Tissue Transglutaminase Is a Negative Regulator of Monomeric Lacritin Bioactivity

PURPOSE. Molar accounting of bioactive fluids can expose new regulatory mechanisms in the growing proteomic focus on epithelial biology. Essential for the viability of the surface epithelium of the eye and for normal vision is the thin, but protein-rich, tear film in which the small tear glycoprotein lacritin appears to play a prominent prosecretory, cytoprotective, and mitogenic role. Although optimal bioactive levels in cell culture are 1 to 10 nM over a biphasic dose optimum, ELISA suggests a sustained tear lacritin concentration in the midmicromolar range in healthy adults. Here we identify a reconciling mechanism. METHODS. Monoclonal anti-lacritin 1F5 antibody was generated, and applied together with a new anti-C-terminal polyclonal antibody to tear and tissue Western blotting. In vitro tissue transglutaminase (Tgm2) cross-linking was monitored and characterized by mass spectrometry. RESULTS. Blotting for lacritin in human tears or saliva surprisingly detected immunoreactive material with a higher molecular weight and prominence equal or exceeding the~23 to 25 kDa band of monomeric glycosylated lacritin. Exogenous Tgm2 initiated lacritin cross-linking within 1 minute and was complete by 90 minutes-even with as little as 0.1 nM lacritin, and involved the donors lysine 82 and 85 and the acceptor glutamine 106 in the syndecan-1 binding domain. Lacritin spiked into lacritin-depleted tears formed multimers, in keeping with~0.6 lM TGM2 in tears. Cross-linking was absent when Tgm2 was inactive, and cross-linked lacritin, unlike recombinant monomer, bound syndecan-1 poorly. Enhanced TGM2 expression correlates with reduced cell viability, caspase activation, TNF receptor clustering, 7 and mitochondrial dysfunction 8 associated with hyperosmolar stress in dry eye. 14 Could TGM2 in tears regulate ocular surface biology? Lacritin is a 12.3 kDa tear prosecretory mitogen 15 with glutamine and lysine residues suitable for TGM2 catalyzed cross-linking. Lacritin promotes corneal epithelial cell survival (Zimmerman K, et al. IOVS 2012;53:ARVO E-Abstract 4231) and proliferation

Heparanase deglycanation of syndecan-1 is required for binding of the epithelial-restricted prosecretory mitogen lacritin

Cell surface heparan sulfate (HS) proteoglycans are carbohydrate-rich regulators of cell migratory, mitogenic, secretory, and inflammatory activity that bind and present soluble heparin-binding growth factors (e.g., fibroblast growth factor, Wnt, Hh, transforming growth factor β, amphiregulin, and hepatocyte growth factor) to their respective signaling receptors. We demonstrate that the deglycanated core protein of syndecan-1 (SDC1) and not HS chains nor SDC2 or -4, appears to target the epithelial selective prosecretory mitogen lacritin. An important and novel step in this mechanism is that binding necessitates prior partial or complete removal of HS chains by endogenous heparanase. This limits lacritin activity to sites where heparanase appears to predominate, such as sites of exocrine cell migration, secretion, renewal, and inflammation. Binding is mutually specified by lacritin's C-terminal mitogenic domain and SDC1's N terminus. Heparanase modification of the latter transforms a widely expressed HS proteoglycan into a highly selective surface-binding protein. This novel example of cell specification through extracellular modification of an HS proteoglycan has broad implications in development, homeostasis, and disease

Causes of death and associated conditions (Codac): a utilitarian approach to the classification of perinatal deaths.

A carefully classified dataset of perinatal mortality will retain the most significant information on the causes of death. Such information is needed for health care policy development, surveillance and international comparisons, clinical services and research. For comparability purposes, we propose a classification system that could serve all these needs, and be applicable in both developing and developed countries. It is developed to adhere to basic concepts of underlying cause in the International Classification of Diseases (ICD), although gaps in ICD prevent classification of perinatal deaths solely on existing ICD codes.We tested the Causes of Death and Associated Conditions (Codac) classification for perinatal deaths in seven populations, including two developing country settings. We identified areas of potential improvements in the ability to retain existing information, ease of use and inter-rater agreement. After revisions to address these issues we propose Version II of Codac with detailed coding instructions.The ten main categories of Codac consist of three key contributors to global perinatal mortality (intrapartum events, infections and congenital anomalies), two crucial aspects of perinatal mortality (unknown causes of death and termination of pregnancy), a clear distinction of conditions relevant only to the neonatal period and the remaining conditions are arranged in the four anatomical compartments (fetal, cord, placental and maternal).For more detail there are 94 subcategories, further specified in 577 categories in the full version. Codac is designed to accommodate both the main cause of death as well as two associated conditions. We suggest reporting not only the main cause of death, but also the associated relevant conditions so that scenarios of combined conditions and events are captured.The appropriately applied Codac system promises to better manage information on causes of perinatal deaths, the conditions associated with them, and the most common clinical scenarios for future study and comparisons.RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are

Autophagy in the eye:from physiology to pathophysology

Autophagy is a catabolic self-degradative pathway that promotes the degradation and recycling of intracellular material through the lysosomal compartment. Although first believed to function in conditions of nutritional stress, autophagy is emerging as a critical cellular pathway, involved in a variety of physiological and pathophysiological processes. Autophagy dysregulation is associated with an increasing number of diseases, including ocular diseases. On one hand, mutations in autophagy-related genes have been linked to cataracts, glaucoma, and corneal dystrophy; on the other hand, alterations in autophagy and lysosomal pathways are a common finding in essentially all diseases of the eye. Moreover, LC3-associated phagocytosis, a form of non-canonical autophagy, is critical in promoting visual cycle function. This review collects the latest understanding of autophagy in the context of the eye. We will review and discuss the respective roles of autophagy in the physiology and/or pathophysiology of each of the ocular tissues, its diurnal/circadian variation, as well as its involvement in diseases of the eye

Subacromial decompression surgery for adults with shoulder pain : a clinical practice guideline

Clinical question Do adults with atraumatic shoulder pain for more than 3 months diagnosed as subacromial pain syndrome (SAPS), also labelled as rotator cuff disease, benefit from subacromial decompression surgery? This guideline builds on to two recent high quality trials of shoulder surgery. Current practice SAPS is the common diagnosis for shoulder pain with several first line treatment options, including analgesia, exercises, and injections. Surgeons frequently perform arthroscopic subacromial decompression for prolonged symptoms, with guidelines providing conflicting recommendations. Recommendation The guideline panel makes a strong recommendation against surgery. How this guideline was created A guideline panel including patients, clinicians, and methodologists produced this recommendation in adherence with standards for trustworthy guidelines and the GRADE system. The recommendation is based on two linked systematic reviews on (a) the benefits and harms of subacromial decompression surgery and (b) the minimally important differences for patient reported outcome measures. Recommendations are made actionable for clinicians and their patients through visual overviews. These provide the relative and absolute benefits and harms of surgery in multilayered evidence summaries and decision aids available in MAGIC (www.magicapp.org) to support shared decisions and adaptation. The evidence Surgery did not provide important improvements in pain, function, or quality of life compared with placebo surgery or other options. Frozen shoulder may be more common with surgery. Understanding the recommendation The panel concluded that almost all informed patients would choose to avoid surgery because there is no benefit but there are harms and it is burdensome. Subacromial decompression surgery should not be offered to patients with SAPS. However, there is substantial uncertainty in what alternative treatment is best.Peer reviewe

A genetic variation map for chicken with 2.8 million single-nucleotide polymorphisms

We describe a genetic variation map for the chicken genome containing 2.8 million single-nucleotide polymorphisms ( SNPs). This map is based on a comparison of the sequences of three domestic chicken breeds ( a broiler, a layer and a Chinese silkie) with that of their wild ancestor, red jungle fowl. Subsequent experiments indicate that at least 90% of the variant sites are true SNPs, and at least 70% are common SNPs that segregate in many domestic breeds. Mean nucleotide diversity is about five SNPs per kilobase for almost every possible comparison between red jungle fowl and domestic lines, between two different domestic lines, and within domestic lines - in contrast to the notion that domestic animals are highly inbred relative to their wild ancestors. In fact, most of the SNPs originated before domestication, and there is little evidence of selective sweeps for adaptive alleles on length scales greater than 100 kilobases

A Structure-Based Approach for Detection of Thiol Oxidoreductases and Their Catalytic Redox-Active Cysteine Residues

Cysteine (Cys) residues often play critical roles in proteins, for example, in the formation of structural disulfide bonds, metal binding, targeting proteins to the membranes, and various catalytic functions. However, the structural determinants for various Cys functions are not clear. Thiol oxidoreductases, which are enzymes containing catalytic redox-active Cys residues, have been extensively studied, but even for these proteins there is little understanding of what distinguishes their catalytic redox Cys from other Cys functions. Herein, we characterized thiol oxidoreductases at a structural level and developed an algorithm that can recognize these enzymes by (i) analyzing amino acid and secondary structure composition of the active site and its similarity to known active sites containing redox Cys and (ii) calculating accessibility, active site location, and reactivity of Cys. For proteins with known or modeled structures, this method can identify proteins with catalytic Cys residues and distinguish thiol oxidoreductases from the enzymes containing other catalytic Cys types. Furthermore, by applying this procedure to Saccharomyces cerevisiae proteins containing conserved Cys, we could identify the majority of known yeast thiol oxidoreductases. This study provides insights into the structural properties of catalytic redox-active Cys and should further help to recognize thiol oxidoreductases in protein sequence and structure databases

Consensus-based care recommendations for adults with myotonic dystrophy type 1

Purpose of review Myotonic dystrophy type 1 (DM1) is a severe, progressive genetic disease that affects between 1 in 3,000 and 8,000 individuals globally. No evidence-based guideline exists to inform the care of these patients, and most do not have access to multidisciplinary care centers staffed by experienced professionals, creating a clinical care deficit. Recent findings The Myotonic Dystrophy Foundation (MDF) recruited 66 international clinicians experienced in DM1 patient care to develop consensus-based care recommendations. MDF created a 2-step methodology for the project using elements of the Single Text Procedure and the Nominal Group Technique. The process generated a 4-page Quick Reference Guide and a comprehensive, 55-page document that provides clinical care recommendations for 19 discrete body systems and/or care considerations. Summary The resulting recommendations are intended to help standardize and elevate care for this patient population and reduce variability in clinical trial and study environments. Described as “one of the more variable diseases found in medicine,” myotonic dystrophy type 1 (DM1) is an autosomal dominant, triplet-repeat expansion disorder that affects somewhere between 1:3,000 and 1:8,000 individuals worldwide.1 There is a modest association between increased repeat expansion and disease severity, as evidenced by the average age of onset and overall morbidity of the condition. An expansion of over 35 repeats typically indicates an unstable and expanding mutation. An expansion of 50 repeats or higher is consistent with a diagnosis of DM1. DM1 is a multisystem and heterogeneous disease characterized by distal weakness, atrophy, and myotonia, as well as symptoms in the heart, brain, gastrointestinal tract, endocrine, and respiratory systems. Symptoms may occur at any age. The severity of the condition varies widely among affected individuals, even among members of the same family. Comprehensive evidence-based guidelines do not currently exist to guide the treatment of DM1 patients. As a result, the international patient community reports varied levels of care and care quality, and difficulty accessing care adequate to manage their symptoms, unless they have access to multidisciplinary neuromuscular clinics. Consensus-based care recommendations can help standardize and improve the quality of care received by DM1 patients and assist clinicians who may not be familiar with the significant variability, range of symptoms, and severity of the disease. Care recommendations can also improve the landscape for clinical trial success by eliminating some of the inconsistencies in patient care to allow more accurate understanding of the benefit of potential therapies

Commercially Available Outbred Mice for Genome-Wide Association Studies

Genome-wide association studies using commercially available outbred mice can detect genes involved in phenotypes of biomedical interest. Useful populations need high-frequency alleles to ensure high power to detect quantitative trait loci (QTLs), low linkage disequilibrium between markers to obtain accurate mapping resolution, and an absence of population structure to prevent false positive associations. We surveyed 66 colonies for inbreeding, genetic diversity, and linkage disequilibrium, and we demonstrate that some have haplotype blocks of less than 100 Kb, enabling gene-level mapping resolution. The same alleles contribute to variation in different colonies, so that when mapping progress stalls in one, another can be used in its stead. Colonies are genetically diverse: 45% of the total genetic variation is attributable to differences between colonies. However, quantitative differences in allele frequencies, rather than the existence of private alleles, are responsible for these population differences. The colonies derive from a limited pool of ancestral haplotypes resembling those found in inbred strains: over 95% of sequence variants segregating in outbred populations are found in inbred strains. Consequently it is possible to impute the sequence of any mouse from a dense SNP map combined with inbred strain sequence data, which opens up the possibility of cataloguing and testing all variants for association, a situation that has so far eluded studies in completely outbred populations. We demonstrate the colonies' potential by identifying a deletion in the promoter of H2-Ea as the molecular change that strongly contributes to setting the ratio of CD4+ and CD8+ lymphocytes