Indeed, genetic and/or dietary manipulations aimed at reducing whole-body CoA synthesis have not produced a desirable PKAN model, and this has greatly hindered the discovery of a treatment for the disease.

Objective, Methods, Results and Conclusions

Cellular CoA levels are tightly regulated by a balance between synthesis and degradation. CoA degradation is catalyzed by two peroxisomal nudix hydrolases, Nudt7 and Nudt19. In this study we sought to reduce neuronal CoA in mice through the alternative approach of increasing Nudt7-mediated CoA degradation. This was achieved by combining the use of an adeno-associated virus-based expression system with the synapsin (Syn) promoter. We show that mice with neuronal overexpression of a cytosolic version of Nudt7 (scAAV9-Syn-Nudt7cyt) exhibit a significant decrease in brain CoA levels in conjunction with a reduction in motor coordination. These results strongly support the existence of a link between CoA levels and neuronal function and show that scAAV9-Syn-Nudt7cyt mice can be used to model PKAN.     

Neural Correlates of Emotional Interference in Social Anxiety Disorder

Disorder-relevant but task-unrelated stimuli impair cognitive performance in social anxiety disorder (SAD); however, time course and neural correlates of emotional interference are unknown. The present study investigated time course and neural basis of emotional interference in SAD using event-related functional magnetic resonance imaging (fMRI). Patients with SAD and healthy controls performed an emotional stroop task which allowed examining interference effects on the current and the succeeding trial. Reaction time data showed an emotional interference effect in the current trial, but not the succeeding trial, specifically in SAD. FMRI data showed greater activation in the left amygdala, bilateral insula, medial prefrontal cortex (mPFC), dorsal anterior cingulate cortex (ACC), and left opercular part of the inferior frontal gyrus during emotional interference of the current trial in SAD patients. Furthermore, we found a positive correlation between patients’ interference scores and activation in the mPFC, dorsal ACC and left angular/supramarginal gyrus. Taken together, results indicate a network of brain regions comprising amygdala, insula, mPFC, ACC, and areas strongly involved in language processing during the processing of task-unrelated threat in SAD. However, specifically the activation in mPFC, dorsal ACC, and left angular/supramarginal gyrus is associated with the strength of the interference effect, suggesting a cognitive network model of attentional bias in SAD. This probably comprises exceeded allocation of attentional resources to disorder-related information of the presented stimuli and increased self-referential and semantic processing of threat words in SAD.     

The Impact II,a Very High-Resolution Quadrupole Time-of-Flight Instrument (QTOF) for Deep Shotgun Proteomics

Hybrid quadrupole time-of-flight (QTOF) mass spectrometry is one of the two major principles used in proteomics. Although based on simple fundamentals, it has over the last decades greatly evolved in terms of achievable resolution, mass accuracy, and dynamic range. The Bruker impact platform of QTOF instruments takes advantage of these developments and here we develop and evaluate the impact II for shotgun proteomics applications. Adaption of our heated liquid chromatography system achieved very narrow peptide elution peaks. The impact II is equipped with a new collision cell with both axial and radial ion ejection, more than doubling ion extraction at high tandem MS frequencies. The new reflectron and detector improve resolving power compared with the previous model up to 80%, i.e. to 40,000 at m/z 1222. We analyzed the ion current from the inlet capillary and found very high transmission (>80%) up to the collision cell. Simulation and measurement indicated 60% transfer into the flight tube. We adapted MaxQuant for QTOF data, improving absolute average mass deviations to better than 1.45 ppm. More than 4800 proteins can be identified in a single run of HeLa digest in a 90 min gradient. The workflow achieved high technical reproducibility (R2 > 0.99) and accurate fold change determination in spike-in experiments in complex mixtures. Using label-free quantification we rapidly quantified haploid against diploid yeast and characterized overall proteome differences in mouse cell lines originating from different tissues. Finally, after high pH reversed-phase fractionation we identified 9515 proteins in a triplicate measurement of HeLa peptide mixture and 11,257 proteins in single measurements of cerebellum—the highest proteome coverage reported with a QTOF instrument so far.Building on the fundamental advance of the soft ionization techniques electrospray ionization and matrix-assisted laser desorption/ionization (1, 2), MS-based proteomics has advanced tremendously over the last two decades (3–6). Bottom-up, shotgun proteomics is usually performed in a liquid chromatography-tandem MS (LC-MS/MS)¹ format, where nanoscale liquid chromatography is coupled through electrospray ionization to an instrument capable of measuring a mass spectrum and fragmenting the recognized precursor peaks on the chromatographic time scale. Fundamental challenges of shotgun proteomics include the very large numbers of peptides that elute over relatively short periods and peptide abundances that vary by many orders of magnitude. Developments in mass spectrometers toward higher sensitivity, sequencing speed, and resolution were needed and helped to address these critical challenges (7, 8). Especially the introduction of the Orbitrap mass analyzers has advanced the state of the art of the field because of their very high resolution and mass accuracy (9, 10). A popular configuration couples a quadrupole mass filter for precursor selection to the Orbitrap analyzer in a compact benchtop format (11–13).In addition to the improvements in MS instrumentation, there have been key advances in the entire proteomics workflow, from sample preparation through improved LC systems and in computational proteomics (14–16). Together, such advances are making shotgun proteomics increasingly comprehensive and deep analyses can now be performed in a reasonable time (13, 17–19). Nevertheless, complete analysis of all expressed proteins in a complex system remains extremely challenging and complete measurement of all the peptides produced in shotgun proteomics may not even be possible in principle (20, 21). Therefore, an urgent need for continued improvements in proteomics technology remains.Besides the Orbitrap analyzer and other ion trap technologies, the main alternative MS technology is time-of-flight, a technology that has been used for many decades in diverse fields. The configuration employed in proteomics laboratories combines a quadrupole mass filter via a collision cell and orthogonal acceleration unit to a reflectron and a multichannel plate (MCP) detector (22). TOF scans are generated in much less than a millisecond (ms), and a number of these “pulses” are added to obtain an MS or MS/MS spectrum with the desired signal to noise ratio. Our own laboratory has used such a quadrupole time-of-flight (QTOF) instrument as the main workhorse in proteomics for many years, but then switched to high-resolution trapping instruments because of their superior resolution and mass accuracy. However, TOF technology has fundamental attractions, such as the extremely high scan speed and the absence of space charge, which limits the number of usable ions in all trapping instruments. In principle, the high spectra rate makes TOF instruments capable of making use of the majority of ions, thus promising optimal sensitivity, dynamic range and hence quantification. It also means that TOF can naturally be interfaced with ion mobility devices, which typically separate ions on the ms time scale. Data independent analysis strategies such as MS^E, in which all precursors are fragmented simultaneously (23, 24) or SWATH, in which the precursor ion window is rapidly cycled through the entire mass range (25), also make use of the high scanning speed offered by QTOF instruments. It appears that QTOFs are set to make a comeback in proteomics with recent examples showing impressive depth of coverage of complex proteomes. For instance, using a variant of the MS^E method, identification of 5468 proteins was reported in HeLa cells in single shots and small sample amounts (26). In another report, employing ion mobility for better transmission of fragment ions to the detector led to the identification of up to 7548 proteins in human ovary tissue (27).In this paper, we describe the impact II™, a benchtop QTOF instrument from Bruker Daltonics, and its use in shotgun proteomics. This QTOF instrument is a member of an instrument family first introduced in 2008, which consists of the compact, the impact, and the maXis. The original impact was introduced in 2011 and was followed by the impact HD, which was equipped with a better digitizer, expanding the dynamic range of the detector. With the impact II, which became commercially available in 2014, we aimed to achieve a resolution and sequencing speed adequate for demanding shotgun proteomics experiments. To achieve this we developed an improved collision cell, orthogonal accelerator scheme, reflectron, and detector. Here we measure ion transmission characteristics of this instrument and the actually realized resolution and mass accuracy in typical proteomics experiments. Furthermore, we investigated the attainable proteome coverage in single shot analysis and we ask if QTOF performance is now sufficient for very deep characterization of complex cell line and tissue proteomes.     

Activation of Salmonella Typhi-Specific Regulatory T Cells in Typhoid Disease in a Wild-Type S. Typhi Challenge Model

Salmonella Typhi (S. Typhi), the causative agent of typhoid fever, causes significant morbidity and mortality worldwide. Currently available vaccines are moderately efficacious, and identification of immunological responses associated with protection or disease will facilitate the development of improved vaccines. We investigated S. Typhi-specific modulation of activation and homing potential of circulating regulatory T cells (T_reg) by flow and mass cytometry using specimens obtained from a human challenge study. Peripheral blood mononuclear cells were obtained from volunteers pre- and at multiple time-points post-challenge with wild-type S. Typhi. We identified differing patterns of S. Typhi-specific modulation of the homing potential of circulating T_reg between volunteers diagnosed with typhoid (TD) and those who were not (No TD). TD volunteers demonstrated up-regulation of the gut homing molecule integrin α4ß7 pre-challenge, followed by a significant down-regulation post-challenge consistent with T_reg homing to the gut. Additionally, S. Typhi-specific T_reg from TD volunteers exhibited up-regulation of activation molecules post-challenge (e.g., HLA-DR, LFA-1). We further demonstrate that depletion of T_reg results in increased S. Typhi-specific cytokine production by CD8+ T_EM in vitro. These results suggest that the tissue distribution of activated T_reg, their characteristics and activation status may play a pivotal role in typhoid fever, possibly through suppression of S. Typhi-specific effector T cell responses. These studies provide important novel insights into the regulation of immune responses that are likely to be critical in protection against typhoid and other enteric infectious diseases.