169 research outputs found
Translational science in chronic tendinopathies
Chronic tendinopathies involve majority of patients in clinical practice of orthopaedic surgeons and sports physicians. Translational medicine confers an emerging medical advance efficiently towards the clinician directly from scientists which may be used as a targeted therapy. The main objective of translational research from âbench to bedsideâ is to test novel inventions in humans. Our purpose in this article to understand the translational medicine approach for chronic tendinopathies in clinical aspects. Translational research in chronic tendinopathies is required certainly due to plenty of reasons. Newer advances and targeted approach to these tendon disorders may curtail the further degenerative process. It aids in earlier diagnosis and prevention of morbidity, early occupancy of occupational activity, lack of economical as well as recreational failure. Pre-disease level activity is ultimate goal of any therapy. Tendon pathophysiology is constantly evolving researched topic in both biochemical as well as molecular aspect. The basic fundamental understanding of complex process of tendon healing and regeneration is necessary for formulating a newer guideline. The cornerstone of treatment of tendinopathies is still non-operative management. Physical therapy, better pain control, NSAIDS are still primary choice for these conditions. Various biological therapy whenever used one should combined them with other appropriate options to obtain an optimum outcome
FANCD2âFANCI is a clamp stabilized on DNA by monoubiquitination of FANCD2 during DNA repair
Vertebrate DNA crosslink repair excises toxic replication-blocking DNA crosslinks. Numerous factors involved in crosslink repair have been identified, and mutations in their corresponding genes cause Fanconi anemia (FA). A key step in crosslink repair is monoubiquitination of the FANCD2-FANCI heterodimer, which then recruits nucleases to remove the DNA lesion. Here, we use cryo-EM to determine the structures of recombinant chicken FANCD2 and FANCI complexes. FANCD2-FANCI adopts a closed conformation when the FANCD2 subunit is monoubiquitinated, creating a channel that encloses double-stranded DNA (dsDNA). Ubiquitin is positioned at the interface of FANCD2 and FANCI, where it acts as a covalent molecular pin to trap the complex on DNA. In contrast, isolated FANCD2 is a homodimer that is unable to bind DNA, suggestive of an autoinhibitory mechanism that prevents premature activation. Together, our work suggests that FANCD2-FANCI is a clamp that is locked onto DNA by ubiquitin, with distinct interfaces that may recruit other DNA repair factors
Quark-lepton mass relation and CKM mixing in an A(4) extension of the minimal supersymmetric standard model
An interesting mass relation between down-type quarks and charged leptons has been recently predicted within a supersymmetric SU(3)(c) circle times SU(2)(L) circle times U(1)(Y) model based on the A(4) flavor symmetry. Here we propose a simple extension which provides an adequate full description of the quark sector. By adding a pair of vectorlike up quarks, we show how the CKM entries V-ub, V-cb, V-td and V-ts arise from deviations of the unitarity. We perform an analysis including the most relevant observables in the quark sector, such as oscillations and rare decays of kaons, B-d and B-s mesons. In the lepton sector, the model predicts an inverted hierarchy for the neutrino masses, leading to a potentially observable rate of neutrinoless double beta decay
Endogenous Formaldehyde Is a Hematopoietic Stem Cell Genotoxin and Metabolic Carcinogen
Endogenous formaldehyde is produced by numerous biochemical pathways fundamental to life, and it can crosslink both DNA and proteins. However, the consequences of its accumulation are unclear. Here we show that endogenous formaldehyde is removed by the enzyme alcohol dehydrogenase 5 (ADH5/GSNOR), and Adh5â/â mice therefore accumulate formaldehyde adducts in DNA. The repair of this damage is mediated by FANCD2, a DNA crosslink repair protein. Adh5â/âFancd2â/â mice reveal an essential requirement for these protection mechanisms in hematopoietic stem cells (HSCs), leading to their depletion and precipitating bone marrow failure. More widespread formaldehyde-induced DNA damage also causes karyomegaly and dysfunction of hepatocytes and nephrons. Bone marrow transplantation not only rescued hematopoiesis but, surprisingly, also preserved nephron function. Nevertheless, all of these animals eventually developed fatal malignancies. Formaldehyde is therefore an important source of endogenous DNA damage that is counteracted in mammals by a conserved protection mechanism.Medical Research Council de Reino Unido. MC_U105178811Instituto de Salud Carlos III (ISCIII) de España. CP12/03273Ministerio de EconomĂa y Competitividad de España. BFU2013-041457-PNational Institute of Environmental Health Sciences (NIEHS) de los Estados Unidos. P42 ES005948 y P30 ES010126Texas Commission for Environmental Quality. Estados Unidos. 582-12-2186
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Repurposing cancer drugs, batimastat and marimastat, to inhibit the activity of a group I metalloprotease from the venom of the Western Diamondback rattlesnake, Crotalus atrox
Snakebite envenomation causes over 140,000 deaths every year predominantly in developing countries. As a result, it is one of the most lethal neglected tropical diseases. It is associated with an incredibly complex pathophysiology due to the vast number of unique toxins/proteins found in the venoms of diverse snake species found worldwide. Here, we report the purification and functional characteristics of a group I metalloprotease (CAMP-2) from the venom of the western diamondback rattlesnake, Crotalus atrox. Its sensitivity to matrix metalloprotease inhibitors (batimastat and marimastat) was established using specific in vitro experiments and in silico molecular docking analysis. CAMP-2 shows high sequence homology to atroxase from the venom of Crotalus atrox and exhibits collagenolytic, fibrinogenolytic and mild haemolytic activities. It exerts a mild inhibitory effect on agonist-induced platelet aggregation in the absence of plasma proteins. Its collagenolytic activity was completely inhibited by batimastat and marimastat. Zinc chloride also inhibits the collagenolytic activity of CAMP-2 by around 75% at 50 M, while it is partially potentiated by calcium chloride. Molecular docking studies demonstrate that batimastat and marimastat are able to bind strongly to the active site residues of CAMP-2. This study demonstrates the impact of matrix metalloprotease inhibitors in the modulation of a purified, group I metalloprotease activities in comparison to the whole venom. By improving our understanding of snake venom metalloproteases and their sensitivity to small molecule inhibitors, we can begin to develop novel and improved treatment strategies for snakebites
The genetic and biochemical basis of FANCD2 Monoubiquitination
Fanconi anaemia (FA) is a cancer predisposition syndrome characterized by cellular sensitivity to DNA interstrand crosslinkers. The molecular defect in FA is an impaired DNA repair pathway. The critical event in activating this pathway is monoubiquitination of FANCD2. In vivo, a multisubunit FA core complex catalyzes this step, but its mechanism is unclear. Here, we report purification of a native avian FA core complex and biochemical reconstitution of FANCD2 monoubiquitination. This demonstrates that the catalytic FANCL E3 ligase subunit must be embedded within the complex for maximal activity and site specificity. We genetically and biochemically define a minimal subcomplex comprising just three proteins (FANCB, FANCL, and FAAP100) that functions as the monoubiquitination module. Residual FANCD2 monoubiquitination activity is retained in cells defective for other FA core complex subunits. This work describes the in vitro reconstitution and characterization of this multisubunit monoubiquitin E3 ligase, providing key insight into the conserved FA DNA repair pathway
Xpf suppresses the mutagenic consequences of phagocytosis in Dictyostelium
As time passes, mutations accumulate in the genomes of all living
organisms. These changes promote genetic diversity, but also
precipitate ageing and the initiation of cancer. Food is a common
source of mutagens, but little is known about how nutritional factors
cause lasting genetic changes in the consuming organism. Here, we
describe an unusual genetic interaction between DNA repair in the
unicellular amoeba Dictyostelium discoideum and its natural bacterial
food source. We found that Dictyostelium deficient in the DNA repair
nuclease Xpf (xpfâ) display a severe and specific growth defect when
feeding on bacteria. Despite being proficient in the phagocytosis and
digestion of bacteria, over time, xpfâ Dictyostelium feeding on bacteria
cease to grow and in many instances die. The Xpf nuclease activity
is required for sustained growth using a bacterial food source.
Furthermore, the ingestion of this food source leads to a striking
accumulation of mutations in the genome of xpfâ Dictyostelium. This
work therefore establishes Dictyostelium as a model genetic system to
dissect nutritional genotoxicity, providing insight into how phagocytosis
can induce mutagenesis and compromise survival fitness.Medical Research Council (MRC) de Reino Unido. MC_U105178811 y MC_U105115237Wellcome Trust de Reino Unido. WT10620
Aldehyde-mediated inhibition of asparagine biosynthesis has implications for diabetes and alcoholism
Patients with alcoholism and type 2 diabetes manifest altered metabolism, including elevated aldehyde levels and unusually low asparagine levels. We show that asparagine synthetase B (ASNS), the only human asparagine-forming enzyme, is inhibited by disease-relevant reactive aldehydes, including formaldehyde and acetaldehyde. Cellular studies show non-cytotoxic amounts of reactive aldehydes induce a decrease in asparagine levels. Biochemical analyses reveal inhibition results from reaction of the aldehydes with the catalytically important N-terminal cysteine of ASNS. The combined cellular and biochemical results suggest a possible mechanism underlying the low asparagine levels in alcoholism and diabetes. The results will stimulate research on the biological consequences of the reactions of aldehydes with nucleophilic residues
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Discovery of a mammalian splice variant of myostatin that stimulates myogenesis
Myostatin plays a fundamental role in regulating the size of skeletal muscles. To date, only a single myostatin gene and no splice variants have been identified in mammals. Here we describe the splicing of a cryptic intron that removes the coding sequence for the receptor binding moiety of sheep myostatin. The deduced polypeptide sequence of the myostatin splice variant (MSV) contains a 256 amino acid N-terminal domain, which is common to myostatin, and a unique C-terminus of 65 amino acids. Western immunoblotting demonstrated that MSV mRNA is translated into protein, which is present in skeletal muscles. To determine the biological role of MSV, we developed an MSV over-expressing C2C12 myoblast line and showed that it proliferated faster than that of the control line in association with an increased abundance of the CDK2/Cyclin E complex in the nucleus. Recombinant protein made for the novel C-terminus of MSV also stimulated myoblast proliferation and bound to myostatin with high affinity as determined by surface plasmon resonance assay. Therefore, we postulated that MSV functions as a binding protein and antagonist of myostatin. Consistent with our postulate, myostatin protein was
co-immunoprecipitated from skeletal muscle extracts with an MSV-specific antibody. MSV over-expression in C2C12 myoblasts blocked myostatin-induced Smad2/3-dependent signaling, thereby confirming that MSV antagonizes the
canonical myostatin pathway. Furthermore, MSV over expression increased the abundance of MyoD, Myogenin and MRF4 proteins (P,0.05), which indicates that MSV stimulates myogenesis through the induction of myogenic regulatory factors. To help elucidate a possible role in vivo, we observed that MSV protein was more abundant during early post-natal muscle development, while myostatin remained unchanged, which suggests that MSV may promote the growth of skeletal muscles. We conclude that MSV represents a unique example of intra-genic regulation in which a splice variant directly antagonizes the biological activity of the canonical gene product
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Amino acid dependent formaldehyde metabolism in mammals
Abstract: Aldehyde dehydrogenase class 3, encoded by ADH5 in humans, catalyzes the glutathione dependent detoxification of formaldehyde. Here we show that ADH5 deficient cells turn over formaldehyde using alternative pathways starting from the reaction of formaldehyde with free amino acids. When mammalian cells are exposed to formaldehyde, the levels of the reaction products of formaldehyde with the amino acids cysteine and histidine - timonacic and spinacine - are increased. These reactions take place spontaneously and the formation of timonacic is reversible. The levels of timonacic are higher in the plasma of Adh5â/â mice relative to controls and they are further increased upon administration of methanol. We conclude that mammals possess pathways of cysteine and histidine dependent formaldehyde metabolism and that timonacic is a formaldehyde reservoir
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