Electrostatic Contribution to the Photo-Assisted Piezoresponse Force Microscopy by Photo-Induced Surface Charge.

The surging interest in manipulating the polarization of piezo/ferroelectric materials by means of light has driven an increasing number of studies toward their light-polarization interaction. One way to investigate such interaction is by performing piezoresponse force microscopy (PFM) while/after the sample is exposed to light illumination. However, caution must be exercised when analyzing and interpreting the data, as demonstrated in this paper, because sizeable photo-response observed in the PFM amplitude image of the sample is shown to be caused by the electrostatic interaction between the photo-induced surface charge and tip. Through photo-assisted Kelvin probe force microscopy (KPFM), positive surface potential is found to be developed near the sample's surface under 405 nm light illumination, whose effects on the measured PFM signal is revealed by the comparative studies on its amplitude curves that are obtained using PFM spectroscopy mode with/without illumination. This work exemplifies the need for complementary use of KPFM, PFM imaging mode, and PFM spectroscopy mode in order to distinguish real behavior from artifacts.

A Genome-wide CRISPR Screen Reveals a Role for the Non-canonical Nucleosome-Remodeling BAF Complex in Foxp3 Expression and Regulatory T Cell Function.

Regulatory T (Treg) cells play a pivotal role in suppressing auto-reactive T cells and maintaining immune homeostasis. Treg cell development and function are dependent on the transcription factor Foxp3. Here, we performed a genome-wide CRISPR loss-of-function screen to identify Foxp3 regulators in mouse primary Treg cells. Foxp3 regulators were enriched in genes encoding subunits of the SWI/SNF nucleosome-remodeling and SAGA chromatin-modifying complexes. Among the three SWI/SNF-related complexes, the Brd9-containing non-canonical (nc) BAF complex promoted Foxp3 expression, whereas the PBAF complex was repressive. Chemical-induced degradation of Brd9 led to reduced Foxp3 expression and reduced Treg cell function in vitro. Brd9 ablation compromised Treg cell function in inflammatory disease and tumor immunity in vivo. Furthermore, Brd9 promoted Foxp3 binding and expression of a subset of Foxp3 target genes. Our findings provide an unbiased analysis of the genetic networks regulating Foxp3 and reveal ncBAF as a target for therapeutic manipulation of Treg cell function.

Utility of directional high-density mapping catheter (AdvisorTM HD Grid) in complex scar-related atrial tachycardia.

Mapping of scar-related atrial tachycardias (AT) can be challenging even with the use of high-density (HD) mapping catheter. AdvisorTM HD Grid is the only directional HD mapping catheter which not only identify local electrical signal but more importantly capture the direction of wave front propagation especially in low voltage zone. Accordingly, we present a case of complex scar-related AT with the use of AdvisorTM HD Grid which showed clear fractionated signal at isthmus area as compare to the absence of signal on ablation catheter at the same area despite adequate contact force. Ablation at this area terminated the tachycardia.

The nuclear receptor REV-ERBα modulates Th17 cell-mediated autoimmune disease.

T helper 17 (Th17) cells produce interleukin-17 (IL-17) cytokines and drive inflammatory responses in autoimmune diseases such as multiple sclerosis. The differentiation of Th17 cells is dependent on the retinoic acid receptor-related orphan nuclear receptor RORγt. Here, we identify REV-ERBα (encoded by Nr1d1), a member of the nuclear hormone receptor family, as a transcriptional repressor that antagonizes RORγt function in Th17 cells. REV-ERBα binds to ROR response elements (RORE) in Th17 cells and inhibits the expression of RORγt-dependent genes including Il17a and Il17f Furthermore, elevated REV-ERBα expression or treatment with a synthetic REV-ERB agonist significantly delays the onset and impedes the progression of experimental autoimmune encephalomyelitis (EAE). These results suggest that modulating REV-ERBα activity may be used to manipulate Th17 cells in autoimmune diseases.

The lysolipid transporter Mfsd2a regulates lipogenesis in the developing brain.

Brain development requires a massive increase in brain lipogenesis and accretion of the essential omega-3 fatty acid docosahexaenoic acid (DHA). Brain acquisition of DHA is primarily mediated by the transporter Major Facilitator Superfamily Domain containing 2a (Mfsd2a) expressed in the endothelium of the blood-brain barrier (BBB) and other abundant cell types within the brain. Mfsd2a transports DHA and other polyunsaturated fatty acids (PUFAs) esterified to lysophosphatidylcholine (LPC-DHA). However, the function of Mfsd2a and DHA in brain development is incompletely understood. Here, we demonstrate, using vascular endothelial-specific and inducible vascular endothelial-specific deletion of Mfsd2a in mice, that Mfsd2a is uniquely required postnatally at the BBB for normal brain growth and DHA accretion, with DHA deficiency preceding the onset of microcephaly. In Mfsd2a-deficient mouse models, a lipidomic signature was identified that is indicative of increased de novo lipogenesis of PUFAs. Gene expression profiling analysis of these DHA-deficient brains indicated that sterol regulatory-element binding protein (Srebp)-1 and Srebp-2 pathways were highly elevated. Mechanistically, LPC-DHA treatment of primary neural stem cells down-regulated Srebp processing and activation in a Mfsd2a-dependent fashion, resulting in profound effects on phospholipid membrane saturation. In addition, Srebp regulated the expression of Mfsd2a. These data identify LPC-DHA transported by Mfsd2a as a physiological regulator of membrane phospholipid saturation acting in a feedback loop on Srebp activity during brain development.

Platelet-derived growth factor (PDGF) signaling directs cardiomyocyte movement toward the midline during heart tube assembly.

Communication between neighboring tissues plays a central role in guiding organ morphogenesis. During heart tube assembly, interactions with the adjacent endoderm control the medial movement of cardiomyocytes, a process referred to as cardiac fusion. However, the molecular underpinnings of this endodermal-myocardial relationship remain unclear. Here, we show an essential role for platelet-derived growth factor receptor alpha (Pdgfra) in directing cardiac fusion. Mutation of pdgfra disrupts heart tube assembly in both zebrafish and mouse. Timelapse analysis of individual cardiomyocyte trajectories reveals misdirected cells in zebrafish pdgfra mutants, suggesting that PDGF signaling steers cardiomyocytes toward the midline during cardiac fusion. Intriguingly, the ligand pdgfaa is expressed in the endoderm medial to the pdgfra-expressing myocardial precursors. Ectopic expression of pdgfaa interferes with cardiac fusion, consistent with an instructive role for PDGF signaling. Together, these data uncover a novel mechanism through which endodermal-myocardial communication can guide the cell movements that initiate cardiac morphogenesis.

STIM2 regulates PKA-dependent phosphorylation and trafficking of AMPARs.

STIMs (STIM1 and STIM2 in mammals) are transmembrane proteins that reside in the endoplasmic reticulum (ER) and regulate store-operated Ca(2+) entry (SOCE). The function of STIMs in the brain is only beginning to be explored, and the relevance of SOCE in nerve cells is being debated. Here we identify STIM2 as a central organizer of excitatory synapses. STIM2, but not its paralogue STIM1, influences the formation of dendritic spines and shapes basal synaptic transmission in excitatory neurons. We further demonstrate that STIM2 is essential for cAMP/PKA-dependent phosphorylation of the AMPA receptor (AMPAR) subunit GluA1. cAMP triggers rapid migration of STIM2 to ER-plasma membrane (PM) contact sites, enhances recruitment of GluA1 to these ER-PM junctions, and promotes localization of STIM2 in dendritic spines. Both biochemical and imaging data suggest that STIM2 regulates GluA1 phosphorylation by coupling PKA to the AMPAR in a SOCE-independent manner. Consistent with a central role of STIM2 in regulating AMPAR phosphorylation, STIM2 promotes cAMP-dependent surface delivery of GluA1 through combined effects on exocytosis and endocytosis. Collectively our results point to a unique mechanism of synaptic plasticity driven by dynamic assembly of a STIM2 signaling complex at ER-PM contact sites.

Quantitative apical membrane proteomics reveals vasopressin-induced actin dynamics in collecting duct cells.

In kidney collecting duct cells, filamentous actin (F-actin) depolymerization is a critical step in vasopressin-induced trafficking of aquaporin-2 to the apical plasma membrane. However, the molecular components of this response are largely unknown. Using stable isotope-based quantitative protein mass spectrometry and surface biotinylation, we identified 100 proteins that showed significant abundance changes in the apical plasma membrane of mouse cortical collecting duct cells in response to vasopressin. Fourteen of these proteins are involved in actin cytoskeleton regulation, including actin itself, 10 actin-associated proteins, and 3 regulatory proteins. Identified were two integral membrane proteins (Clmn, Nckap1) and one actin-binding protein (Mpp5) that link F-actin to the plasma membrane, five F-actin end-binding proteins (Arpc2, Arpc4, Gsn, Scin, and Capzb) involved in F-actin reorganization, and two actin adaptor proteins (Dbn1, Lasp1) that regulate actin cytoskeleton organization. There were also protease (Capn1), protein kinase (Cdc42bpb), and Rho guanine nucleotide exchange factor 2 (Arhgef2) that mediate signal-induced F-actin changes. Based on these findings, we devised a live-cell imaging method to observe vasopressin-induced F-actin dynamics in polarized mouse cortical collecting duct cells. In response to vasopressin, F-actin gradually disappeared near the center of the apical plasma membrane while consolidating laterally near the tight junction. This F-actin peripheralization was blocked by calcium ion chelation. Vasopressin-induced apical aquaporin-2 trafficking and forskolin-induced water permeability increase were blocked by F-actin disruption. In conclusion, we identified a vasopressin-regulated actin network potentially responsible for vasopressin-induced apical F-actin dynamics that could explain regulation of apical aquaporin-2 trafficking and water permeability increase.

Fully-automated image processing software to analyze calcium traces in populations of single cells.

Advances in fluorescence live cell imaging over the last decade have revolutionized cell biology by providing access to single-cell information in space and time. One current limitation of live-cell imaging is the lack of automated procedures to analyze single-cell data in large cell populations. Most commercially available image processing softwares do not have built-in image segmentation tools that can automatically and accurately extract single-cell data in a time series. Consequently, individual cells are usually identified manually, a process which is time consuming and inherently low-throughput. We have developed a MATLAB-based image segmentation algorithm that reliably detects individual cells in dense populations and measures their fluorescence intensity over time. To demonstrate the value of this algorithm, we measured store-operated calcium entry (SOCE) in hundreds of individual cells. Rapid access to single-cell calcium signals in large populations allowed us to precisely determine the relationship between SOCE activity and STIM1 levels, a key component of SOCE. Our image processing tool can in principle be applied to a wide range of live-cell imaging modalities and cell-based drug screening platforms.

Obstetric care of Jehovah's Witnesses: a 14-year observational study.

Over a 14-year period, the obstetric outcome of Jehovah's Witnesses in an inner city hospital was reviewed and the effect of refusal of blood on morbidity and mortality evaluated. Ninety women had 116 deliveries and of these, 24% were delivered by caesarean section, 10% had instrumental deliveries and 66% were normal vaginal deliveries. Postpartum haemorrhage of >1,000 mls occurred in 6% and postpartum anaemia was the commonest complication. The mean postdelivery haemoglobin (11.10 +/- 1.15 g/dl) was not significantly less from the mean predelivery haemoglobin level (11.81 +/- 1.62 g/dl) (P > 0.05, paired t test). The single maternal death occurred after caesarean hysterectomy, which when extrapolated, resulted in a 65-fold increased risk of maternal death compared to the national rate. The optimum management of pregnant women who decline transfusion is discussed.

Application of invariant grey scale features for analysis of porous minerals.

Three-dimensional (3D) texture analysis can be used to differentiate similar materials which have a complex structural nature that is not easily reduced to geometric primitives. A method which extends the concept of invariant grey scale features to non-structured 3D textures is introduced and applied to the study of five processed mineral carbon materials which are characteristically similar but derive from different industrial sources. X-ray microtomography (XRMT) was used to obtain 3D tomographic data with isotropic voxel spacing of 9.8 microm. These data were used to construct invariant features for 3D texture measurement via Monte Carlo based sampling routines and integrals of grey scale relational kernel functions. The procedure produced multi-component texture vectors, which were successfully tested against texture samples as a classification-recognition tool. Identification accuracies ranging from 69% to approximately 84% were achieved for the five material sources examined. This result provides a sound basis for quantitative analysis of these materials which to date have proved very difficult to examine using traditional image analysis tools because of their complex natural structure.