Identification of serpins specific for activated protein C using a lysate-based screening assay.

Activated protein C (APC) is a powerful anticoagulant enzyme that proteolytically inactivates the cofactors of the Xase and prothrombinase complexes, factors VIIIa and Va. A common mutation in factor V, fVLeiden, confers resistance to APC leading to an increased risk of thrombosis in the normal population. However, when coinherited with haemophilia, fVLeiden reduces bleeding severity, suggesting that inhibition of APC may be a useful strategy for treatment of haemophilia. We previously reported on serpins that were rationally designed for improved specificity for APC over other coagulation serine proteases. Based on structural differences in the substrate binding pockets to either side of the P1 Arg, we mutated the P2 and P1' residues to Lys. Although this approach achieved APC specificity, it resulted in a reduction in the rate of APC inhibition relative to the parent containing only the P1 Arg. Here we conduct site-specific random mutagenesis at the P2 and P1' positions to determine if improvements could be made in the rate of APC inhibition. In addition to our original Lys mutations, we found that Arg and Gln also confer specificity for APC. However, in all cases specificity for APC resulted in a reduction in inhibition rate

Ixodes ricinus Salivary Serpin Iripin-8 Inhibits the Intrinsic Pathway of Coagulation and Complement.

Tick saliva is a rich source of antihemostatic, anti-inflammatory, and immunomodulatory molecules that actively help the tick to finish its blood meal. Moreover, these molecules facilitate the transmission of tick-borne pathogens. Here we present the functional and structural characterization of Iripin-8, a salivary serpin from the tick Ixodes ricinus, a European vector of tick-borne encephalitis and Lyme disease. Iripin-8 displayed blood-meal-induced mRNA expression that peaked in nymphs and the salivary glands of adult females. Iripin-8 inhibited multiple proteases involved in blood coagulation and blocked the intrinsic and common pathways of the coagulation cascade in vitro. Moreover, Iripin-8 inhibited erythrocyte lysis by complement, and Iripin-8 knockdown by RNA interference in tick nymphs delayed the feeding time. Finally, we resolved the crystal structure of Iripin-8 at 1.89 Å resolution to reveal an unusually long and rigid reactive center loop that is conserved in several tick species. The P1 Arg residue is held in place distant from the serpin body by a conserved poly-Pro element on the P' side. Several PEG molecules bind to Iripin-8, including one in a deep cavity, perhaps indicating the presence of a small-molecule binding site. This is the first crystal structure of a tick serpin in the native state, and Iripin-8 is a tick serpin with a conserved reactive center loop that possesses antihemostatic activity that may mediate interference with host innate immunity

Northeast Folklore volume 2 numbers 1-4

Description The second issue of Northeast Folklore was published in the spring of 1959 under the editorship of Edward D. Ives (known as Sandy) and Bacil F. Kirtley through the Department of English at the University of Maine. The four editions that year were later bound into a single volume. Table of Contents Number 1 (Spring): Two Songs from Martha\u27s Vineyard by E.G. Huntington The Deer Isle Hoax by James J. Flynn and Charles A. Huguenin Folklore from Aroostook County, Maine, and Neighboring Canada edited by Bacil F. Kirtley Notes and Queries Number 2 (Summer): Bibliography of New England-Maritimes Folklore Crooked Brook : A Song of the Maine Woods by Edward D. Ives Folklore from Aroostook County, Maine, and Neighboring Canada by Bacil F. Kirtley Record Reviews: Songs of a New York Lumberjack (Steckert) by Norman Cazden Timber-r-r! (Clayton) by Frank A. Hoffmann Folksongs of Martha\u27s Vineyard (Huntington) by Evelyn K. Wells Number 3 (Fall): Folklore in Rhode Island by Horace P. Beck Larry Gorman and Old Henry by Edward D. Ives Folklore from Aroostook County, Maine, and Neighboring Canada edited by Bacil F. Kirtley Number 4 (Winter): A New England Folklore Weekend at Old Sturbridge Village More Notes on the Burning Ship of Northumberland Strait Folklore from Aroostook County, Maine, and Neighboring Canada edited by Bacil F. Kirtley The Lumberman in Town by Edward D. Ives Notes and Queries Book Reviews: The Abelard Folk Song Book (Cazden) by Helen Creighton.https://digitalcommons.library.umaine.edu/nf/1002/thumbnail.jp

The Strategic Shuffle: Ethnic Geography, the Internal Security Apparatus, and Elections in Kenya

For autocrats facing elections, officers in the internal security apparatus play a crucial role by engaging in coercion on behalf of the incumbent. Yet reliance on these officers introduces a principal‐agent problem: Officers can shirk from the autocrat’s demands. To solve this problem, autocrats strategically post officers to different areas based on an area’s importance to the election and the expected loyalty of an individual officer, which is a function of the officer’s expected benefits from the president winning reelection. Using a data set of 8,000 local security appointments within Kenya in the 1990s, one of the first of its kind for any autocracy, I find that the president’s coethnic officers were sent to, and the opposition’s coethnic officers were kept away from, swing areas. This article demonstrates how state institutions from a country’s previous authoritarian regime can persist despite the introduction of multi‐party elections and thus prevent full democratization.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/136510/1/ajps12279_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/136510/2/ajps12279.pd

RNA-Seq identifies SPGs as a ventral skeletal patterning cue in sea urchins

The sea urchin larval skeleton offers a simple model for formation of developmental patterns. The calcium carbonate skeleton is secreted by primary mesenchyme cells (PMCs) in response to largely unknown patterning cues expressed by the ectoderm. To discover novel ectodermal cues, we performed an unbiased RNA-Seq-based screen and functionally tested candidates; we thereby identified several novel skeletal patterning cues. Among these, we show that SLC26a2/7 is a ventrally expressed sulfate transporter that promotes a ventral accumulation of sulfated proteoglycans, which is required for ventral PMC positioning and skeletal patterning. We show that the effects of SLC perturbation are mimicked by manipulation of either external sulfate levels or proteoglycan sulfation. These results identify novel skeletal patterning genes and demonstrate that ventral proteoglycan sulfation serves as a positional cue for sea urchin skeletal patterning

Phosphatidylethanolamine critically supports internalization of cell-penetrating protein C inhibitor

Although their contribution remains unclear, lipids may facilitate noncanonical routes of protein internalization into cells such as those used by cell-penetrating proteins. We show that protein C inhibitor (PCI), a serine protease inhibitor (serpin), rapidly transverses the plasma membrane, which persists at low temperatures and enables its nuclear targeting in vitro and in vivo. Cell membrane translocation of PCI necessarily requires phosphatidylethanolamine (PE). In parallel, PCI acts as a lipid transferase for PE. The internalized serpin promotes phagocytosis of bacteria, thus suggesting a function in host defense. Membrane insertion of PCI depends on the conical shape of PE and is associated with the formation of restricted aqueous compartments within the membrane. Gain- and loss-of-function mutations indicate that the transmembrane passage of PCI requires a branched cavity between its helices H and D, which, according to docking studies, precisely accommodates PE. Our findings show that its specific shape enables cell surface PE to drive plasma membrane translocation of cell-penetrating PCI

Performance of the 12-item WHODAS 2.0 in prodromal Huntington disease

ACKNOWLEDGEMENTS We thank the PREDICT-HD sites, the study participants, the National Research Roster for Huntington Disease Patients and Families, the Huntington’s Disease Society of America and the Huntington Study Group. This publication was supported by the National Center for Advancing Translational Sciences, and the National Institutes of Health (NIH), through Grant 2 UL1 TR000442-06. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH. This research is supported by the National Institutes of Health, National Institute of Neurological Disorders and Stroke (5R01NS040068) awarded to Dr Paulsen, CHDI Foundation, Inc (A3917) awarded to Dr Paulsen, Cognitive and Functional Brain Changes in Preclinical Huntington’s Disease (HD) (5R01NS054893) awarded to Dr Paulsen, 4D Shape Analysis for Modeling Spatiotemporal Change Trajectories in Huntington’s (1U01NS082086), Functional Connectivity in Premanifest Huntington’s Disease (1U01NS082083), and Basal Ganglia Shape Analysis and Circuitry in Huntington’s Disease (1U01NS082085).Peer reviewedPublisher PD