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Cell Biology
No significant enrichment of rare functionally defective CPA1 variants in a large Chinese idiopathic chronic pancreatitis cohort
May 12, 2017   Human Mutation
Wu H, Zhou DZ, Berki D, Geisz A, Zou WB,   . . . . . .   , Férec C, Chen JM, Li ZS, Sahin-Tóth M, Liao Z
No significant enrichment of rare functionally defective CPA1 variants in a large Chinese idiopathic chronic pancreatitis cohort
May 12, 2017
Human Mutation
Rare functionally defective carboxypeptidase A1 (CPA1) variants have been reported to predispose to nonalcoholic chronic pancreatitis, mainly the idiopathic subtype. However, independent replication has so far been lacking, particularly in Asian cohorts where initial studies employed small sample sizes. Herein we performed targeted next-generation sequencing of the CPA1 gene in 1112 Han Chinese idiopathic chronic pancreatitis (ICP) patients - the largest ICP cohort so far analyzed in a single population - and 1580 controls. Sanger sequencing was used to validate called variants, and the CPA1 activity and secretion of all newly found variants were measured. A total of 18 rare CPA1 variants were characterized, 11 of which have not been previously described. However, no significant association was noted with ICP irrespective of whether all rare variants [20/1112 (1.8%) in patients vs. 24/1580 (1.52%) in controls; P = 0.57] or functionally impaired variants [3/1112 (0.27%) in patients vs. 2/1580 (0.13%) in controls; P = 0.68] were considered. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.
Top quality blastocyst formation rates in relation to progesterone levels on the day of oocyte maturation in GnRH antagonist IVF/ICSI cycles
May 18, 2017   PloS One
Vanni VS, Somigliana E, Reschini M, Pagliardini L, Marotta E, Faulisi S, Paffoni A, Vigano' P, Vegetti W, Candiani M, Papaleo E
Top quality blastocyst formation rates in relation to progesterone levels on the day of oocyte maturation in GnRH antagonist IVF/ICSI cycles
May 18, 2017
PloS One
Cycles with progesterone elevation during controlled ovarian stimulation (COS) for IVF/ICSI are commonly managed with a "freeze-all" strategy, due to a well-recognized detrimental effect of high progesterone levels on endometrial receptivity. However, also a detrimental effect of elevated progesterone on day-3 embryo quality has recently been found with regards to top quality embryo formation rate. Because blastocyst culture and cryopreservation are largely adopted, we deemed relevant to determine whether this detrimental effect is also seen on blastocyst quality on day 5-6. This issue was investigated through a large two-center retrospective study including 986 GnRH antagonist IVF/ICSI cycles and using top quality blastocyst formation rate as the main outcome. Results showed that on multivariate analysis sperm motility (p1.49 ng/ml as the best cut-off for identification of patients at risk for the absence of top quality blastocysts (AUC 0.55, p
Co-inheritance of glucose-6-phosphate dehydrogenase deficiency mutations and hemoglobin E in a Kachin population in a malaria-endemic region of Southeast Asia
May 22, 2017   PloS One
Deng Z, Yang F, Bai Y, He L, Li Q, Wu Y, Luo L, Li H, Ma L, Yang Z, He Y, Cui L
Co-inheritance of glucose-6-phosphate dehydrogenase deficiency mutations and hemoglobin E in a Kachin population in a malaria-endemic region of Southeast Asia
May 22, 2017
PloS One
Glucose-6-phosphate dehydrogenase (G6PD) deficiency and hemoglobin E (HbE, β26 Glu-Lys) are two common red cell disorders in Southeast Asia. G6PD deficiency produces hemolytic anemia, which can be triggered by certain drugs or infections. HbE is asymptomatic or is manifested as microcytic, minimally hemolytic anemia. The association between G6PD deficiency and HbE is little understood. This study aimed to investigate G6PD deficiency and HbE in a Kachin ethnic group in the China-Myanmar border area. G6PD enzyme activity was measured using a quantitative G6PD assay, G6PD variants genotyped by the SNaPshot assay, and an HbE gene mutation identified by an amplification refractory mutation system and subsequently confirmed by using a reverse dot blot hybridization assay from 100 unrelated individuals in the study area. G6PD enzyme activity ranged from 0.4 to 24.7 U/g Hb, and six males had severe G6PD deficiency (1.2-4.5 U/g Hb). Among the 24 G6PD-deficient subjects, 22 (92%) had the Mahidol 487G>A mutation (12 male hemizygotes, one female homozygote, and nine female heterozygotes), while the G6PD genotypes in two female subjects were unknown. HbE was identified in 39 subjects (20 males and 19 females), including 15 HbEE (seven males and eight females) and 24 HbAE (13 males and 11 females). Twenty-three subjects co-inherited both G6PD deficiency and HbE (22 with HbAE and one with HbEE). Whereas mean Hb levels were not significantly different between the HbA and HbE groups, G6PD-deficient males had significantly lower Hb levels than G6PD-normal males (P < 0.05, t-test). However, it is noteworthy that two G6PD-deficient hemizygous males with HbAE were severely anemic with Hb levels below 50 g/L. This study revealed high prevalence of co-inheritance of G6PD deficiency with HbAE in the Kachin ethnicity, and a potential interaction of the G6PD Mahidol 487G>A and HbAE in males leading to severe anemia. The presence of 6% males with severe G6PD deficiency raised a major concern in the use of primaquine for radical cure of vivax malaria.
The complex genetics of hypoplastic left heart syndrome
May 22, 2017   Nature Genetics Add nature.com free-link Cancel
Liu X, Yagi H, Saeed S, Bais AS, Gabriel GC,   . . . . . .   , Tsang M, Martin LJ, Woodrow Benson D, Aronow BJ, Lo CW
The complex genetics of hypoplastic left heart syndrome
May 22, 2017
Nature Genetics
Congenital heart disease (CHD) affects up to 1% of live births. Although a genetic etiology is indicated by an increased recurrence risk, sporadic occurrence suggests that CHD genetics is complex. Here, we show that hypoplastic left heart syndrome (HLHS), a severe CHD, is multigenic and genetically heterogeneous. Using mouse forward genetics, we report what is, to our knowledge, the first isolation of HLHS mutant mice and identification of genes causing HLHS. Mutations from seven HLHS mouse lines showed multigenic enrichment in ten human chromosome regions linked to HLHS. Mutations in Sap130 and Pcdha9, genes not previously associated with CHD, were validated by CRISPR-Cas9 genome editing in mice as being digenic causes of HLHS. We also identified one subject with HLHS with SAP130 and PCDHA13 mutations. Mouse and zebrafish modeling showed that Sap130 mediates left ventricular hypoplasia, whereas Pcdha9 increases penetrance of aortic valve abnormalities, both signature HLHS defects. These findings show that HLHS can arise genetically in a combinatorial fashion, thus providing a new paradigm for the complex genetics of CHD.
Mutations in DZIP1L, which encodes a ciliary-transition-zone protein, cause autosomal recessive polycystic kidney disease
May 22, 2017   Nature Genetics Add nature.com free-link Cancel
Lu H, Galeano MCR, Ott E, Kaeslin G, Kausalya PJ,   . . . . . .   , Zerres K, Hildebrandt F, Roy S, Wicking C, Bergmann C
Mutations in DZIP1L, which encodes a ciliary-transition-zone protein, cause autosomal recessive polycystic kidney disease
May 22, 2017
Nature Genetics
Autosomal recessive polycystic kidney disease (ARPKD), usually considered to be a genetically homogeneous disease caused by mutations in PKHD1, has been associated with ciliary dysfunction. Here, we describe mutations in DZIP1L, which encodes DAZ interacting protein 1-like, in patients with ARPKD. We further validated these findings through loss-of-function studies in mice and zebrafish. DZIP1L localizes to centrioles and to the distal ends of basal bodies, and interacts with septin2, a protein implicated in maintenance of the periciliary diffusion barrier at the ciliary transition zone. In agreement with a defect in the diffusion barrier, we found that the ciliary-membrane translocation of the PKD proteins polycystin-1 and polycystin-2 is compromised in DZIP1L-mutant cells. Together, these data provide what is, to our knowledge, the first conclusive evidence that ARPKD is not a homogeneous disorder and further establish DZIP1L as a second gene involved in ARPKD pathogenesis.
Dynamic Palmitoylation Targets MAP6 to the Axon to Promote Microtubule Stabilization during Neuronal Polarization
May 18, 2017   Neuron
Tortosa E, Adolfs Y, Fukata M, Pasterkamp RJ, Kapitein LC, Hoogenraad CC
Dynamic Palmitoylation Targets MAP6 to the Axon to Promote Microtubule Stabilization during Neuronal Polarization
May 18, 2017
Neuron
Microtubule-associated proteins (MAPs) are main candidates to stabilize neuronal microtubules, playing an important role in establishing axon-dendrite polarity. However, how MAPs are selectively targeted to specific neuronal compartments remains poorly understood. Here, we show specific localization of microtubule-associated protein 6 (MAP6)/stable tubule-only polypeptide (STOP) throughout neuronal maturation and its role in axonal development. In unpolarized neurons, MAP6 is present at the Golgi complex and in secretory vesicles. As neurons mature, MAP6 is translocated to the proximal axon, where it binds and stabilizes microtubules. Further, we demonstrate that dynamic palmitoylation, mediated by the family of α/β Hydrolase domain-containing protein 17 (ABHD17A-C) depalmitoylating enzymes, controls shuttling of MAP6 between membranes and microtubules and is required for MAP6 retention in axons. We propose a model in which MAP6's palmitoylation mediates microtubule stabilization, allows efficient organelle trafficking, and controls axon maturation in vitro and in situ. Copyright © 2017 Elsevier Inc. All rights reserved.
Long chain saturated and unsaturated fatty acids exert opposing effects on viability and function of GLP-1-producing cells: Mechanisms of lipotoxicity
May 18, 2017   PloS One
Thombare K, Ntika S, Wang X, Krizhanovskii C
Long chain saturated and unsaturated fatty acids exert opposing effects on viability and function of GLP-1-producing cells: Mechanisms of lipotoxicity
May 18, 2017
PloS One
Fatty acids acutely stimulate GLP-1 secretion from L-cells in vivo. However, a high fat diet has been shown to reduce the density of L-cells in the mouse intestine and a positive correlation has been indicated between L-cell number and GLP-1 secretion. Thus, the mechanism of fatty acid-stimulated GLP-1 secretion, potential effects of long-term exposure to elevated levels of different fatty acid species, and underlying mechanisms are not fully understood. In the present study, we sought to determine how long-term exposure to saturated (16:0) and unsaturated (18:1) fatty acids, by direct effects on GLP-1-producing cells, alter function and viability, and the underlying mechanisms. GLP-1-secreting GLUTag cells were cultured in the presence/absence of saturated (16:0) and unsaturated (18:1) fatty acids (0.125 mM for 48 h, followed by analyses of viability and apoptosis, as well as involvement of fatty acid oxidation, free fatty acid receptors (FFAR1) and ceramide synthesis. In addition, effects on the expression of proglucagon, prohormone convertase 1/3 (PC1/3), free fatty acid receptors (FFAR1, FFAR3), sodium glucose co-transporter (SGLT) and subsequent secretory response were determined. Saturated (16:0) and unsaturated (18:1) fatty acids exerted opposing effects on the induction of apoptosis (1.4-fold increase in DNA fragmentation by palmitate and a 0.5-fold reduction by oleate; p
Development of an R4 dual-site (R4DS) gateway cloning system enabling the efficient simultaneous cloning of two desired sets of promoters and open reading frames in a binary vector for plant research
May 18, 2017   PloS One
Aboulela M, Tanaka Y, Nishimura K, Mano S, Nishimura M, Ishiguro S, Kimura T, Nakagawa T
Development of an R4 dual-site (R4DS) gateway cloning system enabling the efficient simultaneous cloning of two desired sets of promoters and open reading frames in a binary vector for plant research
May 18, 2017
PloS One
Vast numbers of proteins work cooperatively to exert their functions in various cells. In order to understand the functions and molecular mechanisms of these proteins in plants, analyses of transgenic plants that concomitantly express two protein-coding genes are often required. We developed a novel Gateway cloning technology-compatible binary vector system, the R4 dual-site (R4DS) Gateway cloning system, which enables the easy and efficient cloning of two desired sets of promoters and open reading frames (ORFs) into a binary vector using promoter and ORF entry clones. In this system, C-terminal fusions with 17 kinds of tags including visible reporters and epitope tags are available for each ORF, and selection by four kinds of resistance markers is possible. We verified that the R4DS Gateway cloning system functioned well in Arabidopsis thaliana by observing the expression and localization patterns of fluorescent proteins fused with organelle-targeting signals and driven by stomatal-lineage specific promoters. We also confirmed that the two cloning sites in the R4DS Gateway cloning system were equivalent and independently regulated. The results obtained indicate that the R4DS Gateway cloning system facilitates detailed comparisons of the expression patterns of two promoters as well as co-localization and interaction analyses of two proteins in specific cells in plants.
KDM1A/LSD1 regulates the differentiation and maintenance of spermatogonia in mice
May 12, 2017   PloS One
Myrick DA, Christopher MA, Scott AM, Simon AK, Donlin-Asp PG, Kelly WG, Katz DJ
KDM1A/LSD1 regulates the differentiation and maintenance of spermatogonia in mice
May 12, 2017
PloS One
The proper regulation of spermatogenesis is crucial to ensure the continued production of sperm and fertility. Here, we investigated the function of the H3K4me2 demethylase KDM1A/LSD1 during spermatogenesis in developing and adult mice. Conditional deletion of Kdm1a in the testis just prior to birth leads to fewer spermatogonia and germ cell loss before 3 weeks of age. These results demonstrate that KDM1A is required for spermatogonial differentiation, as well as germ cell survival, in the developing testis. In addition, inducible deletion of Kdm1a in the adult testis results in the abnormal accumulation of meiotic spermatocytes, as well as apoptosis and progressive germ cell loss. These results demonstrate that KDM1A is also required during adult spermatogenesis. Furthermore, without KDM1A, the stem cell factor OCT4 is ectopically maintained in differentiating germ cells. This requirement for KDM1A is similar to what has been observed in other stem cell populations, suggesting a common function. Taken together, we propose that KDM1A is a key regulator of spermatogenesis and germ cell maintenance in the mouse.
DNA recognition by an RNA-guided bacterial Argonaute
May 18, 2017   PloS One
Doxzen KW, Doudna JA
DNA recognition by an RNA-guided bacterial Argonaute
May 18, 2017
PloS One
Argonaute (Ago) proteins are widespread in prokaryotes and eukaryotes and share a four-domain architecture capable of RNA- or DNA-guided nucleic acid recognition. Previous studies identified a prokaryotic Argonaute protein from the eubacterium Marinitoga piezophila (MpAgo), which binds preferentially to 5'-hydroxylated guide RNAs and cleaves single-stranded RNA (ssRNA) and DNA (ssDNA) targets. Here we present a 3.2 Å resolution crystal structure of MpAgo bound to a 21-nucleotide RNA guide and a complementary 21-nucleotide ssDNA substrate. Comparison of this ternary complex to other target-bound Argonaute structures reveals a unique orientation of the N-terminal domain, resulting in a straight helical axis of the entire RNA-DNA heteroduplex through the central cleft of the protein. Additionally, mismatches introduced into the heteroduplex reduce MpAgo cleavage efficiency with a symmetric profile centered around the middle of the helix. This pattern differs from the canonical mismatch tolerance of other Argonautes, which display decreased cleavage efficiency for substrates bearing sequence mismatches to the 5' region of the guide strand. This structural analysis of MpAgo bound to a hybrid helix advances our understanding of the diversity of target recognition mechanisms by Argonaute proteins.
Identification of preoptic sleep neurons using retrograde labelling and gene profiling
May 17, 2017   Nature Add nature.com free-link Cancel
Chung S, Weber F, Zhong P, Tan CL, Nguyen TN,   . . . . . .   , Cetin A, Zeng H, Knight ZA, Luo L, Dan Y
Identification of preoptic sleep neurons using retrograde labelling and gene profiling
May 17, 2017
Nature
In humans and other mammalian species, lesions in the preoptic area of the hypothalamus cause profound sleep impairment, indicating a crucial role of the preoptic area in sleep generation. However, the underlying circuit mechanism remains poorly understood. Electrophysiological recordings and c-Fos immunohistochemistry have shown the existence of sleep-active neurons in the preoptic area, especially in the ventrolateral preoptic area and median preoptic nucleus. Pharmacogenetic activation of c-Fos-labelled sleep-active neurons has been shown to induce sleep. However, the sleep-active neurons are spatially intermingled with wake-active neurons, making it difficult to target the sleep neurons specifically for circuit analysis. Here we identify a population of preoptic area sleep neurons on the basis of their projection target and discover their molecular markers. Using a lentivirus expressing channelrhodopsin-2 or a light-activated chloride channel for retrograde labelling, bidirectional optogenetic manipulation, and optrode recording, we show that the preoptic area GABAergic neurons projecting to the tuberomammillary nucleus are both sleep active and sleep promoting. Furthermore, translating ribosome affinity purification and single-cell RNA sequencing identify candidate markers for these neurons, and optogenetic and pharmacogenetic manipulations demonstrate that several peptide markers (cholecystokinin, corticotropin-releasing hormone, and tachykinin 1) label sleep-promoting neurons. Together, these findings provide easy genetic access to sleep-promoting preoptic area neurons and a valuable entry point for dissecting the sleep control circuit.
Plant biology: An immunity boost combats crop disease
May 17, 2017   Nature Add nature.com free-link Cancel
Bailey-Serres J, Ma W
Architecture of the human interactome defines protein communities and disease networks
May 17, 2017   Nature Add nature.com free-link Cancel
Huttlin EL, Bruckner RJ, Paulo JA, Cannon JR, Ting L,   . . . . . .   , Guruharsha KG, Li K, Artavanis-Tsakonas S, Gygi SP, Harper JW
Architecture of the human interactome defines protein communities and disease networks
May 17, 2017
Nature
The physiology of a cell can be viewed as the product of thousands of proteins acting in concert to shape the cellular response. Coordination is achieved in part through networks of protein-protein interactions that assemble functionally related proteins into complexes, organelles, and signal transduction pathways. Understanding the architecture of the human proteome has the potential to inform cellular, structural, and evolutionary mechanisms and is critical to elucidating how genome variation contributes to disease. Here we present BioPlex 2.0 (Biophysical Interactions of ORFeome-derived complexes), which uses robust affinity purification-mass spectrometry methodology to elucidate protein interaction networks and co-complexes nucleated by more than 25% of protein-coding genes from the human genome, and constitutes, to our knowledge, the largest such network so far. With more than 56,000 candidate interactions, BioPlex 2.0 contains more than 29,000 previously unknown co-associations and provides functional insights into hundreds of poorly characterized proteins while enhancing network-based analyses of domain associations, subcellular localization, and co-complex formation. Unsupervised Markov clustering of interacting proteins identified more than 1,300 protein communities representing diverse cellular activities. Genes essential for cell fitness are enriched within 53 communities representing central cellular functions. Moreover, we identified 442 communities associated with more than 2,000 disease annotations, placing numerous candidate disease genes into a cellular framework. BioPlex 2.0 exceeds previous experimentally derived interaction networks in depth and breadth, and will be a valuable resource for exploring the biology of incompletely characterized proteins and for elucidating larger-scale patterns of proteome organization.
PD-1 expression by tumour-associated macrophages inhibits phagocytosis and tumour immunity
May 17, 2017   Nature Add nature.com free-link Cancel
Gordon SR, Maute RL, Dulken BW, Hutter G, George BM, McCracken MN, Gupta R, Tsai JM, Sinha R, Corey D, Ring AM, Connolly AJ, Weissman IL
PD-1 expression by tumour-associated macrophages inhibits phagocytosis and tumour immunity
May 17, 2017
Nature
Programmed cell death protein 1 (PD-1) is an immune checkpoint receptor that is upregulated on activated T cells for the induction of immune tolerance. Tumour cells frequently overexpress the ligand for PD-1, programmed cell death ligand 1 (PD-L1), facilitating their escape from the immune system. Monoclonal antibodies that block the interaction between PD-1 and PD-L1, by binding to either the ligand or receptor, have shown notable clinical efficacy in patients with a variety of cancers, including melanoma, colorectal cancer, non-small-cell lung cancer and Hodgkin's lymphoma. Although it is well established that PD-1-PD-L1 blockade activates T cells, little is known about the role that this pathway may have in tumour-associated macrophages (TAMs). Here we show that both mouse and human TAMs express PD-1. TAM PD-1 expression increases over time in mouse models of cancer and with increasing disease stage in primary human cancers. TAM PD-1 expression correlates negatively with phagocytic potency against tumour cells, and blockade of PD-1-PD-L1 in vivo increases macrophage phagocytosis, reduces tumour growth and lengthens the survival of mice in mouse models of cancer in a macrophage-dependent fashion. This suggests that PD-1-PD-L1 therapies may also function through a direct effect on macrophages, with substantial implications for the treatment of cancer with these agents.
Extracellular matrix nitration alters growth factor release and activates bioactive complement in human retinal pigment epithelial cells
May 15, 2017   PloS One
Fields MA, Bowrey HE, Gong J, Moreira EF, Cai H, Del Priore LV
Extracellular matrix nitration alters growth factor release and activates bioactive complement in human retinal pigment epithelial cells
May 15, 2017
PloS One
We have shown previously that non-enzymatic nitration (NEN) of the extracellular matrix (ECM), which serves as a model of Bruch's membrane (BM) aging, has a profound effect on the behavior of the overlying retinal pigment epithelial (RPE) cells, including altered phagocytic ability, reduced cell adhesion, and inhibition of proliferation. We know that transplanted RPE monolayers will encounter a hostile sub-RPE environment, including age-related alterations in BM that may compromise cell function and survival. Here we use our previous NEN model of BM aging to determine the effects of NEN of the ECM on growth factor release and complement activation in RPE cells. Human induced-pluripotent stem cells (iPSCs) were differentiated into RPE cells, and confirmed by immunohistochemistry, confocal microscopy, and polymerase chain reaction. IPSC-derived RPE cells were plated onto RPE-derived ECM under untreated or nitrite-modified conditions. Cells were cultured for 7 days and barrier function measured by transepithelial resistance (TER). Vascular endothelial growth factor (VEGF), pigment epithelium-derived factor (PEDF), and complement component C3a were measured using enzyme-linked immunosorbent assay (ELISA). On average nitrite-modified ECM increased VEGF release both apically and basally by 0.15 ± 0.014 ng/mL (p
Reduced sensory synaptic excitation impairs motor neuron function via Kv2.1 in spinal muscular atrophy
May 15, 2017   Nature Neuroscience Add nature.com free-link Cancel
Fletcher EV, Simon CM, Pagiazitis JG, Chalif JI, Vukojicic A, Drobac E, Wang X, Mentis GZ
Reduced sensory synaptic excitation impairs motor neuron function via Kv2.1 in spinal muscular atrophy
May 15, 2017
Nature Neuroscience
Behavioral deficits in neurodegenerative diseases are often attributed to the selective dysfunction of vulnerable neurons via cell-autonomous mechanisms. Although vulnerable neurons are embedded in neuronal circuits, the contributions of their synaptic partners to disease process are largely unknown. Here we show that, in a mouse model of spinal muscular atrophy (SMA), a reduction in proprioceptive synaptic drive leads to motor neuron dysfunction and motor behavior impairments. In SMA mice or after the blockade of proprioceptive synaptic transmission, we observed a decrease in the motor neuron firing that could be explained by the reduction in the expression of the potassium channel Kv2.1 at the surface of motor neurons. Chronically increasing neuronal activity pharmacologically in vivo led to a normalization of Kv2.1 expression and an improvement in motor function. Our results demonstrate a key role of excitatory synaptic drive in shaping the function of motor neurons during development and the contribution of its disruption to a neurodegenerative disease.
Surround Integration Organizes a Spatial Map during Active Sensation
May 15, 2017   Neuron
Pluta SR, Lyall EH, Telian GI, Ryapolova-Webb E, Adesnik H
Surround Integration Organizes a Spatial Map during Active Sensation
May 15, 2017
Neuron
During active sensation, sensors scan space in order to generate a representation of the outside world. However, since spatial coding in sensory systems is typically addressed by measuring receptive fields in a fixed, sensor-based coordinate frame, the cortical representation of scanned space is poorly understood. To address this question, we probed spatial coding in the rodent whisker system using a combination of two-photon imaging and electrophysiology during active touch. We found that surround whiskers powerfully transform the cortical representation of scanned space. On the single-neuron level, surround input profoundly alters response amplitude and modulates spatial preference in the cortex. On the population level, surround input organizes the spatial preference of neurons into a continuous map of the space swept out by the whiskers. These data demonstrate how spatial summation over a moving sensor array is critical to generating population codes of sensory space. Copyright © 2017 Elsevier Inc. All rights reserved.
Soluble bone-derived osteopontin promotes migration and stem-like behavior of breast cancer cells
May 12, 2017   PloS One
Pio GM, Xia Y, Piaseczny MM, Chu JE, Allan AL
Soluble bone-derived osteopontin promotes migration and stem-like behavior of breast cancer cells
May 12, 2017
PloS One
Breast cancer is a leading cause of cancer death in women, with the majority of these deaths caused by metastasis to distant organs. The most common site of breast cancer metastasis is the bone, which has been shown to provide a rich microenvironment that supports the migration and growth of breast cancer cells. Additionally, growing evidence suggests that breast cancer cells that do successfully metastasize have a stem-like phenotype including high activity of aldehyde dehydrogenase (ALDH) and/or a CD44+CD24- phenotype. In the current study, we tested the hypothesis that these ALDHhiCD44+CD24- breast cancer cells interact with factors in the bone secondary organ microenvironment to facilitate metastasis. Specifically, we focused on bone-derived osteopontin and its ability to promote the migration and stem-like phenotype of breast cancer cells. Our results indicate that bone-derived osteopontin promotes the migration, tumorsphere-forming ability and colony-forming ability of whole population and ALDHhiCD44+CD24- breast cancer cells in bone marrow-conditioned media (an ex vivo representation of the bone microenvironment) (p≤0.05). We also demonstrate that CD44 and RGD-dependent cell surface integrins facilitate this functional response to bone-derived osteopontin (p≤0.05), potentially through activation of WNK-1 and PRAS40-related pathways. Our findings suggest that soluble bone-derived osteopontin enhances the ability of breast cancer cells to migrate to the bone and maintain a stem-like phenotype within the bone microenvironment, and this may contribute to the establishment and growth of bone metastases.
Notch ligands regulate the muscle stem-like state ex vivo but are not sufficient for retaining regenerative capacity
May 12, 2017   PloS One
Sakai H, Fukuda S, Nakamura M, Uezumi A, Noguchi YT, Sato T, Morita M, Yamada H, Tsuchida K, Tajbakhsh S, Fukada SI
Notch ligands regulate the muscle stem-like state ex vivo but are not sufficient for retaining regenerative capacity
May 12, 2017
PloS One
Myogenic stem cells are a promising avenue for the treatment of muscular disorders. Freshly isolated muscle stem cells have a remarkable engraftment ability in vivo, but their cell number is limited. Current conventional culture conditions do not allow muscle stem cells to expand in vitro with their bona fide engraftment efficiency, requiring the improvement of culture procedures for achieving successful cell-therapy for muscle disorders. Here we expanded mouse muscle stem cells and human myoblasts with Notch ligands, DLL1, DLL4, and JAG1 to activate Notch signaling in vitro and to investigate whether these cells could retain their engraftment efficiency. Notch signaling promotes the expansion of Pax7+MyoD- mouse muscle stem-like cells and inhibits differentiation even after passage in vitro. Treatment with Notch ligands induced the Notch target genes and generated PAX7+MYOD- stem-like cells from human myoblasts previously cultured on conventional culture plates. However, cells treated with Notch ligands exhibit a stem cell-like state in culture, yet their regenerative ability was less than that of freshly isolated cells in vivo and was comparable to that of the control. These unexpected findings suggest that artificial maintenance of Notch signaling alone is insufficient for improving regenerative capacity of mouse and human donor-muscle cells and suggest that combinatorial events are critical to achieve muscle stem cell and myoblast engraftment potential.
Psychosine enhances the shedding of membrane microvesicles: Implications in demyelination in Krabbe's disease
May 22, 2017   PloS One
D'Auria L, Reiter C, Ward E, Moyano AL, Marshall MS, Nguyen D, Scesa G, Hauck Z, van Breemen R, Givogri MI, Bongarzone ER
Psychosine enhances the shedding of membrane microvesicles: Implications in demyelination in Krabbe's disease
May 22, 2017
PloS One
In prior studies, our laboratory showed that psychosine accumulates and disrupts lipid rafts in brain membranes of Krabbe's disease. A model of lipid raft disruption helped explaining psychosine's effects on several signaling pathways important for oligodendrocyte survival and differentiation but provided more limited insight in how this sphingolipid caused demyelination. Here, we have studied how this cationic inverted coned lipid affects the fluidity, stability and structure of myelin and plasma membranes. Using a combination of cutting-edge imaging techniques in non-myelinating (red blood cell), and myelinating (oligodendrocyte) cell models, we show that psychosine is sufficient to disrupt sphingomyelin-enriched domains, increases the rigidity of localized areas in the plasma membrane, and promotes the shedding of membranous microvesicles. The same physicochemical and structural changes were measured in myelin membranes purified from the mutant mouse Twitcher, a model for Krabbe's disease. Areas of higher rigidity were measured in Twitcher myelin and correlated with higher levels of psychosine and of myelin microvesiculation. These results expand our previous analyses and support, for the first time a pathogenic mechanism where psychosine's toxicity in Krabbe disease involves deregulation of cell signaling not only by disruption of membrane rafts, but also by direct local destabilization and fragmentation of the membrane through microvesiculation. This model of membrane disruption may be fundamental to introduce focal weak points in the myelin sheath, and consequent diffuse demyelination in this leukodystrophy, with possible commonality to other demyelinating disorders.
Myelin-Associated Glycoprotein (MAG) inhibits Schwann cell migration and induces their death
May 19, 2017   The Journal Of Neuroscience : The Official Journal Of The Society For Neuroscience
Chaudhry N, Bachelin C, Zujovic V, Hilaire M, Baldwin KT, Follis RM, Giger R, Carter BD, Evercooren AB, Filbin MT
Myelin-Associated Glycoprotein (MAG) inhibits Schwann cell migration and induces their death
May 19, 2017
The Journal Of Neuroscience : The Official Journal Of The Society For Neuroscience
Remyelination of central nervous system (CNS) axons by Schwann cells (SC) is not efficient, in part due to their poor migration into the adult CNS. Although it is known that migrating SC avoid white matter tracts, the molecular mechanisms underlying this exclusion has never been elucidated. We now demonstrate that myelin-associated glycoprotein (MAG), a well-known inhibitor of neurite outgrowth, inhibits rat SC migration and induces their death via γ secretase-dependent regulated intramembrane proteolysis (RIP) of the p75 neurotrophin receptor (also known as p75 cleavage). Blocking p75 cleavage using inhibitor X (Inh X), a compound that inhibits γ-secretase activity before exposing to MAG or CNS myelin improves SC migration and survival in vitro Furthermore, mouse SC pre-treated with Inh X migrate extensively in the demyelinated mouse spinal cord and re-myelinate axons. These results suggest a novel role for MAG/myelin in poor SC-myelin interaction and identify p75 cleavage as a mechanism that can be therapeutically targeted to enhance SC-mediated axon remyelination in the adult CNS.SIGNIFICANCE STATEMENTNumerous studies have used Schwann cells, the myelin-making cells of the peripheral nervous system (PNS) to remyelinate adult central nervous system (CNS) axons. Indeed, these transplanted cells successfully remyelinate axons, but unfortunately they do not migrate far and so remyelinate only a few axons in the vicinity of the transplant site. It is believed that if Schwann cells could be induced to migrate further and survive better, they may represent a valid therapy for remyelination. We show that myelin-associated glycoprotein (MAG) or CNS myelin in general, inhibit rodent Schwann cell migration and induce their death via cleavage of the neurotrophin receptor, p75. Blockade of p75 cleavage using a specific inhibitor significantly improves migration and survival of the transplanted Schwann cells in vivo. Copyright © 2017 the authors.
Pattern fusion analysis by adaptive alignment of multiple heterogeneous omics data
May 18, 2017   Bioinformatics (Oxford, England)
Shi Q, Zhang C, Peng M, Yu X, Zeng T, Liu J, Chen L
Pattern fusion analysis by adaptive alignment of multiple heterogeneous omics data
May 18, 2017
Bioinformatics (Oxford, England)
Integrating different omics profiles is a challenging task, which provides a comprehensive way to understand complex diseases in a multi-view manner. One key for such an integration is to extract intrinsic patterns in concordance with data structures, so as to discover consistent information across various data types even with noise pollution. Thus, we proposed a novel framework called "pattern fusion analysis" (PFA), which performs automated information alignment and bias correction, to fuse local sample-patterns (e.g., from each data type) into a global sample-pattern corresponding to phenotypes (e.g., across most data types). In particular, PFA can identify significant sample-patterns from different omics profiles by optimally adjusting the effects of each data type to the patterns, thereby alleviating the problems to process different platforms and different reliability levels of heterogeneous data. To validate the effectiveness of our method, we first tested PFA on various synthetic datasets, and found that PFA can not only capture the intrinsic sample clustering structures from the multi-omics data in contrast to the state-of-the-art methods, such as iClusterPlus, SNF and moCluster, but also provide an automatic weight-scheme to measure the corresponding contributions by data types or even samples. In addition, the computational results show that PFA can reveal shared and complementary sample-patterns across data types with distinct signal-to-noise ratios in Cancer Cell Line Encyclopedia (CCLE) data sets, and outperforms over other works at identifying clinically distinct cancer subtypes in The Cancer Genome Atlas (TCGA) data sets. PFA has been implemented as a Matlab package, which is available at http://www.sysbio.ac.cn/cb/chenlab/images/PFApackage_0.1.rar . lnchen@sibs.ac.cn. Supplementary data are available at Bioinformatics online.
Accelerating high dimensional clustering with lossless data reduction
May 18, 2017   Bioinformatics (Oxford, England)
Qaqish BF, O'Brien JJ, Hibbard JC, Clowers KJ
Accelerating high dimensional clustering with lossless data reduction
May 18, 2017
Bioinformatics (Oxford, England)
For cluster analysis, high dimensional data is associated with instability, decreased classification accuracy and high computational burden. The latter challenge can be eliminated as a serious concern. For applications where dimension reduction techniques are not implemented, we propose a temporary transformation which accelerates computations with no loss of information. The algorithm can be applied for any statistical procedure depending only on Euclidean distances and can be implemented sequentially to enable analyses of data that would otherwise exceed memory limitations. The method is easily implemented in common statistical software as a standard pre-processing step. The benefit of our algorithm grows with the dimensionality of the problem and the complexity of the analysis. Consequently, our simple algorithm not only decreases the computation time for routine analyses, it opens the door to performing calculations that may have otherwise been too burdensome to attempt. R, Matlab and SAS/IML code for implementing lossless data reduction is freely available in the online supplement. obrienj@hms.harvard.edu. Supplementary data are available at Bioinformatics online.
TWEAK mediates inflammation in experimental atopic dermatitis and psoriasis
May 22, 2017   Nature Communications
Sidler D, Wu P, Herro R, Claus M, Wolf D, Kawakami Y, Kawakami T, Burkly L, Croft M
TWEAK mediates inflammation in experimental atopic dermatitis and psoriasis
May 22, 2017
Nature Communications
Atopic dermatitis (AD) and psoriasis are driven by alternate type 2 and type 17 immune responses, but some proteins might be critical to both diseases. Here we show that a deficiency of the TNF superfamily molecule TWEAK (TNFSF12) in mice results in defective maintenance of AD-specific T helper type 2 (Th2) and psoriasis-specific Th17 cells in the skin, and impaired expression of disease-characteristic chemokines and cytokines, such as CCL17 and TSLP in AD, and CCL20 and IL-19 in psoriasis. The TWEAK receptor, Fn14, is upregulated in keratinocytes and dermal fibroblasts, and TWEAK induces these cytokines and chemokines alone and in synergy with the signature T helper cytokines of either disease, IL-13 and IL-17. Furthermore, subcutaneous injection of recombinant TWEAK into naive mice induces cutaneous inflammation with histological and molecular signs of both diseases. TWEAK is therefore a critical contributor to skin inflammation and a possible therapeutic target in AD and psoriasis.
Hit and go CAS9 delivered through a lentiviral based self-limiting circuit
May 22, 2017   Nature Communications
Petris G, Casini A, Montagna C, Lorenzin F, Prandi D, Romanel A, Zasso J, Conti L, Demichelis F, Cereseto A
Hit and go CAS9 delivered through a lentiviral based self-limiting circuit
May 22, 2017
Nature Communications
In vivo application of the CRISPR-Cas9 technology is still limited by unwanted Cas9 genomic cleavages. Long-term expression of Cas9 increases the number of genomic loci non-specifically cleaved by the nuclease. Here we develop a Self-Limiting Cas9 circuit for Enhanced Safety and specificity (SLiCES) which consists of an expression unit for Streptococcus pyogenes Cas9 (SpCas9), a self-targeting sgRNA and a second sgRNA targeting a chosen genomic locus. The self-limiting circuit results in increased genome editing specificity by controlling Cas9 levels. For its in vivo utilization, we next integrate SLiCES into a lentiviral delivery system (lentiSLiCES) via circuit inhibition to achieve viral particle production. Upon delivery into target cells, the lentiSLiCES circuit switches on to edit the intended genomic locus while simultaneously stepping up its own neutralization through SpCas9 inactivation. By preserving target cells from residual nuclease activity, our hit and go system increases safety margins for genome editing.

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