Corneal transplantation is the current gold standard treatment to restore visual acuity to patients with severe corneal diseases and injuries. Due to severe donor tissue shortage, efforts to develop ...a corneal equivalent have been made but the challenge remains unmet. Another issue of concern in ocular surgery is the difficult instillation and fast drainage of antibiotic ocular eye drops as bacterial infections can jeopardize implant success by delaying or impairing tissue healing. In this study, we developed antimicrobial silk‐based hydrogels that have the potential to be photoactivated in situ, fully adapting to the corneal injury shape. Gentamicin‐loaded methacrylated‐silk (SilkMA) hydrogels were prepared within minutes using low UV intensity (3 mW/cm2). SilkMA gels provided a Young's modulus between 21 and 79 kPa together with a light transmittance spectrum and water content (83%–90%) similar to the human cornea. Polymer concentration (15%–25%) was found to offer a tool for tailoring the physical properties of the hydrogels. We confirmed that the methacrylation did not affect the material's in vitro degradation and biocompatibility by observing fibroblast adhesion and proliferation. Importantly, agar diffusion tests showed that the synthesized hydrogels were able to inhibit Staphylococcus aureus and Pseudomonas aeruginosa growth for 72 h. These characteristics along with their injectability and viscoelasticity demonstrate the potential of SilkMA hydrogels to be applied in several soft tissue engineering fields. As such, for the first time we demonstrate the potential of photocurable antimicrobial SilkMA hydrogels as a novel biomaterial to facilitate corneal regeneration.
The Arctic marine environment is undergoing a transition from thick multi-year to first-year sea-ice cover with coincident lengthening of the melt season. Such changes are evident in the Baffin ...Bay-Davis Strait-Labrador Sea (BDL) region where melt onset has occurred ~8 days decade−1 earlier from 1979 to 2015. A series of anomalously early events has occurred since the mid-1990s, overlapping a period of increased upper-air ridging across Greenland and the northwestern North Atlantic. We investigate an extreme early melt event observed in spring 2013. (~6σ below the 1981–2010 melt climatology), with respect to preceding sub-seasonal mid-tropospheric circulation conditions as described by a daily Greenland Blocking Index (GBI). The 40-days prior to the 2013 BDL melt onset are characterized by a persistent, strong 500 hPa anticyclone over the region (GBI >+1 on >75% of days). This circulation pattern advected warm air from northeastern Canada and the northwestern Atlantic poleward onto the thin, first-year sea ice and caused melt ~50 days earlier than normal. The episodic increase in the ridging atmospheric pattern near western Greenland as in 2013, exemplified by large positive GBI values, is an important recent process impacting the atmospheric circulation over a North Atlantic cryosphere undergoing accelerated regional climate change.
Epithelia can eliminate apoptotic cells by apical extrusion. This is a complex morphogenetic event where expulsion of the apoptotic cell is accompanied by rearrangement of its immediate neighbors to ...form a rosette. A key mechanism for extrusion is constriction of an actomyosin network that neighbor cells form at their interface with the apoptotic cell. Here we report a complementary process of cytoskeletal relaxation that occurs when cortical contractility is down-regulated at the junctions between those neighbor cells themselves. This reflects a mechanosensitive Src family kinase (SFK) signaling pathway that is activated in neighbor cells when the apoptotic cell relaxes shortly after injury. Inhibiting SFK signaling blocks both the expulsion of apoptotic cells and the rosette formation among their neighbor cells. This reveals the complex pattern of spatially distinct contraction and relaxation that must be established in the neighboring epithelium for apoptotic cells to be extruded.
Dysfunction of caveolae is involved in human muscle disease, although the underlying molecular mechanisms remain unclear. In this paper, we have functionally characterized mouse and zebrafish models ...of caveolae-associated muscle disease. Using electron tomography, we quantitatively defined the unique three-dimensional membrane architecture of the mature muscle surface. Caveolae occupied around 50% of the sarcolemmal area predominantly assembled into multilobed rosettes. These rosettes were preferentially disassembled in response to increased membrane tension. Caveola-deficient cavin-1(-/-) muscle fibers showed a striking loss of sarcolemmal organization, aberrant T-tubule structures, and increased sensitivity to membrane tension, which was rescued by muscle-specific Cavin-1 reexpression. In vivo imaging of live zebrafish embryos revealed that loss of muscle-specific Cavin-1 or expression of a dystrophy-associated Caveolin-3 mutant both led to sarcolemmal damage but only in response to vigorous muscle activity. Our findings define a conserved and critical role in mechanoprotection for the unique membrane architecture generated by the caveolin-cavin system.
Common Missense Variant in the Glucokinase Regulatory Protein Gene Is Associated With Increased Plasma Triglyceride and C-Reactive
Protein but Lower Fasting Glucose Concentrations
Marju Orho-Melander ...1 ,
Olle Melander 1 ,
Candace Guiducci 2 ,
Pablo Perez-Martinez 3 4 5 ,
Dolores Corella 5 ,
Charlotta Roos 1 ,
Ryan Tewhey 2 ,
Mark J. Rieder 6 ,
Jennifer Hall 7 ,
Goncalo Abecasis 8 ,
E. Shyong Tai 9 ,
Cullan Welch 7 ,
Donna K. Arnett 10 ,
Valeriya Lyssenko 1 ,
Eero Lindholm 1 ,
Richa Saxena 2 ,
Paul I.W. de Bakker 2 ,
Noel Burtt 2 ,
Benjamin F. Voight 2 ,
Joel N. Hirschhorn 2 ,
Katherine L. Tucker 11 ,
Thomas Hedner 12 ,
Tiinamaija Tuomi 13 14 ,
Bo Isomaa 14 ,
Karl-Fredrik Eriksson 1 ,
Marja-Riitta Taskinen 13 ,
Björn Wahlstrand 12 ,
Thomas E. Hughes 15 ,
Laurence D. Parnell 4 ,
Chao-Qiang Lai 4 ,
Göran Berglund 16 ,
Leena Peltonen 17 ,
Erkki Vartiainen 18 ,
Pekka Jousilahti 18 ,
Aki S. Havulinna 18 ,
Veikko Salomaa 18 ,
Peter Nilsson 1 ,
Leif Groop 1 13 ,
David Altshuler 2 19 20 ,
Jose M. Ordovas 4 and
Sekar Kathiresan 2 21
1 Department of Clinical Sciences, University Hospital Malmö, Clinical Research Center, Lund University, Malmö, Sweden
2 Program in Medical and Population Genetics, Broad Institute of the Massachusetts Institute of Technology and Harvard University,
Cambridge, Massachusetts
3 Lipids and Atherosclerosis Research Unit, Reina Sofia University Hospital, University of Cordoba, Cordoba, Spain
4 Nutrition and Genomics Laboratory, Jean Mayer-U.S. Department of Agriculture Human Nutrition Research Center on Aging at Tufts
University, Boston, Massachusetts
5 Genetic and Molecular Epidemiology Unit and CIBER Fisiopatología de la Obesidad y Nutrición, School of Medicine University
of Valencia, Valencia, Spain
6 Department of Genome Sciences, University of Washington, Seattle, Washington
7 Lillehei Heart Institute, Department of Medicine, University of Minnesota, Minneapolis, Minnesota
8 Center for Statistical Genetics, Department of Biostatistics, University of Michigan School of Public Health, Ann Arbor, Michigan
9 Department of Endocrinology, Singapore General Hospital, Singapore
10 Dietary Assessment and Epidemiology Research Program, Jean Mayer-U.S. Department of Agriculture Human Nutrition Research Center
on Aging, Tufts University, Boston, Massachusetts
11 Department of Epidemiology, University of Alabama, Birmingham, Alabama
12 Department of Clinical Pharmacology, Sahlgrenska Academy, Göteborg, Sweden
13 Department of Medicine, Helsinki University Hospital, University of Helsinki, Helsinki, Finland
14 Folkhälsan Research Center, Helsinki, Finland
15 Novartis Institutes for BioMedical Research, Cambridge, Massachusetts
16 Department of Clinical Sciences, Medicine, Lund University, Malmö, Sweden
17 Department of Molecular Medicine, National Public Health Institute, Biomedicum, Helsinki, Finland
18 Department of Epidemiology and Health Promotion, National Public Health Institute, Helsinki, Finland
19 Center for Human Genetic Research and Department of Molecular Biology, Massachusetts General Hospital, Boston, Massachusetts
20 Department of Genetics, Harvard Medical School, Boston, Massachusetts
21 Cardiovascular Disease Prevention Center, Cardiology Division, Department of Medicine, Massachusetts General Hospital, Harvard
Medical School, Boston, Massachusetts
Corresponding author: Marju Orho-Melander, marju.orho-melander{at}med.lu.se
Abstract
OBJECTIVE— Using the genome-wide association approach, we recently identified the glucokinase regulatory protein gene ( GCKR , rs780094) region as a novel quantitative trait locus for plasma triglyceride concentration in Europeans. Here, we sought
to study the association of GCKR variants with metabolic phenotypes, including measures of glucose homeostasis, to evaluate the GCKR locus in samples of non-European ancestry and to fine- map across the associated genomic interval.
RESEARCH DESIGN AND METHODS— We performed association studies in 12 independent cohorts comprising >45,000 individuals representing several ancestral groups
(whites from Northern and Southern Europe, whites from the U.S., African Americans from the U.S., Hispanics of Caribbean origin,
and Chinese, Malays, and Asian Indians from Singapore). We conducted genetic fine-mapping across the ∼417-kb region of linkage
disequilibrium spanning GCKR and 16 other genes on chromosome 2p23 by imputing untyped HapMap single nucleotide polymorphisms (SNPs) and genotyping 104
SNPs across the associated genomic interval.
RESULTS— We provide comprehensive evidence that GCKR rs780094 is associated with opposite effects on fasting plasma triglyceride ( P meta = 3 × 10 −56 ) and glucose ( P meta = 1 × 10 −13 ) concentrations. In addition, we confirmed recent reports that the same SNP is associated with C-reactive protein (CRP) level
( P = 5 × 10 −5 ). Both fine-mapping approaches revealed a common missense GCKR variant (rs1260326, Pro446Leu, 34% frequency, r 2 = 0.93 with rs780094) as the strongest association signal in the region.
CONCLUSIONS— These findings point to a molecular mechanism in humans by which higher triglycerides and CRP can be coupled with lower plasma
glucose concentrations and position GCKR in central pathways regulating both hepatic triglyceride and glucose metabolism.
Footnotes
Published ahead of print at http://diabetes.diabetesjournals.org on 4 August 2008.
Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work
is not altered. See http://creativecommons.org/licenses/by-nc-nd/3.0/ for details.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore
be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Accepted July 29, 2008.
Received April 17, 2008.
DIABETES
A quantitative understanding of the dynamics of biological neural networks is fundamental to gaining insight into information processing in the brain. While techniques exist to measure spatial or ...temporal properties of these networks, it remains a significant challenge to resolve the neural dynamics with subcellular spatial resolution. In this work we consider a fundamentally new form of wide-field imaging for neuronal networks based on the nanoscale magnetic field sensing properties of optically active spins in a diamond substrate. We analyse the sensitivity of the system to the magnetic field generated by an axon transmembrane potential and confirm these predictions experimentally using electronically-generated neuron signals. By numerical simulation of the time dependent transmembrane potential of a morphologically reconstructed hippocampal CA1 pyramidal neuron, we show that the imaging system is capable of imaging planar neuron activity non-invasively at millisecond temporal resolution and micron spatial resolution over wide-fields.
Abstract
Here we describe a short feasibility study and methodological framework for the production of stable, CRISPR/Cas9-based, large genomic deletions in zebrafish, ranging from several base pairs ...(bp) to hundreds of kilobases (kb). Using a cocktail of four single guide RNAs (sgRNAs) targeting a single genomic region mixed with a marker-sgRNA against the pigmentation gene tyrosinase, we demonstrate that one can easily and accurately excise genomic regions such as promoters, protein domains, specific exons, or whole genes. We exemplify this technique with a complex gene family, neurexins, composed of three duplicated genes with multiple promoters and intricate splicing processes leading to thousands of isoforms. We precisely deleted small regions such as their transmembrane domains (150 bp deletion in average) to their entire genomic locus (300 kb deletion for nrxn1a for instance). We find that both the concentration and ratio of Cas9/sgRNAs are critical for the successful generation of these large deletions and, interestingly, that in our study, their transmission frequency does not seem to decrease with increasing distance between sgRNA target sites. Considering the growing reports and debate about genetically compensated small indel mutants, the use of large-deletion approaches is likely to be widely adopted in studies of gene function. This strategy will also be key to the study of non-coding genomic regions. Note that we are also describing here a custom method to produce the sgRNAs, which proved to be faster and more robust than the ones traditionally used in the community to date.
Plasma membrane rupture can result in catastrophic cell death. The skeletal muscle fiber plasma membrane, the sarcolemma, provides an extreme example of a membrane subject to mechanical stress since ...these cells specifically evolved to generate contraction and movement. A quantitative model correlating ultrastructural remodeling of surface architecture with tissue changes in vivo is required to understand how membrane domains contribute to the shape changes associated with tissue deformation in whole animals. We and others have shown that loss of caveolae, small invaginations of the plasma membrane particularly prevalent in the muscle sarcolemma, renders the plasma membrane more susceptible to rupture during stretch.1,2,3 While it is thought that caveolae are able to flatten and be absorbed into the bulk membrane to buffer local membrane expansion, a direct demonstration of this model in vivo has been unachievable since it would require measurement of caveolae at the nanoscale combined with detailed whole-animal morphometrics under conditions of perturbation. Here, we describe the development and application of the “active trapping model” where embryonic zebrafish are immobilized in a curved state that mimics natural body axis curvature during an escape response. The model is amenable to multiscale, multimodal imaging including high-resolution whole-animal three-dimensional quantitative electron microscopy. Using the active trapping model, we demonstrate the essential role of caveolae in maintaining sarcolemmal integrity and quantify the specific contribution of caveolar-derived membrane to surface expansion. We show that caveolae directly contribute to an increase in plasma membrane surface area under physiologically relevant membrane deformation conditions.
•“Active trapping” of zebrafish provides an in vivo model of membrane stretch•Membrane stretch results in disassembly of muscle caveolae•Genetic ablation of caveolae results in membrane rupture under stretch•Membrane released by disassembly matches that required for surface area expansion
Animal cells can be subject to rapid and dramatic shape changes during growth, development, and normal function, but plasma membrane rupture results in catastrophic cell death. Hall et al. use a flexing fish model to show how muscle fibers under stretch buffer sarcolemmal tension using small membrane pits called caveolae.
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