A novel auxiliary subunit critical to BK channel function in caenorhabditis elegans

The BK channel is a Ca(2+)- and voltage-gated potassium channel with many important physiological functions. To identify proteins important to its function in vivo, we screened for C. elegans mutants that suppressed a lethargic phenotype caused by expressing a gain-of-function (gf) isoform of the BK channel α-subunit SLO-1. BKIP-1, a small peptide with no significant homology to any previously characterized molecules was thus identified. BKIP-1 and SLO-1 showed similar expression and subcellular localization patterns, and appeared to interact physically through discrete domains. bkip-1 loss-of-function (lf) mutants phenocopied slo-1(lf) mutants in behavior and synaptic transmission, and suppressed the lethargy, egg-laying defect, and deficient neurotransmitter release caused by SLO-1(gf). In heterologous expression systems, BKIP-1 decreased the activation rate and shifted the conductance-voltage (G-V) relationship of SLO-1 in a Ca(2+)-dependent manner, and increased SLO-1 surface expression. Thus, BKIP-1 is a novel auxiliary subunit critical to SLO-1 function in vivo

Clinical practice guidelines of remote ischemic conditioning for the management of cerebrovascular diseases

Remote ischemic conditioning (RIC) using transient limb ischemia and reperfusion has been shown in small clinical studies to reduce myocardial injury and infarction in cardiac patients, although larger clinical outcome studies have been neutral. Experimental and emerging clinical studies have also reported beneficial effects of limb RIC in a number of different settings of cerebrovascular disease including stroke (ischemic and hemorrhagic), carotid artery stenosis, intracranial artery stenosis, aneurysms, small vessel disease, and vascular cognitive impairment. Although limb RIC has many advantages, in that it is non-invasive, easy to administer, relatively innocuous, cost-effective, has few or no contraindications, and may be deployed under various circumstances (e.g., home, ambulance, and hospital), several questions remain regarding its clinical application for cerebrovascular disease. Therefore, in this document, we aim to provide practicing clinicians with a coherent synthesis of the latest scientific evidence, and we propose several recommendations to help facilitate the clinical application of limb RIC for the management of cerebrovascular disease

Separable Bilayer Microfiltration Device for Viable Label-free Enrichment of Circulating Tumour Cells

The analysis of circulating tumour cells (CTCs) in cancer patients could provide important information for therapeutic management. Enrichment of viable CTCs could permit performance of functional analyses on CTCs to broaden understanding of metastatic disease. However, this has not been widely accomplished. Addressing this challenge, we present a separable bilayer (SB) microfilter for viable size-based CTC capture. Unlike other single-layer CTC microfilters, the precise gap between the two layers and the architecture of pore alignment result in drastic reduction in mechanical stress on CTCs, capturing them viably. Using multiple cancer cell lines spiked in healthy donor blood, the SB microfilter demonstrated high capture efficiency (78–83%), high retention of cell viability (71–74%), high tumour cell enrichment against leukocytes (1.7–2 × 10^3), and widespread ability to establish cultures post-capture (100% of cell lines tested). In a metastatic mouse model, SB microfilters successfully enriched viable mouse CTCs from 0.4–0.6 mL whole mouse blood samples and established in vitro cultures for further genetic and functional analysis. Our preliminary studies reflect the efficacy of the SB microfilter device to efficiently and reliably enrich viable CTCs in animal model studies, constituting an exciting technology for new insights in cancer research

Limb development genes underlie variation in human fingerprint patterns

Fingerprints are of long-standing practical and cultural interest, but little is known about the mechanisms that underlie their variation. Using genome-wide scans in Han Chinese cohorts, we identified 18 loci associated with fingerprint type across the digits, including a genetic basis for the long-recognized “pattern-block” correlations among the middle three digits. In particular, we identified a variant near EVI1 that alters regulatory activity and established a role for EVI1 in dermatoglyph patterning in mice. Dynamic EVI1 expression during human development supports its role in shaping the limbs and digits, rather than influencing skin patterning directly. Trans-ethnic meta-analysis identified 43 fingerprint-associated loci, with nearby genes being strongly enriched for general limb development pathways. We also found that fingerprint patterns were genetically correlated with hand proportions. Taken together, these findings support the key role of limb development genes in influencing the outcome of fingerprint patterning

Fully automatic landmarking of 2D photographs identifies novel genetic loci influencing facial features

We report a genome-wide association study for facial features in > 6,000 Latin Americans. We placed 106 landmarks on 2D frontal photographs using the cloud service platform Face++. After Procrustes superposition, genome-wide association testing was performed for 301 inter-landmark distances. We detected nominally significant association (P-value < 5×10− 8) for 42 genome regions. Of these, 9 regions have been previously reported in GWAS of facial features. In follow-up analyses, we replicated 26 of the 33 novel regions (in East Asians or Europeans). The replicated regions include 1q32.3, 3q21.1, 8p11.21, 10p11.1, and 22q12.1, all comprising strong candidate genes involved in craniofacial development. Furthermore, the 1q32.3 region shows evidence of introgression from archaic humans. These results provide novel biological insights into facial variation and establish that automatic landmarking of standard 2D photographs is a simple and informative approach for the genetic analysis of facial variation, suitable for the rapid analysis of large population samples.- Introduction - Results And Discussion -- Study sample and phenotyping -- Trait/covariate correlation and heritability -- Overview of GWAS results and integration with the literature -- Follow-up of genomic regions newly associated with facial features: Replication in two human cohorts -- Follow-up of genomic regions newly associated with facial features: effects in the mouse -- Genome annotations at associated loci - Conclusion - Methods -- Study subjects -- Genotype data -- Phenotyping -- Statistical genetic analysis -- Interaction of EDAR with other genes -- Expression analysis for significant SNPs -- Detection of archaic introgression near ATF3 and association with facial features -- Annotation of SNPs in FUMA -- Shape GWAS in outbred mic

Newly formed dust within the circumstellar environment of SN Ia-CSM 2018evt

Dust associated with various stellar sources in galaxies at all cosmic epochs remains a controversial topic, particularly whether supernovae play an important role in dust production. We report evidence of dust formation in the cold, dense shell behind the ejecta–circumstellar medium (CSM) interaction in the Type Ia-CSM supernova (SN) 2018evt three years after the explosion, characterized by a rise in mid-infrared emission accompanied by an accelerated decline in the optical radiation of the SN. Such a dust-formation picture is also corroborated by the concurrent evolution of the profiles of the Hα emission line. Our model suggests enhanced CSM dust concentration at increasing distances from the SN as compared to what can be expected from the density profile of the mass loss from a steady stellar wind. By the time of the last mid-infrared observations at day +1,041, a total amount of 1.2 ± 0.2 × 10−2 M⊙ of new dust has been formed by SN 2018evt, making SN 2018evt one of the most prolific dust factories among supernovae with evidence of dust formation. The unprecedented witness of the intense production procedure of dust may shed light on the perceptions of dust formation in cosmic history

PR1 Leukemia-Associated Antigen Is Cross-Presented by B Cells from Soluble Serum Proteases

Abstract PR1 (VLQELNVTV) is an HLA-A*0201-binding leukemia-associated peptide epitope within proteinase 3 (PRTN3) and neutrophil elastase (ELA2), and PR1-specific CD8+ cytotoxic T lymphocytes (PR1-CTL) contribute to cytogenetic remission in some patients with chronic myelogenous leukemia. It is not understood how immunity is induced to the PR1 self-antigen in leukemia patients. Normally, myeloid dendritic cells (mDC) take up exogenous antigen and peptides are cross-presented on MHC-I to induce CTL immunity. However, in myeloid leukemia patients, mDC do not function normally and they are reduced in number. On the other hand, B cells, and potentially plasmacytoid DC (pDC), are uninvolved in the malignant process but they have been implicated in tolerance induction. Indeed, PRTN3 and ELA2 are present in serum and cross-presentation of soluble antigens can lead to cross-tolerance. To investigate whether B cells can cross-present PRTN3 or ELA2, both genes were first cloned from normal bone marrow and individually cloned into the human B cell line HMy.CIR-A2 cell (previously transfected with HLA-A*0201), which doesn’t normally express the proteins. Transfectant-induced proliferation of 25-day old PR1-CTL cell lines, measured by BrDU incorporation, was used to assess sustained PR1 antigen presentation. PRTN3- and ELA2-transfected HMy.CIR-A2 cells induced HLA-A2-restricted PR1-specific CTL proliferation compared to control sham transfectants with/without HLA-A*0201. PR1 presentation was proteasome-dependent and involved transport from the ER to the cell surface since PR1-CL proliferation was abrogated by treating the transfectants with lactacystin and Brefeldin A, respectively. Western blotting (WB) of cell supernatants showed that transfected HMy.CIR-A2 cells also secreted PRTN3 and ELA2. Furthermore, endocytosis of ELA2 by HMy.CIR-A2 and normal CD19+ B cells occurred within 30 minutes, by intracellular FACS staining. Early endocytosis of ELA2 was partly mediated by binding to LOX-1, a class E scavenger receptor on B cells and DC, since poly (I) and anti-LOX-1 antibody blocked uptake, but did not involve the scavenger receptors SR-A, CD91, or CD36 (no expression by FACS analysis). Confocal microscopy demonstrated that soluble ELA2 co-localized primarily to LAMP-2+ vesicles and to a lesser extent to cytoplasm of CD19+ B cells after 2 hours co-incubation. In contrast, freshly isolated mDC (CD11c+ CD14-) expressed ELA2 exclusively in lysosomes, shown by LAMP-2 co-localization. Like B cells, freshly isolated pDC did not express ELA2 transcripts (by RT-PCR) or protein (by WB), and confocal microscopy confirmed endocytosis of soluble ELA2 with similar lysosome + cytoplasm localization. Finally, cross-presentation of soluble ELA2 by HMy.CIR-A2 cells to PR1-CTL could be blocked by pre-treating B cells with lactacystin. In summary, soluble ELA2 and PRTN3 are taken up by normal B cells and ELA2 by pDC, in part via the LOX-1 scavenger receptor, and are localized primarily to lysosomes and to cytoplasm to a lesser extent. We previously showed that both proteins bind to ubiquitin and to the chaperone protein gp96 in protein-pulsed B cells, and the proteasome is required for PR1 processing. Thus, this is the first evidence that soluble tumor antigens can be cross-presented by B cells using the “phagosome-to-lysosome” pathway, and it suggests how immunity to the PR1 antigen could occur in leukemia patients

Aberrant Subcellular Localization of Azurophil Granule Proteins in Myeloid Leukemia Favors Peptide Antigen Presentation on MHC-I and Susceptibility to Killing by Cytotoxic T Lymphocytes

Abstract The recognition of leukemia-associated antigens (LAA) by immune cells is essential for generating anti-leukemia responses. Whether elicited by vaccine or hematopoietic stem cell transplantation (HSCT), anti-leukemia immunity requires that cytotoxic T lymphocytes (CTL) recognize and mount an effective response against leukemia cells. PR1 is a nonomeric HLA-A2-restricted peptide (VLQELNVTV) derived from the myeloid-restricted serine proteases neutrophil elastase (ELA2) and proteinase-3 (PRTN3), recognized azurophil granule proteins. PR1-specific cytotoxic T lymphocytes (PR1-CTL) have been shown to preferentially recognize and kill myeloid leukemia cells. We hypothesized that aberrant protein expression in target leukemia cells predisposes to CTL killing. To determine the subcellular localization of ELA2 and PRTN3 in leukemia and in healthy donor (HD) neutrophils, high-speed ultracentrifugation of leukapheresed cells from patients with AML and CML in blast crises yielded 4 fractions: cytoplasmic, nuclear, membrane and golgi/endoplasmic reticulum (ER). Western blot analyses identified higher levels of ELA2 and PRTN3 in cytoplasmic, nuclear, and golgi/ER fractions in leukemia, compared to HD granulocytes. Confocal microscopy of leukapheresed myeloid blasts and HD granulocytes confirmed these findings. We further hypothesized that cross-presentation of PR1 is necessary for priming anti-leukemia immunity. Confocal microscopy of the human B-cell line HMy.CIR-A2, co-incubated with either purified or recombinant ELA2 and PRTN3 demonstrated co-localization of both proteins with LAMP-2, a marker of lysosomes and early endosomes, and small distribution in cytoplasm after 2 hours. Uptake of ELA2 and PRTN3 by HMy.CIR-A2 cells was additionally confirmed by Western blots. Priming of naive PR1-CTL by co-culture of HD lymphocytes with PRTN3 or ELA2-transfected HMy.CIR-A2 cells was confirmed by cell proliferation assays using BrDU. This could be abrogated by addition of lactacystin and cyclohexamide to cell culture. Together, these results demonstrate that ELA2 and PRTN3 are aberrantly localized preferentially in gogli/ER and cytoplasmic fractions in leukemia, rather than azurophil granules, which are often absent or deficient in myeloid leukemia. Similar forced subcellular localization of these proteins in B cells leads to their susceptibility to PR1-CTL killing. Therefore, we conclude that the natural mistrafficking of PRTN3 and ELA2 in myeloid leukemia is necessary to render cells susceptible to CTL attack and that cross-presentation of ELA2 and PRTN3 contributes to priming of a PR1-CTL response