Tyrosine cross-linking of extracellular matrix is catalyzed by Duox, a multidomain oxidase/peroxidase with homology to the phagocyte oxidase subunit gp91phox

High molecular weight homologues of gp91phox, the superoxide-generating subunit of phagocyte nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase, have been identified in human (h) and Caenorhabditis elegans (Ce), and are termed Duox for “dual oxidase” because they have both a peroxidase homology domain and a gp91phox domain. A topology model predicts that the enzyme will utilize cytosolic NADPH to generate reactive oxygen, but the function of the ecto peroxidase domain was unknown. Ce-Duox1 is expressed in hypodermal cells underlying the cuticle of larval animals. To investigate function, RNA interference (RNAi) was carried out in C. elegans. RNAi animals showed complex phenotypes similar to those described previously in mutations in collagen biosynthesis that are known to affect the cuticle, an extracellular matrix. Electron micrographs showed gross abnormalities in the cuticle of RNAi animals. In cuticle, collagen and other proteins are cross-linked via di- and trityrosine linkages, and these linkages were absent in RNAi animals. The expressed peroxidase domains of both Ce-Duox1 and h-Duox showed peroxidase activity and catalyzed cross-linking of free tyrosine ethyl ester. Thus, Ce-Duox catalyzes the cross-linking of tyrosine residues involved in the stabilization of cuticular extracellular matrix

Редкое наблюдение двустороннего пневмоторакса у пациента с буллезной эмфиземой единственного легкого и медиастинальной грыжей после пневмонэктомии

Background. Pneumothorax is the most common surgical complication of bullous emphysema, and extremely rare in patients with a single lung. We present our clinical study of a patient with bilateral pneumothorax and bullous emphysema of the single lung and mediastinal hernia diagnosed 40 years after pneumonectomy.Methods. A case report of a patient with bilateral pneumothorax is presented. Patient F., 44 years old, was admitted with complaints on thoracic pain syndrome and respiratory failure. In the previous history of the patient there were mentions of surgery on the left lung without documentary clarification. Routine radiography of the lungs made it possible to diagnose bilateral pneumothorax and postoperative changes of the left hemithorax. Further examination, including computed tomography of the chest and fiber-bronchoscopy, revealed that the patient has emphysematous single right lung, mediastinal pulmonary hernia with the formation of a single pleural cavity, bilateral pneumothorax as a complication and consequence of two previous conditions and due to pneumonectomy in the childhood. The patient underwent a re-draining of the pleural cavity with the elimination of pneumothorax and clinical recovery. The patient refused of antirelapse surgery. Subsequently, he was at the dispensary observation.Results. The most frequent and regular evolution of the residual pleural cavity after pneumonectomy is to fill it with an effusion with subsequent obliteration. The formation of a mediastinal hernia is an extremely rare phenomenon, that has not yet been studied. The tactics of treatment of such patients should be individual, taking into account the revealed changes, the functional state of the respiratory and cardiovascular systems, and the dynamic of the disease.Conlusions. Our study demonstrated a rare clinical case of mediastinal pulmonary hernia after pneumonectomy and the development of bilateral pneumothorax after the rupture of a single lung bull. Modern image diagnostics make it possible to identify the nature of existing changes and clarify their nature even in the absence of accurate anamnestic data.Введение. Пневмоторакс является самым распространенным хирургическим осложнением буллезной эмфиземы легких, у пациентов с единственным легким он встречается крайне редко. Представляем свое клиническое наблюдение пациента с двусторонним пневмотораксом вследствие буллезной эмфиземы единственного легкого и медиастинальной грыжей, диагностированными через 40 лет после пневмонэктомии. Приведено клиническое наблюдение пациента с двусторонним пневмотораксом. Пациент Ф., 44 лет, поступил с клиникой торакального болевого синдрома и дыхательной недостаточности. В анамнезе у пациента есть указания на оперативное вмешательство на левом легком без документального уточнения. Рутинная рентгенография легких позволила диагностировать двусторонний пневмоторакс и послеоперационные изменения левого гемиторакса. При углубленном обследовании, включающем компьютерную томографию грудной клетки и фибробронхоскопию, выявлено, что у пациента имеется эмфизематозно измененное единственное правое легкое, медиастинальная легочная грыжа с формированием единой плевральной полости, двусторонний пневмоторакс как осложнение и следствие двух предыдущих состояний и перенесенной в детстве пневмонэктомии. Пациенту было проведено редренирование плевральной полости с ликвидацией пневмоторакса и клиническим выздоровлением. От противорецидивной операции пациент отказался. В последующем находился на диспансерном наблюдении.Обсуждение результатов. Наиболее частой и закономерной эволюцией остаточной плевральной полости после пневмонэктомии является заполнение ее выпотом с последующей облитерацией. Формирование медиастинальной грыжи является крайне редким феноменом, причина которого до конца не установлена. Тактика лечения подобных пациентов должна быть индивидуальной с учетом выявленных изменений, функционального состояния респираторной и сердечно-сосудистой систем, динамики течения заболевания.Выводы. В представленном наблюдении продемонстрирована редкая клиническая ситуация в виде медиастинальной легочной грыжи после пневмонэктомии и развитии на этом фоне двустороннего пневмоторакса вследствие разрыва булл единственного легкого. Современная имидж-диагностика позволяет выявить природу имеющихся изменений и уточнить их характер даже при отсутствии точных анамнестических данных

Large Isoforms of UNC-89 (Obscurin) Are Required for Muscle Cell Architecture and Optimal Calcium Release in Caenorhabditis elegans

Calcium, a ubiquitous intracellular signaling molecule, controls a diverse array of cellular processes. Consequently, cells have developed strategies to modulate the shape of calcium signals in space and time. The force generating machinery in muscle is regulated by the influx and efflux of calcium ions into the muscle cytoplasm. In order for efficient and effective muscle contraction to occur, calcium needs to be rapidly, accurately and reliably regulated. The mechanisms underlying this highly regulated process are not fully understood. Here, we show that the Caenorhabditis elegans homolog of the giant muscle protein obscurin, UNC-89, is required for normal muscle cell architecture. The large immunoglobulin domain-rich isoforms of UNC-89 are critical for sarcomere and sarcoplasmic reticulum organization. Furthermore, we have found evidence that this structural organization is crucial for excitation-contraction coupling in the body wall muscle, through the coordination of calcium signaling. Thus, our data implicates UNC-89 in maintaining muscle cell architecture and that this precise organization is essential for optimal calcium mobilization and efficient and effective muscle contraction

Eye Absence Does Not Regulate Planarian Stem Cells during Eye Regeneration

Dividing cells called neoblasts contain pluripotent stem cells and drive planarian flatworm regeneration from diverse injuries. A long-standing question is whether neoblasts directly sense and respond to the identity of missing tissues during regeneration. We used the eye to investigate this question. Surprisingly, eye removal was neither sufficient nor necessary for neoblasts to increase eye progenitor production. Neoblasts normally increase eye progenitor production following decapitation, facilitating regeneration. Eye removal alone, however, did not induce this response. Eye regeneration following eye-specific resection resulted from homeostatic rates of eye progenitor production and less cell death in the regenerating eye. Conversely, large head injuries that left eyes intact increased eye progenitor production. Large injuries also non-specifically increased progenitor production for multiple uninjured tissues. We propose a model for eye regeneration in which eye tissue production by planarian stem cells is not directly regulated by the absence of the eye itself. Keywords: planarian; regeneration; stem cell; eye; tissue turnover; target blind; progenitor; neoblastNational Institutes of Health (U.S.) (Grant R01GM080639

Evolution of Susceptibility to Ingested Double-Stranded RNAs in Caenorhabditis Nematodes

International audienceBACKGROUND: The nematode Caenorhabditis elegans is able to take up external double-stranded RNAs (dsRNAs) and mount an RNA interference response, leading to the inactivation of specific gene expression. The uptake of ingested dsRNAs into intestinal cells has been shown to require the SID-2 transmembrane protein in C. elegans. By contrast, C. briggsae was shown to be naturally insensitive to ingested dsRNAs, yet could be rendered sensitive by transgenesis with the C. elegans sid-2 gene. Here we aimed to elucidate the evolution of the susceptibility to external RNAi in the Caenorhabditis genus. PRINCIPAL FINDINGS: We study the sensitivity of many new species of Caenorhabditis to ingested dsRNAs matching a conserved actin gene sequence from the nematode Oscheius tipulae. We find ample variation in the Caenorhabditis genus in the ability to mount an RNAi response. We map this sensitivity onto a phylogenetic tree, and show that sensitivity or insensitivity have evolved convergently several times. We uncover several evolutionary losses in sensitivity, which may have occurred through distinct mechanisms. We could render C. remanei and C. briggsae sensitive to ingested dsRNAs by transgenesis of the Cel-sid-2 gene. We thus provide tools for RNA interference studies in these species. We also show that transgenesis by injection is possible in many Caenorhabditis species. CONCLUSIONS: The ability of animals to take up dsRNAs or to respond to them by gene inactivation is under rapid evolution in the Caenorhabditis genus. This study provides a framework and tools to use RNA interference and transgenesis in various Caenorhabditis species for further comparative and evolutionary studies

Трудности диагностики и лечения при пневмотораксе и гигантских буллах

Spontaneous pneumothorax is the most common acute chest disease. Often, giant bullae give the impression of the presence of air in the pleural cavity. Inadequate differential diagnosis leads to vain drainage of the pleural cavity, damage to the lung with its collapse and pneumothorax.The aim. Analyze diagnostic and tactical mistakes in patients with pulmonary emphysema, which manifests with giant bullae, and outline the ways to prevent complications.Methods. The analysis of the treatment of 1,636 patients with pulmonary emphysema and its complications undergoing treatment in the thoracic surgical department of the Samara Regional Clinical Hospital named after V.D.Seredavin in the period from 2001 to 2018 is presented.Results. Giant bulla were diagnosed in 35 (2.1%) patients, 16 of them were hospitalized ungently. In 6 patients, the diagnosis of a giant bulla of the lung was correct, and the patients were referred to the thoracic surgical department. In 10 patients, a giant bulla of the lung was regarded as pneumothorax, and pleural drainage was performed before referral to the thoracic surgical department.Conclusion. The correct interpretation of the radiological data and comparison with the clinical picture allows avoiding diagnostic errors and the associated danger and complications.Спонтанный пневмоторакс является самым частым острым заболеванием органов грудной клетки. Нередко гигантские буллы создают впечатление о наличии воздуха в плевральной полости. Неадекватная дифференциальная диагностика приводит к напрасному дренированию плевральной полости, повреждению легкого с его спадением и возникновением пневмоторакса.Целью исследования явился анализ диагностических и тактических ошибок у пациентов с буллезной эмфиземой легких, представленной гигантскими буллами, и обозначение путей профилактики осложнений.Материалы и методы. Проведен анализ лечения пациентов (n = 1 636) с эмфиземой легких и ее осложнениями, госпитализированных в хирургическое торакальное отделение Государственного бюджетного учреждения здравоохранения «Самарская областная клиническая больница имени В.Д.Середавина» Министерства здравоохранения Самарской области (2001–2018).Результаты. Гигантские буллы диагностированы у 35 (2,1%) пациентов, 16 из которых госпитализированы в экстренном порядке. Верный диагноз гигантская булла легкого установлен в 6 случаях, больные направлены в хирургическое торакальное отделение. В 10 случаях гигантская булла легкого расценена как пневмоторакс и до поступления в хирургическое торакальное отделение больным проведено дренирование плевральной полости.Заключение. Верная интерпретация рентгенологических данных и сопоставление с клинической картиной позволяет избежать диагностических ошибок и связанных с этим дальнейших опасностей и осложнений

Calpains Mediate Integrin Attachment Complex Maintenance of Adult Muscle in Caenorhabditis elegans

Two components of integrin containing attachment complexes, UNC-97/PINCH and UNC-112/MIG-2/Kindlin-2, were recently identified as negative regulators of muscle protein degradation and as having decreased mRNA levels in response to spaceflight. Integrin complexes transmit force between the inside and outside of muscle cells and signal changes in muscle size in response to force and, perhaps, disuse. We therefore investigated the effects of acute decreases in expression of the genes encoding these multi-protein complexes. We find that in fully developed adult Caenorhabditis elegans muscle, RNAi against genes encoding core, and peripheral, members of these complexes induces protein degradation, myofibrillar and mitochondrial dystrophies, and a movement defect. Genetic disruption of Z-line– or M-line–specific complex members is sufficient to induce these defects. We confirmed that defects occur in temperature-sensitive mutants for two of the genes: unc-52, which encodes the extra-cellular ligand Perlecan, and unc-112, which encodes the intracellular component Kindlin-2. These results demonstrate that integrin containing attachment complexes, as a whole, are required for proper maintenance of adult muscle. These defects, and collapse of arrayed attachment complexes into ball like structures, are blocked when DIM-1 levels are reduced. Degradation is also blocked by RNAi or drugs targeting calpains, implying that disruption of integrin containing complexes results in calpain activation. In wild-type animals, either during development or in adults, RNAi against calpain genes results in integrin muscle attachment disruptions and consequent sub-cellular defects. These results demonstrate that calpains are required for proper assembly and maintenance of integrin attachment complexes. Taken together our data provide in vivo evidence that a calpain-based molecular repair mechanism exists for dealing with attachment complex disruption in adult muscle. Since C. elegans lacks satellite cells, this mechanism is intrinsic to the muscles and raises the question if such a mechanism also exists in higher metazoans

Binding of myomesin to obscurin-like-1 to the muscle M-band provides a strategy for isoform-specific mechanical protection

The sarcomeric cytoskeleton is a network of modular proteins that integrate mechanical and signalling roles. Obscurin, or its homolog obscurin-like-1, bridges the giant ruler titin and the myosin crosslinker myomesin at the M-band. Yet, the molecular mechanisms underlying the physical obscurin(-like-1):myomesin connection, important for mechanical integrity of the M-band, remained elusive. Here, using a combination of structural, cellular, and single-molecule force spectroscopy techniques, we decode the architectural and functional determinants defining the obscurin(-like-1): myomesin complex. The crystal structure reveals a trans-complementation mechanism whereby an incomplete immunoglobulin-like domain assimilates an isoform-specific myomesin interdomain sequence. Crucially, this unconventional architecture provides mechanical stability up to forces of 135 pN. A cellular competition assay in neonatal rat cardiomyocytes validates the complex and provides the rationale for the isoform specificity of the interaction. Altogether, our results reveal a novel binding strategy in sarcomere assembly, which might have implications on muscle nanomechanics and overall M-band organization.We thank the Diamond Light Source and the European Synchrotron Radiation Laboratory for access to MX and SAXS beamlines, respectively. This work was supported by a British Heart Foundation grant (PG/10/67/28527) awarded to R.A.S. and M.G. as well as MRC grant MR/J010456/1 to M.G. and a British Heart Foundation grant (PG/13/50/30426) and EPSRC Fellowship (K00641X/1) to S.G.-M

Cytoskeletal protein kinases: titin and its relations in mechanosensing

Titin, the giant elastic ruler protein of striated muscle sarcomeres, contains a catalytic kinase domain related to a family of intrasterically regulated protein kinases. The most extensively studied member of this branch of the human kinome is the Ca2+–calmodulin (CaM)-regulated myosin light-chain kinases (MLCK). However, not all kinases of the MLCK branch are functional MLCKs, and about half lack a CaM binding site in their C-terminal autoinhibitory tail (AI). A unifying feature is their association with the cytoskeleton, mostly via actin and myosin filaments. Titin kinase, similar to its invertebrate analogue twitchin kinase and likely other “MLCKs”, is not Ca2+–calmodulin-activated. Recently, local protein unfolding of the C-terminal AI has emerged as a common mechanism in the activation of CaM kinases. Single-molecule data suggested that opening of the TK active site could also be achieved by mechanical unfolding of the AI. Mechanical modulation of catalytic activity might thus allow cytoskeletal signalling proteins to act as mechanosensors, creating feedback mechanisms between cytoskeletal tension and tension generation or cellular remodelling. Similar to other MLCK-like kinases like DRAK2 and DAPK1, TK is linked to protein turnover regulation via the autophagy/lysosomal system, suggesting the MLCK-like kinases have common functions beyond contraction regulation