bluuebunny/crossref_metadata_2025 huggingface dataset:
Heparin Resistance and Anticoagulation Failure in a Challenging Case of Cerebral Venous Sinus Thrombosis
Authors: King Adam B. , O’Duffy Anne E. , Kumar Avinash B.
Published: June 2015

We report a challenging case of cerebral venous sinus thrombosis (multiple etiologic factors) that was complicated by heparin resistance secondary to suspected antithrombin III (ATIII) deficiency. A 20-year-old female previously healthy and currently 8 weeks pregnant presented with worsening headaches, nausea, and decreasing Glasgow Coma Scale/Score (GCS), necessitating mechanical ventilatory support. Imaging showed extensive clots in multiple cerebral venous sinuses including the superior sagittal sinus, transverse, sigmoid, jugular veins, and the straight sinus. She was started on systemic anticoagulation and underwent mechanical clot removal and catheter-directed endovascular thrombolysis with limited success. Complicating the intensive care unit care was the development of heparin resistance, with an inability to reach the target partial thomboplastin time (PTT) of 60 to 80 seconds. At her peak heparin dose, she was receiving >35 000 units/24 h, and her PTT was subtherapeutic at <50 seconds. Deficiency of ATIII was suspected as a possible etiology of her heparin resistance. Fresh frozen plasma was administered for ATIII level repletion. Given her high thrombogenic risk and challenges with conventional anticoagulation regimens, we transitioned to argatroban for systemic anticoagulation. Heparin produces its major anticoagulant effect by inactivating thrombin and factor X through an AT-dependent mechanism. For inhibition of thrombin, heparin must bind to both the coagulation enzyme and the AT. A deficiency of AT leads to a hypercoagulable state and decreased efficacy of heparin that places patients at high risk of thromboembolism. Heparin resistance, especially in the setting of critical illness, should raise the index of suspicion for AT deficiency. Argatroban is an alternate agent for systemic anticoagulation in the setting of heparin resistance.
Nonenhancing Leptomeningeal Metastases
Authors: Hatzoglou Vaios , Karimi Sasan , Diamond Eli L. , Lis Eric , Krol George , Holodny Andrei I. , Young Robert J.
Published: June 2015

The diagnosis of leptomeningeal metastasis (LM) has increased in frequency, as new therapies have lengthened the survival of patients with cancer. Early diagnosis and intervention help improve quality of life and prevent further neurological deterioration in LM. The detection of LM is often established by magnetic resonance imaging examinations, cerebrospinal fluid analysis, or both. We present a series of cases where LM was identified on fluid-attenuated inversion recovery or T2-weighted image but was nonenhancing on the traditionally more sensitive postcontrast T1-weighted sequences. Nonenhancing LM is unusual and not yet fully understood but should be considered in the appropriate clinical context and may become more common with increased utilization of antiangiogenic therapies.
Faculty Perceptions of Race to the Top Policy Influence on a University-Based Preparation Program Partnership
Authors: Reyes-Guerra Daniel , Lochmiller Chad R.
Published: June 2015

Florida’s Race to the Top (RTTT) competition invited university−district partnerships to compete for funds aimed at improving principal preparation programs. In this article, we report findings from a qualitative case study focused on one program partnership funded by RTTT. Drawing upon interviews with faculty and relevant documents, we conducted a thematic analysis to determine how faculty perceived RTTT, the state, and the district influenced the development of the program. Three themes were identified and suggest that faculty perceived the influence of RTTT was filtered by the state’s existing reform priorities. Implications for preparation and directions for future research are provided.
Repairing Osteochondral Defects of Critical Size Using Multiple Costal Grafts
Authors: Du Dajiang , Sugita Norihiko , Liu Zhen , Moriguchi Yu , Nakata Ken , Myoui Akira , Yoshikawa Hideki
Published: June 2015

Objective:

To investigate the feasibility of repairing osteochondral defects of critical size by performing mosaicplasty using multiple sliced costal cartilage grafts, which enables repair of extensively injured knees using grafts from a single rib.

Design:

Critical osteochondral defects were prepared on the femoral groove of skeletally mature Japanese white rabbits. Costal cartilage grafts from a single rib were harvested and sliced into multiple segments (approximately 3-5 mm in length). The defects were left untreated or repaired by performing mosaicplasty using costal cartilage grafts (with or without a longitudinal cut along the middle). At 4 and 12 weeks after transplantation, International Cartilage Repair Society macroscopic and histological grading was performed.

Results:

The macroscopic score and visual histological score were significantly higher in the repaired groups than in the untreated group at 4 and 12 weeks after surgery. Histological continuous integration between grafted costal cartilage and host bone was observed in both repaired groups.

Conclusions:

The findings suggest that costal cartilage might be a useful alternative source for chondral grafting. We were able to repair large osteochondral defects by performing mosaicplasty using multiple sliced costal cartilage grafts from a single rib.
Stent-graft treatment for extrahepatic portal vein hemorrhage after pancreaticoduodenectomy
Authors: Suzuki Kojiro , Igami Tsuyoshi , Komada Tomohiro , Mori Yoshine , Yokoyama Yukihiro , Ebata Tomoki , Naganawa Shinji , Nagino Masato
Published: June 2015

We report a case of intraperitoneal hemorrhage from the extrahepatic portal vein after pancreaticoduodenectomy for distal bile duct carcinoma. A stent-graft was deployed from the superior mesenteric vein to the main portal vein using a transhepatic approach. After the procedure, the patient remained free of intraperitoneal hemorrhage and was discharged 2 months later.
Sliding wear behaviour and its mechanisms of carbonitrided AISI 8620 steel at 100°C under unlubricated conditions
Authors: Sankaran R. , Rajamani D. , Natarajan S. , Thirugnanasambantham K. G.
Published: June 2015

This paper deals with high temperature dry sliding wear behaviour and its mechanism of carbonitrided AISI 8620 steel. The sliding wear behaviour of the carbonitrided AISI 8620 steel was investigated using a pin on disc equipment at 100°C with varying parameters like normal load and sliding velocity. The scanning electron microscopy features of the worn samples reveal that abrasion with shallow and narrower wear track is the dominant wear mechanism of carbonitrided AISI 8620 at 50 N, while at 150 N, plastic deformation is caused by high friction sliding, delamination, spalling, pitting and deep grooving. The analysis of wear track was performed using high resolution universal tribometer.
PI3K Mediated Activation of GSK-3β Reduces At-Level Primary Afferent Growth Responses Associated with Excitotoxic Spinal Cord Injury Dysesthesias
Authors: Bareiss Sonja K , Dugan Elizabeth , Brewer Kori L
Published: June 2015

Background

Neuropathic pain and sensory abnormalities are a debilitating secondary consequence of spinal cord injury (SCI). Maladaptive structural plasticity is gaining recognition for its role in contributing to the development of post SCI pain syndromes. We previously demonstrated that excitotoxic induced SCI dysesthesias are associated with enhanced dorsal root ganglia (DRG) neuronal outgrowth. Although glycogen synthase kinase-3β (GSK-3β) is a known intracellular regulator neuronal growth, the potential contribution to primary afferent growth responses following SCI are undefined. We hypothesized that SCI triggers inhibition of GSK-3β signaling resulting in enhanced DRG growth responses, and that PI3K mediated activation of GSK-3β can prevent this growth and the development of at-level pain syndromes.

Results

Excitotoxic SCI using intraspinal quisqualic acid (QUIS) resulted in inhibition of GSK-3β in the superficial spinal cord dorsal horn and adjacent DRG. Double immunofluorescent staining showed that GSK-3βP was expressed in DRG neurons, especially small nociceptive, CGRP and IB4-positive neurons. Intrathecal administration of a potent PI3-kinase inhibitor (LY294002), a known GSK-3β activator, significantly decreased GSK-3βP expression levels in the dorsal horn. QUIS injection resulted in early (3 days) and sustained (14 days) DRG neurite outgrowth of small and subsequently large fibers that was reduced with short term (3 days) administration of LY294002. Furthermore, LY294002 treatment initiated on the date of injury, prevented the development of overgrooming, a spontaneous at-level pain related dysesthesia.

Conclusions

QUIS induced SCI resulted in inhibition of GSK-3β in primary afferents and enhanced at-level DRG intrinsic growth (neurite elongation and initiation). Early PI3K mediated activation of GSK-3β attenuated QUIS-induced DRG neurite outgrowth and prevented the development of at-level dysesthesias.
Middle-down electron capture dissociation and electron transfer dissociation for histone analysis
Authors: Moradian Annie , Franco Catarina , Sweredoski Michael J. , Hess Sonja
Published: June 2015

The post-translational modifications (PTMs) of histones play a major role in activating or silencing gene transcription. To gain better understanding of the interplay between the PTMs that occur on histones, they are extensively studied using mass spectrometry techniques. Due to the abundance of lysines and arginines, the typical trypsin digestion has been found less favorable and GluC-digests have been explored as an alternative to yield larger peptides amenable to middle-down approaches. In addition, the use of weak cation exchange hydrophilic interaction liquid chromatography (WCX-HILIC) and the use of electron-based fragmentation techniques were found to be advantageous for the in-depth characterization of histone variants containing multiple PTMs.

As a test model, we used histones from MEL (murine erythroleukemia) cells treated with butyric acid or DMSO. After acid extraction, histone pellets were dried and fractionated using a reversed-phase C3 column. For middle-down analysis, selected histone fractions were digested using GluC. The digested samples were separated on a WCX-HILIC capillary column packed in-house with PolyCAT A resin, coupled to a linear trap quadrupole Fourier transformation ion cyclotron resonance (LTQFT-ICR) instrument. Raw data was acquired on the LTQFT-ICR using electron capture dissociation (ECD). After deconvolution of the raw data, we generated heatmaps to illustrate differential maps between differentially treated histone samples. We also explored the innovative use of Skyline to quantify histone tails. In addition, we report some preliminary data using a synthetic histone peptide acquired on an Orbitrap Fusion using electron transfer dissociation (ETD). Both, ECD and ETD methods are capable of comprehensively analyzing complex histone variations not accessible with conventional techniques.
Critical illness-induced bone loss is related to deficient autophagy and histone hypomethylation
Authors: Owen Helen C , Vanhees Ineke , Gunst Jan , Van Cromphaut Sophie , Van den Berghe Greet
Published: June 2015

Background

Survivors of critical illness are at increased risk of fractures. This may be due to increased osteoclast formation during critical illness, leading to trabecular bone loss. Such bone loss has also been observed in Paget’s disease, and has been related to deficient autophagy. Deficient autophagy has also been documented in vital organs and skeletal muscle of critically ill patients. The objective of this study was to investigate whether deficient autophagy can be linked to critical illness-induced bone loss.

Methods

Osteoclasts grown in vitro and their precursor cells isolated from peripheral blood of critically ill patients and from matched healthy volunteers were analysed for the expression of autophagy genes (SQSTM1, Atg3 and Atg7), and proteins (p62, Atg–5, and microtubule-associated protein light chain 3–II (LC3–II)) and for autophagy and epigenetic signalling factors via PCR arrays and were treated with the autophagy inducer rapamycin. The effect of rapamycin was also investigated at the tissue level in an in vivo rabbit model of critical illness.

Results

Many more osteoclasts formed in vitro from the blood precursor cells isolated from critically ill patients, which accumulated p62, and displayed reduced expression of Atg5, Atg7, and LC3–II compared to healthy controls, suggesting deficient autophagy, whilst addition of rapamycin reduced osteoclast formation. PCR arrays revealed a down-regulation of histone methyltransferases coupled with an up-regulation of negative regulators of autophagy. Critically ill rabbits displayed a reduction in trabecular and cortical bone, which was rescued with rapamycin.

Conclusions

Deficient autophagy in osteoclasts and their blood precursor cells at least partially explained aberrant osteoclast formation during critical illness and was linked to global histone hypomethylation. Treatment with the autophagy activator Rapamycin reduced patient osteoclast formation in vitro and reduced the amount of bone loss in critically ill rabbits in vivo. These findings may help to develop novel therapeutic targets to prevent critical illness-induced bone loss.
Resveratrol increases F508del-CFTR dependent salivary secretion in cystic fibrosis mice
Authors: Dhooghe Barbara , Bouckaert Charlotte , Capron Arnaud , Wallemacq Pierre , Leal Teresinha , Noel Sabrina
Published: June 2015

Cystic fibrosis (CF) is a fatal genetic disease associated with widespread exocrine gland dysfunction. Studies have suggested activating effects of resveratrol, a naturally-occurring polyphenol compound with antioxidant and anti-inflammatory properties, on CF transmembrane conductance regulator (CFTR) protein function. We assayed, in F508del-CFTR homozygous (CF) and in wild-type mice, the effect of resveratrol on salivary secretion in basal conditions, in response to inhibition by atropine (basal β-adrenergic-dependent component) and to stimulation by isoprenaline (CFTR-dependent component). Both components of the salivary secretion were smaller in CF mice than in controls. Two hours after intraperitoneal administration of resveratrol (50 mg/kg) dissolved in DMSO, the compound was detected in salivary glands. As in both CF and in wild-type mice, DMSO alone increased the response to isoprenaline in males but not in females, the effect of resveratrol was only measured in females. In wild-type mice, isoprenaline increased secretion by more than half. In CF mice, resveratrol rescued the response to isoprenaline, eliciting a 2.5-fold increase of β-adrenergic-stimulated secretion. We conclude that the salivary secretion assay is suitable to test DMSO-soluble CFTR modulators in female mice. We show that resveratrol applied in vivo to mice reaches salivary glands and increases β-adrenergic secretion. Immunolabelling of CFTR in human bronchial epithelial cells suggests that the effect is associated with increased CFTR protein expression. Our data support the view that resveratrol is beneficial for treating CF. The salivary secretion assay has a potential application to test efficacy of novel CF therapies.
SNAP23/25 and VAMP2 mediate exocytic event of transferrin receptor-containing recycling vesicles
Authors: Kubo Keiji , Kobayashi Minako , Nozaki Shohei , Yagi Chikako , Hatsuzawa Kiyotaka , Katoh Yohei , Shin Hye-Won , Takahashi Senye , Nakayama Kazuhisa
Published: June 2015

We recently showed that Rab11 is involved not only in formation of recycling vesicles containing the transferrin (Tfn)–transferrin receptor (TfnR) complex at perinuclear recycling endosomes but also in tethering of recycling vesicles to the plasma membrane (PM) in concert with the exocyst tethering complex. We here aimed at identifying SNARE proteins responsible for fusion of Tfn–TfnR-containing recycling vesicles with the PM, downstream of the exocyst. We showed that exocyst subunits, Sec6 and Sec8, can interact with SNAP23 and SNAP25, both of which are PM-localizing Qbc-SNAREs, and that depletion of SNAP23 and/or SNAP25 in HeLa cells suppresses fusion of Tfn–TfnR-containing vesicles with the PM, leading to accumulation of the vesicles at the cell periphery. We also found that VAMP2, an R-SNARE, is colocalized with endocytosed Tfn on punctate endosomal structures, and that its depletion in HeLa cells suppresses recycling vesicle exocytosis. These observations indicate that fusion of recycling vesicles with the PM downstream of the exocyst is mediated by SNAP23/25 and VAMP2, and provide novel insight into non-neuronal roles of VAMP2 and SNAP25.
Myocardium and BMP signaling are required for endocardial differentiation
Authors: Palencia-Desai Sharina , Rost Megan S. , Schumacher Jennifer A. , Ton Quynh V. , Craig Michael P. , Baltrunaite Kristina , Koenig Andrew L. , Wang Jinhu , Poss Kenneth D. , Chi Neil C. , Stainier Didier Y. R. , Sumanas Saulius
Published: June 2015

Endocardial and myocardial progenitors originate in distinct regions of the anterior lateral plate mesoderm and migrate to the midline where they coalesce to form the cardiac tube. Endocardial progenitors acquire a molecular identity distinct from other vascular endothelial cells and initiate expression of specific genes such as nfatc1. Yet the molecular pathways and tissue interactions involved in establishing endocardial identity are poorly understood. The endocardium develops in tight association with cardiomyocytes. To test for a potential role of the myocardium in endocardial morphogenesis, we used two different zebrafish models deficient in cardiomyocytes: the hand2 mutant and a myocardial-specific genetic ablation method. We show that in hand2 mutants endocardial progenitors migrate to the midline but fail to assemble into a cardiac cone and do not express markers of differentiated endocardium. Endocardial differentiation defects were rescued by myocardial but not endocardial-specific expression of hand2. In metronidazole-treated myl7:nitroreductase embryos, myocardial cells were targeted for apoptosis, which resulted in the loss of endocardial nfatc1 expression. However, endocardial cells were present and retained expression of general vascular endothelial markers. We further identified bone morphogenetic protein (BMP) as a candidate myocardium-derived signal required for endocardial differentiation. Chemical and genetic inhibition of BMP signaling at the tailbud stage resulted in severe inhibition of endocardial differentiation while there was little effect on myocardial development. Heat-shock-induced bmp2b expression rescued endocardial nfatc1 expression in hand2 mutants and in myocardium-depleted embryos. Our results indicate that the myocardium is crucial for endocardial morphogenesis and differentiation, and identify BMP as a signal involved in endocardial differentiation.
CHD1 acts via the Hmgpi pathway to regulate mouse early embryogenesis
Authors: Suzuki Shinnosuke , Nozawa Yusuke , Tsukamoto Satoshi , Kaneko Takehito , Manabe Ichiro , Imai Hiroshi , Minami Naojiro
Published: June 2015

The protein CHD1 is a member of the family of ATPase-dependent chromatin remodeling factors. CHD1, which recognizes trimethylated histone H3 lysine 4, has been implicated in transcriptional activation in organisms ranging from yeast to humans. It is required for pre-mRNA maturation, maintenance of mouse embryonic stem cell pluripotency and rapid growth of the mouse epiblast. However, the function(s) of CHD1 in mouse preimplantation embryos has not yet been examined. Here, we show that loss of CHD1 function led to embryonic lethality after implantation. In mouse embryos in which Chd1 was targeted by siRNA microinjection, the expression of the key regulators of cell fate specification Pou5f1 (also known as Oct4), Nanog and Cdx2 was dramatically decreased, starting at mid-preimplantation gene activation (MGA). Moreover, expression of Hmgpi and Klf5, which regulate Pou5f1, Nanog and Cdx2, was also significantly suppressed at zygotic gene activation (ZGA). Suppression of Hmgpi expression in Chd1-knockdown embryos continued until the blastocyst stage, whereas suppression of Klf5 expression was relieved by the morula stage. Next, we rescued HMGPI expression via Hmgpi mRNA microinjection in Chd1-knockdown embryos. Consequently, Pou5f1, Nanog and Cdx2 expression was restored at MGA and live offspring were recovered. These findings indicate that CHD1 plays important roles in mouse early embryogenesis via activation of Hmgpi at ZGA.
A rapid, membrane-dependent pathway directs furrow formation through RalA in the earlyDrosophilaembryo
Authors: Holly Ryan M. , Mavor Lauren M. , Zuo Zhongyuan , Blankenship J. Todd
Published: June 2015

Plasma membrane furrow formation is crucial in cell division and cytokinesis. Furrow formation in early syncytial Drosophila embryos is exceptionally rapid, with furrows forming in as little as 3.75 min. Here, we use 4D imaging to identify furrow formation, stabilization, and regression periods, and identify a rapid, membrane-dependent pathway that is essential for plasma membrane furrow formation in vivo. Myosin II function is thought to provide the ingression force for cytokinetic furrows, but the role of membrane trafficking pathways in guiding furrow formation is less clear. We demonstrate that a membrane trafficking pathway centered on Ras-like protein A (RalA) is required for fast furrow ingression in the early fly embryo. RalA function is absolutely required for furrow formation and initiation. In the absence of RalA and furrow function, chromosomal segregation is aberrant and polyploid nuclei are observed. RalA localizes to syncytial furrows, and mediates the movement of exocytic vesicles to the plasma membrane. Sec5, which is an exocyst complex subunit and localizes to ingressing furrows in wild-type embryos, becomes punctate and loses its cortical association in the absence of RalA function. Rab8 also fails to traffic to the plasma membrane and accumulates aberrantly in the cytoplasm in RalA disrupted embryos. RalA localization precedes F-actin recruitment to the furrow tip, suggesting that membrane trafficking might function upstream of cytoskeletal remodeling. These studies identify a pathway, which stretches from Rab8 to RalA and the exocyst complex, that mediates rapid furrow formation in early Drosophila embryos.
A transport and retention mechanism for the sustained distal localization of Spn-F–IKKε during Drosophila bristle elongation
Authors: Otani Tetsuhisa , Oshima Kenzi , Kimpara Akiyo , Takeda Michiko , Abdu Uri , Hayashi Shigeo
Published: June 2015

Stable localization of the signaling complex is essential for the robust morphogenesis of polarized cells. Cell elongation involves molecular signaling centers that coordinately regulate intracellular transport and cytoskeletal structures. In Drosophila bristle elongation, the protein kinase IKKε is activated at the distal tip of the growing bristle and regulates the shuttling movement of recycling endosomes and cytoskeletal organization. However, how the distal tip localization of IKKε is established and maintained during bristle elongation is unknown. Here, we demonstrate that IKKε distal tip localization is regulated by Spindle-F (Spn-F), which is stably retained at the distal tip and functions as an adaptor linking IKKε to cytoplasmic dynein. We found that Javelin-like (Jvl) is a key regulator of Spn-F retention. In jvl mutant bristles, IKKε and Spn-F initially localize to the distal tip but fail to be retained there. In S2 cells, particles that stain positively for Jvl or Spn-F move in a microtubule-dependent manner, whereas Jvl and Spn-F double-positive particles are immobile, indicating that Jvl and Spn-F are transported separately and, upon forming a complex, immobilize each other. These results suggest that polarized transport and selective retention regulate the distal tip localization of the Spn-F–IKKε complex during bristle cell elongation.
Traffic jam functions in a branched pathway from Notch activation to niche cell fate
Authors: Wingert Lindsey , DiNardo Stephen
Published: June 2015

The niche directs key behaviors of its resident stem cells, and is thus crucial for tissue maintenance, repair and longevity. However, little is known about the genetic pathways that guide niche specification and development. The male germline stem cell niche in Drosophila houses two stem cell populations and is specified within the embryonic gonad, thus making it an excellent model for studying niche development. The hub cells that form the niche are specified early by Notch activation. Over the next few hours, these individual cells then cluster together and take up a defined position before expressing markers of hub cell differentiation. This timing suggests that there are other factors for niche development yet to be defined. Here, we have identified a role for the large Maf transcription factor Traffic jam (Tj) in hub cell specification downstream of Notch. Tj downregulation is the first detectable effect of Notch activation in hub cells. Furthermore, Tj depletion is sufficient to generate ectopic hub cells that can recruit stem cells. Surprisingly, ectopic niche cells in tj mutants remain dispersed in the absence of Notch activation. This led us to uncover a branched pathway downstream of Notch in which Bowl functions to direct hub cell assembly in parallel to Tj downregulation.
The galectin lattice at a glance
Authors: Nabi Ivan R. , Shankar Jay , Dennis James W.
Published: June 2015

Galectins are a family of widely expressed β-galactoside-binding lectins in metazoans. The 15 mammalian galectins have either one or two conserved carbohydrate recognition domains (CRDs), with galectin-3 being able to pentamerize; they form complexes that crosslink glycosylated ligands to form a dynamic lattice. The galectin lattice regulates the diffusion, compartmentalization and endocytosis of plasma membrane glycoproteins and glycolipids. The galectin lattice also regulates the selection, activation and arrest of T cells, receptor kinase signaling and the functionality of membrane receptors, including the glucagon receptor, glucose and amino acid transporters, cadherins and integrins. The affinity of transmembrane glycoproteins to the galectin lattice is proportional to the number and branching of their N-glycans; with branching being mediated by Golgi N-acetylglucosaminyltransferase-branching enzymes and the supply of UDP-GlcNAc through metabolite flux through the hexosamine biosynthesis pathway. The relative affinities of glycoproteins for the galectin lattice depend on the activities of the Golgi enzymes that generate the epitopes of their ligands and, thus, provide a means to analyze biological function of lectins and of the ‘glycome’ more broadly.
Cell-stiffness-induced mechanosignaling – a key driver of leukocyte transendothelial migration
Authors: Schaefer Antje , Hordijk Peter L.
Published: June 2015

The breaching of cellular and structural barriers by migrating cells is a driving factor in development, inflammation and tumor cell metastasis. One of the most extensively studied examples is the extravasation of activated leukocytes across the vascular endothelium, the inner lining of blood vessels. Each step of this leukocyte transendothelial migration (TEM) process is regulated by distinct endothelial adhesion receptors such as the intercellular adhesion molecule 1 (ICAM1). Adherent leukocytes exert force on these receptors, which sense mechanical cues and transform them into localized mechanosignaling in endothelial cells. In turn, the function of the mechanoreceptors is controlled by the stiffness of the endothelial cells and of the underlying substrate representing a positive-feedback loop. In this Commentary, we focus on the mechanotransduction in leukocytes and endothelial cells, which is induced in response to variations in substrate stiffness. Recent studies have described the first key proteins involved in these mechanosensitive events, allowing us to identify common regulatory mechanisms in both cell types. Finally, we discuss how endothelial cell stiffness controls the individual steps in the leukocyte TEM process. We identify endothelial cell stiffness as an important component, in addition to locally presented chemokines and adhesion receptors, which guides leukocytes to sites that permit TEM.
COPI-mediated retrieval of SCAP is critical for regulating lipogenesis under basal and sterol-deficient conditions
Authors: Takashima Kouhei , Saitoh Akina , Funabashi Teruki , Hirose Shohei , Yagi Chikako , Nozaki Shohei , Sato Ryuichiro , Shin Hye-Won , Nakayama Kazuhisa
Published: June 2015

Retrograde trafficking from the Golgi apparatus to endoplasmic reticulum (ER) via COPI-coated vesicles has been implicated in lipid homeostasis. Here we found that a block in COPI-dependent retrograde trafficking promoted processing and nuclear translocation of SREBPs (sterol regulatory element binding proteins) and upregulated expression of downstream genes involved in lipid biosynthesis. This elevation in SREBP processing/activation was not caused by mislocalization of S1P or S2P, two Golgi-resident endoproteases involved in SREBP processing, but instead by increased Golgi residence of SREBPs, leading to their increased susceptibility to processing by the endoproteases. Analyses using a processing-defective SREBP mutant suggested that a fraction of SREBP molecules undergo basal cycling between the ER and Golgi in complex with SCAP (SREBP cleavage-activating protein). Furthermore, we showed that SCAP alone is retrieved back from the Golgi to the ER after processing of SREBP under sterol-deficient conditions. Thus, our observations indicate that COPI-mediated retrograde trafficking is critical for preventing unnecessary SREBP activation through retrieval of the SCAP–SREBP complex that basally escapes from the sterol-regulated ER retention machinery, as well as for reuse of SCAP.
Lamin-A/C/LAP2α/BAF1 protein complex regulates the mitotic spindle assembly and positioning
Authors: Qi Ran , Xu Nan , Wang Gang , Ren He , Li Si , Lei Jun , Lin Qiaoyu , Wang Lihao , Gu Xin , Zhang Hongyin , Jiang Qing , Zhang Chuanmao
Published: June 2015

Some nuclear proteins crucial in interphase relocate to other places during the G2-M phase transition to take their mitotic functions. However, how they perform these functions and the underlying mechanisms remain largely unknown. Here we report that a fraction of the nuclear periphery proteins lamin-A/C, LAP2α and BAF1 relocate to the spindle and the cell cortex in mitosis. RNAi knockdown of these proteins induces short and fluffy spindle formation and disconnection of the spindle with the cell cortex. Disrupting the microtubule assembly leads to accumulation of these proteins in the cell cortex, whereas depolymerizing the actin microfilaments results in the short spindle formation. We further demonstrate that these proteins are part of a stable complex that links the mitotic spindle to the cell cortex and the spindle matrix by binding with the spindle-associated dynein and the actin filaments in the cell cortex and the spindle matrix. Together, our findings unveil a unique mechanism that the nuclear periphery proteins lamin-A/C, LAP2α and BAF1 are assembled into a protein complex in mitosis to regulate the mitotic spindle assembly and positioning.
Fine-tuning of Cdc6 accumulation by Cdk1 and MAP kinase is essential for completion of oocyte meiotic divisions
Authors: Daldello Enrico M. , Le Tran , Poulhe Robert , Jessus Catherine , Haccard Olivier , Dupré Aude
Published: June 2015

Vertebrate oocytes proceed through the 1st and the 2nd meiotic division without intervening S-phase to become haploid. Although DNA replication does not take place, unfertilized oocytes acquire the competence to replicate DNA one hour after the 1st meiotic division, by accumulating an essential factor of the replicative machinery, Cdc6. Here, we discovered that the turnover of Cdc6 is precisely regulated in oocytes to avoid inhibition of Cdk1. At meiosis resumption, Cdc6 starts to be expressed but cannot accumulate due to a degradation mechanism activated through Cdk1. During transition from 1st to 2nd meiotic division, Cdc6 is under antagonistic regulation of Cyclin B, whose interaction with Cdc6 stabilizes the protein, and Mos/MAPK that negatively controls its accumulation. Since overexpressing Cdc6 inhibits Cdk1 reactivation and drives oocytes into a replicative interphasic state, the fine-tuning of Cdc6 accumulation is essential to ensure two meiotic waves of Cdk1 activation and to avoid unscheduled DNA replication during meiotic maturation.
Access of torsinA to the inner nuclear membrane is activity dependent and regulated in the endoplasmic reticulum
Authors: Goodchild Rose E. , Buchwalter Abigail L. , Naismith Teresa V. , Holbrook Kristen , Billion Karolien , Dauer William T. , Liang Chun-Chi , Dear Mary Lynn , Hanson Phyllis I.
Published: June 2015

TorsinA is a membrane-embedded AAA+ ATPase important in the nuclear envelope (NE) lumen. However, most torsinA is localized in the peripheral endoplasmic reticulum (ER) lumen with slow mobility incompatible with free equilibration between ER subdomains. We now find that NE-localized torsinA is on the inner nuclear membrane (INM) and ask how torsinA reaches this subdomain. The ER system contains two transmembrane proteins, LAP1 and LULL1, that reversibly co-assemble with and activate torsinA. Whereas LAP1 localizes on the INM, we show that LULL1 is in the peripheral ER and does not enter the INM. Paradoxically, interaction between torsinA and LULL1 in the ER targets torsinA to the INM. Native gel electrophoresis reveals torsinA oligomeric complexes that are decreased by LULL1. Mutations in torsinA or LULL1 that inhibit ATPase activity reduce torsinA access to the INM. Furthermore, although LULL1 binds torsinA in the ER lumen, its effect on torsinA localization requires cytosolic domain mediated oligomerization. These data suggest that LULL1 oligomerizes to engage and transiently disassemble torsinA oligomers, and is thereby positioned to transduce cytoplasmic signals to the INM via torsinA.
Protein kinase C (PKC)-mediated phosphorylation of PACSIN2 triggers the removal of caveolae from the plasma membrane
Authors: Senju Yosuke , Rosenbaum Eva , Shah Claudio , Hamada-Nakahara Sayaka , Itoh Yuzuru , Yamamoto Kimiko , Hanawa-Suetsugu Kyoko , Daumke Oliver , Suetsugu Shiro
Published: June 2015

PACSIN2, a membrane-sculpting BAR domain protein, localizes to caveolae. Here, we found that PKC phosphorylates PACSIN2 at serine 313, thereby decreasing its membrane binding and tubulation capacities. Concomitantly, phosphorylation decreased the time span for which caveolae could be tracked at the plasma membrane (the 'tracking-duration'). Analyses of the phospho-mimetic S313E mutant suggested that PACSIN2 phosphorylation is sufficient to reduce caveolar tracking-durations. Both hypotonic treatment and isotonic drug-induced PKC activation increased PACSIN2 phosphorylation at serine 313 and shortened caveolar tracking-durations. Caveolar tracking-durations were also reduced upon the expression of other membrane-binding deficient PACSIN2 mutants or RNAi-mediated PACSIN2 depletion, pointing to a role of PACSIN2 levels for the lifetime of caveolae. Interestingly, the decrease in membrane-bound PACSIN2 was inversely correlated with the recruitment and activity of dynamin 2, a GTPase mediating membrane scission. Furthermore, expression of EHD2, which stabilizes caveolae and binds to PACSIN2, restored the tracking-durations of cells with reduced PACSIN2 levels. These findings suggest that the PACSIN2 phosphorylation decreases its membrane-binding activity, thereby decreasing its stabilizing effect on caveolae and triggering dynamin-mediated removal of caveolae.
Serotonin receptor 5-HT7 regulates morphology and migratory properties of dendritic cells
Authors: Holst Katrin , Guseva Daria , Schindler Susann , Sixt Michael , Braun Armin , Chopra Himpriya , Pabst Oliver , Ponimaskin Evgeni
Published: June 2015

Dendritic cells (DCs) are potent antigen-presenting cells endowed with the unique ability to initiate adaptive immune responses upon inflammation. Inflammatory processes are often associated with an increased production of serotonin, which operates by activating specific receptors. However, the functional role of serotonin receptors in regulation of DC functions is poorly understood. Here we demonstrate that expression of serotonin receptor 5-HT7 (5-HT7R) as well as its down-stream effector Cdc42 is upregulated in DCs upon maturation. While DC maturation was independent of 5-HT7R, receptor stimulation affected DC morphology via Cdc42-mediated signaling. In addition, basal activity of 5-HT7R was required for the proper expression of the chemokine receptor CCR7, which is a key factor to control DC migration. Consistently, we observed that 5-HT7R enhances chemotactic motility of DCs in vitro by modulating their directionality and migration velocity. Accordingly, migration of DCs in murine colon explants was abolished after pharmacological receptor blockade. Our results indicate a critical role of 5-HT7R/Cdc42-mediated signaling in regulation of DC morphology and motility, suggesting 5-HT7R as a novel target for treatment of a variety of inflammatory and immune disorders.
An actin–myosin-II interaction is involved in maintaining the contractile ring in fission yeast
Authors: Takaine Masak , Numata Osamu , Nakano Kentaro
Published: June 2015

The actomyosin-based contractile ring (CR), which assembles at the cell equator, maintains its circularity during cytokinesis in many eukaryotic cells, ensuring its efficient constriction. Although consistent maintenance of the ring is one of the mechanisms underpinning cytokinesis, it has not yet been fully addressed. We here investigated the roles of fission yeast myosin-IIs (Myo2 and Myp2/Myo3) in ring maintenance during cytokinesis, with a focus on Myo3. A site-directed mutational analysis showed that the motor properties of Myo3 were involved in its accumulation in the CR. Ring assembly was often deformable and not properly maintained under conditions in which the activities of myosin-IIs localizing to the CR were decreased, leading to inefficient cell division. Moreover, Myo3 appeared to form motile clusters on the ring. We proposed that large assemblies of myosin-IIs consolidated the CR by continuously binding to F-actin in the ring, thereby contributing to its maintenance.
The Drosophila centriole: conversion of doublets to triplets within the stem cell niche
Authors: Gottardo Marco , Callaini Giuliano , Riparbelli Maria Giovanna
Published: June 2015

We report here that two distinct centriole lineages exist in Drosophila: somatic centrioles usually composed by microtubule doublets and germ line centrioles characterized by triplets. Remarkably, the transition from doublets to triplets in the testis occurs within the stem cell niche with the formation of the C-tubule. We demonstrated that the old mother centriole that stays in the apical cytoplasm of the male germline stem cells (GSCs) is invariably composed by triplets, whereas its daughter is always built by mixed doublets and triplets. This difference represents the first documentation of a structural asymmetry between mother and daughter centrioles in Drosophila GSCs and may reflect a correlation between the architecture of parent centrioles and their ability to recruit centrosomal proteins. We also found that the old mother centriole is linked to the cell membrane by distinct projections that may play an important role in keeping its apical position during centrosome separation.
Ciliary Adenylyl Cyclases control the Hedgehog pathway
Authors: Vuolo Laura , Herrera Antonio , Torroba Blanca , Menendez Anghara , Pons Sebastian
Published: June 2015

Protein kinase A (PKA) accumulates at the base of the cilium where it negatively regulates the Hedgehog (Hh) pathway. Although PKA activity is essentially controlled by the cAMP produced by Adenylyl Cyclases (AC), the influence of these enzymes on the Hh pathway remains unclear. Here we show that AC5 and AC6 are the two isoforms most strongly expressed in cerebellar granular neuron precursors (CGNPs) and that that while over-expression of AC5 and AC6 represses the Hh pathway, their knockdown results in the activation of this pathway in CGNPs and in the embryonic neural tube (NT). Indeed, AC5 and AC6 concentrate in the primary cilium, and mutation of a previously undescribed cilium targeting motif in AC5 suppresses its ciliary location, as well as its capacity to inhibit Hh signalling. Stimulatory and inhibitory Gα proteins, that are engaged by the G protein coupled receptors (GPCRs), control AC5 and AC6 activity and regulate Hh pathway in CGNPs and NT. Therefore, we propose that the activity of different ciliary GPCRs converge on AC5 and AC6 to control PKA activity and hence the Hh pathway.
Mitf is a master regulator of the v-ATPase forming an Mitf/v-ATPase/TORC1 control module for cellular homeostasis
Authors: Zhang T. , Zhou Q. , Ogmundsdottir M. H. , Möller K. , Siddaway R. , Larue L. , Hsing M. , Kong S. W. , Goding C. , Palsson A. , Steingrimsson E. , Pignoni F.
Published: June 2015

The v-ATPase is a fundamental eukaryotic enzyme central to cellular homeostasis. Although its impact on key metabolic regulators such as TORC1 is well-documented, our knowledge of mechanisms that regulate v-ATPase activity is limited. Here, we report that the Drosophila transcription factor Mitf is a master regulator of this holoenzyme. Mitf directly controls transcription of all 15 v-ATPase components through M-box cis-sites and this coordinated regulation impacts holoenzyme activity in vivo. In addition, through the v-ATPase, Mitf promotes the activity of TORC1, which in turn negatively regulates Mitf. We provide evidence that Mitf, v-ATPase and TORC1 form a negative regulatory loop that maintains each of these important metabolic regulators in relative balance. Interestingly, direct regulation of v-ATPase genes by human MITF also occurs in cells of the melanocytic lineage, showing mechanistic conservation in the regulation of the v-ATPase by MITF-TFE proteins in fly and mammals. Collectively, this evidence points to an ancient Mitf/v-ATPase/TORC1 module that serves as a dynamic modulator of metabolism for cellular homeostasis.
Genome-wide detection of high abundance N6-methyladenosine sites by microarray
Authors: Li Yue , Wang Yang , Zhang Zhaolei , Zamudio Alicia Viridiana , Zhao Jing Crystal
Published: June 2015

N6-methyladenosine (m6A), the most abundant internal RNA modification, functions in diverse biological processes, including regulation of embryonic stem cell self-renewal and differentiation. As yet, methods to detect m6A in the transcriptome rely on the availability and quality of an m6A antibody and are often associated with a high rate of false positives. Here, based on our observation that m6A interferes with A–T/U pairing, we report a microarray-based technology to map m6A sites in mouse embryonic stem cells. We identified 72 unbiased sites exhibiting high m6A levels from 66 PolyA RNAs. Bioinformatics analyses suggest identified sites are enriched on developmental regulators and may in some contexts modulate microRNA/mRNA interactions. Overall, we have developed microarray-based technology to capture highly enriched m6A sites in the mammalian transcriptome. This method provides an alternative means to identify m6A sites for certain applications.
MS2 coat proteins bound to yeast mRNAs block 5′ to 3′ degradation and trap mRNA decay products: implications for the localization of mRNAs by MS2-MCP system
Authors: Garcia Jennifer F. , Parker Roy
Published: June 2015

Arrays of MS2 binding sites are placed into mRNAs and are commonly used to visualize the localization of mRNAs in vivo by the expression of an MS2-GFP fusion protein. In Saccharomyces cerevisiae, we observed that arrays of MS2 binding sites inhibit 5′ to 3′ degradation of the mRNA in yeast cells and lead to the accumulation of a 3′ mRNA fragment containing the MS2 binding sites. This accumulation can be dependent on the binding of the MS2 stem loops (MS2-SL) by MS2 coat proteins (MCPs). Since such decay fragments can still bind MCP-GFP, the localization of such mRNA fragments can complicate the interpretation of the localization of full-length mRNA in vivo.
The Lsm1-7-Pat1 complex promotes viral RNA translation and replication by differential mechanisms
Authors: Jungfleisch Jennifer , Chowdhury Ashis , Alves-Rodrigues Isabel , Tharun Sundaresan , Díez Juana
Published: June 2015

The Lsm1-7-Pat1 complex binds to the 3′ end of cellular mRNAs and promotes 3′ end protection and 5′–3′ decay. Interestingly, this complex also specifically binds to cis-acting regulatory sequences of viral positive-strand RNA genomes promoting their translation and subsequent recruitment from translation to replication. Yet, how the Lsm1-7-Pat1 complex regulates these two processes remains elusive. Here, we show that Lsm1-7-Pat1 complex acts differentially in these processes. By using a collection of well-characterized lsm1 mutant alleles and a system that allows the replication of Brome mosaic virus (BMV) in yeast we show that the Lsm1-7-Pat1 complex integrity is essential for both, translation and recruitment. However, the intrinsic RNA-binding ability of the complex is only required for translation. Consistent with an RNA-binding-independent function of the Lsm1-7-Pat1 complex on BMV RNA recruitment, we show that the BMV 1a protein, the sole viral protein required for recruitment, interacts with this complex in an RNA-independent manner. Together, these results support a model wherein Lsm1-7-Pat1 complex binds consecutively to BMV RNA regulatory sequences and the 1a protein to promote viral RNA translation and later recruitment out of the host translation machinery to the viral replication complexes.
The Relationship Between Academic Achievement And Nonmarital Teenage Childbearing: Evidence from the Panel Study of Income Dynamics
Authors: Lou Cary , Thomas Adam
Published: June 2015

CONTEXT

Females who do well in school are less likely than those who do poorly to experience a nonmarital teenage birth. However, little is known about which dimensions of academic achievement are the most strongly related to teenage childbearing, or about whether the relationship between achievement and childbearing varies according to the presence of other behavioral problems.

METHODS

Individual‐level and family‐level data from the Panel Study of Income Dynamics, combined with information on contextual state‐level economic and policy measures, were used to study nonmarital childbearing between the ages of 16 and 19 among 701 females who turned 16 between 2000 and 2007. Multivariate logistic regression analyses examined the relationship between the probability of nonmarital teenage childbearing and age‐standardized scores on academic assessments of letter‐word identification, passage comprehension and applied problem‐solving ability.

RESULTS

Scores on the passage comprehension and applied problem‐solving subtests were strongly associated with the probability of experiencing a nonmarital teenage birth among respondents who had relatively few behavioral problems. For this group, an increase of one standard deviation in the score on either assessment was associated with a reduction of about 50% in the risk of experiencing a nonmarital teenage birth. However, no evidence was found of an equivalent relationship among respondents with more pronounced behavioral problems or for the letter‐word identification assessment.

CONCLUSIONS

Future research should continue to explore the possibility that improvements in academic achievement may help to reduce the rate of nonmarital teenage childbearing.