ABSTRACT
Duchenne muscular dystrophy (DMD) is a genetic neuromuscular disease. Although it leads to muscle weakness, affected individuals predominantly die from cardiomyopathy, which remains uncurable. Accumulating evidence suggests that an overexpression of utrophin may counteract some of the pathophysiological outcomes of DMD. The aim of this study was to investigate the role of utrophin in dystrophin-deficient human cardiomyocytes (CMs) and to test whether an overexpression of utrophin, implemented via the CRISPR-deadCas9-VP64 system, can improve their phenotype. We used human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) lacking either dystrophin (DMD) or both dystrophin and utrophin (DMD KO/UTRN(+/-)). We carried out proteome analysis, which revealed considerable differences in the proteins related to muscle contraction, cell-cell adhesion, and extracellular matrix organization. Furthermore, we evaluated the role of utrophin in maintaining the physiological properties of DMD hiPSC-CMs using atomic force microscopy, patch-clamp, and Ca2+ oscillation analysis. Our results showed higher values of afterhyperpolarization and altered patterns of cytosolic Ca2+ oscillations in DMD; the latter was further disturbed in DMD KO/UTRN(+/-) hiPSC-CMs. Utrophin upregulation improved both parameters. Our findings demonstrate for the first time that utrophin maintains the physiological functions of DMD hiPSC-CMs, and that its upregulation can compensate for the loss of dystrophin.
ABSTRACT
Introduction: We present the case of a patient with recurrent bilateral hemothorax. After misdiagnosis despite several histological samples, a pleural manifestation of epithelioid angiosarcoma was diagnosed by further immunohistological staining. Based on this situation, we aim to sensitize the reader to this rare disease. Main concerns and important clinical findings: A 73-year-old fully conscious woman presented with dyspnea for 3 days. She was in stable general condition, pain was denied, she had a history of cigarette smoking, she had no cardiopulmonary events, and she was not receiving any anticoagulation medication. Physical examination revealed decreased breath sounds on the left side, and her hemoglobin level was 7.0â mmol/L. Primary diagnoses interventions and outcomes: The initial chest x-ray showed a left-sided effusion. Hemothorax was then diagnosed. Further investigation revealed no evidence of malignancy (CT, EBUS, cytology, etc.). VATS was performed, and biopsies of pleural lesions did not reveal congruent findings for the hemothorax. Due to recurrent bilateral hemothorax with the need for erythrocyte transfusion, the patient underwent several operations, including histological sampling, without evidence of malignancy. After further processing, an additional pathological report revealed an epithelioid angiosarcoma defined by massively proliferating epithelioid cells strongly positive for ERG and CD31 and negative for CD34. The neoplastic cells coexpressed D2-40 (podoplanin). Finally, due to multiple cerebral metastases, palliative therapy was indicated. Conclusion: Physicians and pathologists treating spontaneous hemothorax need to have broad knowledge of the possible, sometimes rare, etiologies. If the clinical course and intraoperative findings do not agree with the histopathological results, this finding must be questioned, and further immunohistochemical staining is mandatory. Thus, in the case of recurrent hemothorax, angiosarcoma of the pleura should also be considered for differential diagnosis.
ABSTRACT
Morphological studies of skeletal muscle tissue provide insights into the architecture of muscle fibers, the surrounding cells, and the extracellular matrix (ECM). However, a spatial proteomics analysis of the skeletal muscle including the muscle-tendon transition zone is lacking. Here, we prepare cryotome muscle sections of the mouse soleus muscle and measure each slice using short liquid chromatography-mass spectrometry (LC-MS) gradients. We generate 3,000 high-resolution protein profiles that serve as the basis for a network analysis to reveal the complex architecture of the muscle-tendon junction. Among the protein profiles that increase from muscle to tendon, we find proteins related to neuronal activity, fatty acid biosynthesis, and the renin-angiotensin system (RAS). Blocking the RAS in cultured mouse tenocytes using losartan reduces the ECM synthesis. Overall, our analysis of thin cryotome sections provides a spatial proteome of skeletal muscle and reveals that the RAS acts as an additional regulator of the matrix within muscle-tendon junctions.
Subject(s)
Muscle, Skeletal , Proteomics , Tendons , Animals , Proteomics/methods , Muscle, Skeletal/metabolism , Tendons/metabolism , Mice , Extracellular Matrix/metabolism , Male , Mice, Inbred C57BL , Renin-Angiotensin System/physiology , Adaptation, Physiological , Proteome/metabolism , Losartan/pharmacologyABSTRACT
OBJECTIVE: Evaluation of neonatal morbidity after maternal central neurotropic drug exposure. METHODS: Retrospective single-center level-III neonatology cohort analysis of neonates after CND from 2018 to 2021. Control group of neonates born to mothers without CND cared for at the maternity ward. RESULTS: Significantly more frequent therapy need of neonates with CND [OR 23 (95% CI: 7.8-62); RR 14 (95% CI: 5.4-37); p < 0.01]. Neonates after CND had lower Apgar-scores LM 1 [CND 8.1; CG 8.6; p < 0.05]; LM 5 [CND 9; CG 9.7; p < 0.01]; LM 10 [CND 9.6; CG 9.9; p < 0.05]. The first symptom occurred in 95.35% within 24 h (mean: 3.3 h). CND group showed significantly more often preterm delivery [OR 3.5; RR 3.2; p < 0.05], and especially cumulative multiple symptoms [OR 9.4; RR 6.6; p < 0.01] but no correlation to multiple maternal medication use (p = 0.3). CONCLUSIONS: Neonates exposed to CND are at increased risk for postnatal therapy, often due to multiple symptoms. Neonates should be continuously monitored for at least 24 h.
Subject(s)
Infant, Newborn, Diseases , Humans , Female , Infant, Newborn , Retrospective Studies , Pregnancy , Adult , Male , Infant, Newborn, Diseases/epidemiology , Infant, Newborn, Diseases/chemically induced , Central Nervous System Agents/adverse effects , Central Nervous System Agents/therapeutic use , Prenatal Exposure Delayed Effects/epidemiology , Prenatal Exposure Delayed Effects/chemically induced , Case-Control Studies , Maternal Exposure/adverse effects , Pregnancy Complications/drug therapy , Pregnancy Complications/epidemiologyABSTRACT
Complexes of ERLIN1 and ERLIN2 (ER lipid raft-associated 1 and 2) form large ring-like cup-shaped structures on the endoplasmic reticulum (ER) membrane and serve as platforms to bind cholesterol and E3 ubiquitin ligases, potentially defining functional nanodomains. Here, we show that ERLIN scaffolds mediate the interaction between the full-length isoform of TMUB1 (transmembrane and ubiquitin-like domain-containing 1) and RNF170 (RING finger protein 170). We identify a luminal N-terminal conserved region in TMUB1 and RNF170, which is required for this interaction. Three-dimensional modelling shows that this conserved motif binds the stomatin/prohibitin/flotillin/HflKC domain of two adjacent ERLIN subunits at different interfaces. Protein variants that preclude these interactions have been previously linked to hereditary spastic paraplegia. Using omics-based approaches in combination with phenotypic characterization of HeLa cells lacking both ERLINs, we demonstrate a role of ERLIN scaffolds in limiting cholesterol esterification, thereby favouring cholesterol transport from the ER to the Golgi apparatus and regulating Golgi morphology and the secretory pathway.
Subject(s)
Cholesterol , Endoplasmic Reticulum , Golgi Apparatus , Membrane Proteins , Secretory Pathway , Ubiquitin-Protein Ligases , Humans , Membrane Proteins/metabolism , Cholesterol/metabolism , Ubiquitin-Protein Ligases/metabolism , Ubiquitin-Protein Ligases/genetics , Endoplasmic Reticulum/metabolism , HeLa Cells , Golgi Apparatus/metabolism , Protein Binding , Nerve Tissue ProteinsABSTRACT
Uncontrolled secretion of ECM proteins, such as collagen, can lead to excessive scarring and fibrosis and compromise tissue function. Despite the widespread occurrence of fibrotic diseases and scarring, effective therapies are lacking. A promising approach would be to limit the amount of collagen released from hyperactive fibroblasts. We have designed membrane permeant peptide inhibitors that specifically target the primary interface between TANGO1 and cTAGE5, an interaction that is required for collagen export from endoplasmic reticulum exit sites (ERES). Application of the peptide inhibitors leads to reduced TANGO1 and cTAGE5 protein levels and a corresponding inhibition in the secretion of several ECM components, including collagens. Peptide inhibitor treatment in zebrafish results in altered tissue architecture and reduced granulation tissue formation during cutaneous wound healing. The inhibitors reduce secretion of several ECM proteins, including collagens, fibrillin and fibronectin in human dermal fibroblasts and in cells obtained from patients with a generalized fibrotic disease (scleroderma). Taken together, targeted interference of the TANGO1-cTAGE5 binding interface could enable therapeutic modulation of ERES function in ECM hypersecretion, during wound healing and fibrotic processes.
Subject(s)
Cicatrix , Collagen , Fibroblasts , Wound Healing , Zebrafish , Humans , Animals , Fibroblasts/metabolism , Fibroblasts/drug effects , Collagen/metabolism , Wound Healing/drug effects , Cicatrix/metabolism , Cicatrix/pathology , Cicatrix/drug therapy , Skin/metabolism , Skin/pathology , Skin/drug effects , Fibrosis , Peptides/pharmacology , Peptides/metabolism , Endoplasmic Reticulum/metabolism , Endoplasmic Reticulum/drug effects , Scleroderma, Systemic/metabolism , Scleroderma, Systemic/drug therapy , Scleroderma, Systemic/pathology , Extracellular Matrix/metabolism , Extracellular Matrix/drug effectsABSTRACT
Age-dependent changes in the transcription levels of 5-day-old Euglena gracilis cells, which showed positive gravitaxis, 6-day-old cells without gravitactic orientation, and older cells (9- and 11-day-old, which displayed a precise negative gravitaxis) were determined through microarray analysis. Hierarchical clustering of four independent cell cultures revealed pronounced similarities in transcription levels at the same culture age, which proves the reproducibility of the cultivation method. Employing the non-oriented cells from the 6-day-old culture as a reference, about 2779 transcripts were found to be differentially expressed. While positively gravitactic cells (5-day-old culture) showed only minor differences in gene expression compared to the 6-day reference, pronounced changes of mRNAs (mainly an increase) were found in older cells compared to the reference culture. Among others, genes coding for adenylyl cyclases, photosynthesis, and metabolic enzymes were identified to be differentially expressed. The investigated cells were grown in batch cultures, so variations in transcription levels most likely account for factors such as nutrient depletion in the medium and self-shading. Based on these findings, a particular transcript (e.g., transcript 19556) was downregulated using the RNA interference technique. Gravitaxis and phototaxis were impaired in the transformants, indicating the role of this transcript in signal transduction. Results of the experiment are discussed regarding the increasing importance of E. gracilis in biotechnology as a source of valuable products and the possible application of E. gracilis in life-support systems.
Subject(s)
Euglena gracilis , Euglena gracilis/genetics , Reproducibility of Results , Phototaxis , Photosynthesis , Signal TransductionABSTRACT
OBJECTIVES: Solitary fibrous tumours of the pleura (SFTP) are historically considered to be benign soft tissue neoplasms. However, a clinical relevant number of these neoplasms have malignant histological features. The objective of this study was to evaluate the percentage of SFTP presenting unfavourable clinical behaviour in order to predict negative long-term outcome. METHODS: A retrospective review of 74 patients treated at 4 hospitals between 1990 and 2013 was performed. The median follow-up was 10 years (range: 1-20 years). Risk of tumour recurrence and metastases (unfavourable clinical behaviour) with regard to histology using the Kaplan-Meier and Cox proportional hazards methods. RESULTS: The mean age was 61 years (SD 12.75 years). There were 31 male patients (58%) and 43 female patients (42%). Tumour size ranged from 1 to 30 cm (mean 9.09 cm; SD 6.22 cm). Complete resection (R0) was achieved by minimally invasive thoracoscopic resection in 29% and thoracotomy in 57%; 25% of SFTPs showed histological evidence of malignancy, according to England criteria. Recurrence occurred in 21% and 10% of patients had metastases; 83% of patients with metastases and 39% of patients with recurrence died within 5 years. The median recurrence-free survival for histologically benign SFTP was not reached, compared to 8 years for malignant SFTP. The five-year overall survival rate was 84%. Mitotic rate ≥1/10 HPF, high cellularity, nuclear atypia, Ki-67 level >5% and poorly circumscribed (sessile) growth pattern were associated with poor long-term outcome. CONCLUSIONS: Pathological differentiation of SFTP morphology into pedunculated, well circumscribed and poorly circumscribed (sessile) growth pattern is recommended. Due to the misleading classification into histologically benign and malignant, all unpedunculated SFTP should be classified as potentially aggressive. Lifelong follow-up is mandatory.
Subject(s)
Pleural Neoplasms , Solitary Fibrous Tumor, Pleural , Humans , Male , Female , Middle Aged , Pleura/pathology , Solitary Fibrous Tumor, Pleural/surgery , Solitary Fibrous Tumor, Pleural/pathology , Pleural Neoplasms/surgery , Retrospective Studies , Thoracotomy/methodsABSTRACT
BACKGROUND: Human iPSC-derived 3D-tissue-engineered-skeletal muscles (3D-TESMs) offer advanced technology for disease modelling. However, due to the inherent genetic heterogeneity among human individuals, it is often difficult to distinguish disease-related readouts from random variability. The generation of genetically matched isogenic controls using gene editing can reduce variability, but the generation of isogenic hiPSC-derived 3D-TESMs can take up to 6 months, thereby reducing throughput. METHODS: Here, by combining 3D-TESM and shRNA technologies, we developed a disease modelling strategy to induce distinct genetic deficiencies in a single hiPSC-derived myogenic progenitor cell line within 1 week. RESULTS: As proof of principle, we recapitulated disease-associated pathology of Duchenne muscular dystrophy and limb-girdle muscular dystrophy type 2A caused by loss of function of DMD and CAPN3, respectively. shRNA-mediated knock down of DMD or CAPN3 induced a loss of contractile function, disruption of tissue architecture, and disease-specific proteomes. Pathology in DMD-deficient 3D-TESMs was partially rescued by a candidate gene therapy treatment using micro-dystrophin, with similar efficacy compared to animal models. CONCLUSIONS: These results show that isogenic shRNA-based humanized 3D-TESM models provide a fast, cheap, and efficient tool to model muscular dystrophies and are useful for the preclinical evaluation of novel therapies.
Subject(s)
Muscular Dystrophies, Limb-Girdle , Muscular Dystrophy, Duchenne , Animals , Humans , Muscle, Skeletal/metabolism , Muscular Dystrophy, Duchenne/genetics , Muscular Dystrophy, Duchenne/therapy , Muscular Dystrophy, Duchenne/pathology , Muscular Dystrophies, Limb-Girdle/genetics , Muscular Dystrophies, Limb-Girdle/therapy , Muscular Dystrophies, Limb-Girdle/pathology , Muscle Contraction , RNA, Small InterferingABSTRACT
Cancer is defined as a group of diseases characterized by abnormal cell growth, expansion, and progression with metastasis. Various signaling pathways are involved in its development. Malignant tumors exhibit a high morbidity and mortality. Cancer research increased our knowledge about some of the underlying mechanisms, but to this day, our understanding of this disease is unclear. High throughput omics technology and bioinformatics were successful in detecting some of the unknown cancer mechanisms. However, novel groundbreaking research and ideas are necessary. A stay in orbit causes biochemical and molecular biological changes in human cancer cells which are first, and above all, due to microgravity (µg). The µg-environment provides conditions that are not reachable on Earth, which allow researchers to focus on signaling pathways controlling cell growth and metastasis. Cancer research in space already demonstrated how cancer cell-exposure to µg influenced several biological processes being involved in cancer. This novel approach has the potential to fight cancer and to develop future cancer strategies. Space research has been shown to impact biological processes in cancer cells like proliferation, apoptosis, cell survival, adhesion, migration, the cytoskeleton, the extracellular matrix, focal adhesion, and growth factors, among others. This concise review focuses on publications related to genetic, transcriptional, epigenetic, proteomic, and metabolomic studies on tumor cells exposed to real space conditions or to simulated µg using simulation devices. We discuss all omics studies investigating different tumor cell types from the brain and hematological system, sarcomas, as well as thyroid, prostate, breast, gynecologic, gastrointestinal, and lung cancers, in order to gain new and innovative ideas for understanding the basic biology of cancer.
Subject(s)
Lung Neoplasms , Sarcoma , Weightlessness , Humans , Male , Female , Proteomics , CytoskeletonABSTRACT
So far, the mechanisms that impede AAV transduction, especially in the human heart, are poorly understood, hampering the introduction of new, effective gene therapy strategies. Therefore, the aim of this study was to identify and overcome the main cellular barriers to successful transduction in the heart, using induced pluripotent stem cell (iPSC)-derived cardiomyocytes (iPSC-CMs), iPSC-derived cardiac fibroblasts (iPSC-CFs), and primary endothelial cells to model vector-host interactions. Through phosphoproteome analysis we established that casein kinase 2 (CK2) signaling is one of the most significantly affected pathways upon AAV exposure. Transient inhibition of CK2 activity substantially enhanced the transduction rate of AAV2, AAV6, and AAV9 in all tested cell types. In particular, CK2 inhibition improved the trafficking of AAVs through the cytoplasm, impaired DNA damage response through destabilization of MRE11, and altered the RNA processing pathways, which were also highly responsive to AAV transduction. Also, it augmented transgene expression in already transduced iPSC-CFs, which retain AAV genomes in a functional, but probably silent form. In summary, the present study provides new insights into the current understanding of the host-AAV vector interaction, identifying CK2 activity as a key barrier to efficient transduction and transgene expression, which may translate to improving the outcome of AAV-based therapies in the future.
Subject(s)
Casein Kinase II , Endothelial Cells , Humans , Transduction, Genetic , Casein Kinase II/genetics , Casein Kinase II/metabolism , Genetic Therapy , Transgenes , Dependovirus/genetics , Dependovirus/metabolism , Genetic Vectors/geneticsABSTRACT
AIMS: Duchenne muscular dystrophy (DMD)-associated cardiomyopathy is a serious life-threatening complication, the mechanisms of which have not been fully established, and therefore no effective treatment is currently available. The purpose of the study was to identify new molecular signatures of the cardiomyopathy development in DMD. METHODS AND RESULTS: For modelling of DMD-associated cardiomyopathy, we prepared three pairs of isogenic control and dystrophin-deficient human induced pluripotent stem cell (hiPSC) lines. Two isogenic hiPSC lines were obtained by CRISPR/Cas9-mediated deletion of DMD exon 50 in unaffected cells generated from healthy donor and then differentiated into cardiomyocytes (hiPSC-CM). The latter were subjected to global transcriptomic and proteomic analyses followed by more in-depth investigation of selected pathway and pharmacological modulation of observed defects. Proteomic analysis indicated a decrease in the level of mitoNEET protein in dystrophin-deficient hiPSC-CM, suggesting alteration in iron metabolism. Further experiments demonstrated increased labile iron pool both in the cytoplasm and mitochondria, a decrease in ferroportin level and an increase in both ferritin and transferrin receptor in DMD hiPSC-CM. Importantly, CRISPR/Cas9-mediated correction of the mutation in the patient-derived hiPSC reversed the observed changes in iron metabolism and restored normal iron levels in cardiomyocytes. Moreover, treatment of DMD hiPSC-CM with deferoxamine (DFO, iron chelator) or pioglitazone (mitoNEET stabilizing compound) decreased the level of reactive oxygen species in DMD hiPSC-CM. CONCLUSION: To our knowledge, this study demonstrated for the first time impaired iron metabolism in human DMD cardiomyocytes, and potential reversal of this effect by correction of DMD mutation or pharmacological treatment. This implies that iron overload-regulating compounds may serve as novel therapeutic agents in DMD-associated cardiomyopathy.
Subject(s)
Cardiomyopathies , Induced Pluripotent Stem Cells , Muscular Dystrophy, Duchenne , Humans , Cardiomyopathies/metabolism , CRISPR-Cas Systems , Dystrophin , Gene Editing/methods , Homeostasis , Induced Pluripotent Stem Cells/metabolism , Iron/metabolism , Muscular Dystrophy, Duchenne/drug therapy , Muscular Dystrophy, Duchenne/genetics , Myocytes, Cardiac/metabolism , ProteomicsABSTRACT
Digested dietary fats are taken up by enterocytes where they are assembled into pre-chylomicrons in the endoplasmic reticulum followed by transport to the Golgi for maturation and subsequent secretion to the circulation1. The role of mitochondria in dietary lipid processing is unclear. Here we show that mitochondrial dysfunction in enterocytes inhibits chylomicron production and the transport of dietary lipids to peripheral organs. Mice with specific ablation of the mitochondrial aspartyl-tRNA synthetase DARS2 (ref. 2), the respiratory chain subunit SDHA3 or the assembly factor COX10 (ref. 4) in intestinal epithelial cells showed accumulation of large lipid droplets (LDs) in enterocytes of the proximal small intestine and failed to thrive. Feeding a fat-free diet suppressed the build-up of LDs in DARS2-deficient enterocytes, which shows that the accumulating lipids derive mostly from digested fat. Furthermore, metabolic tracing studies revealed an impaired transport of dietary lipids to peripheral organs in mice lacking DARS2 in intestinal epithelial cells. DARS2 deficiency caused a distinct lack of mature chylomicrons concomitant with a progressive dispersal of the Golgi apparatus in proximal enterocytes. This finding suggests that mitochondrial dysfunction results in impaired trafficking of chylomicrons from the endoplasmic reticulum to the Golgi, which in turn leads to storage of dietary lipids in large cytoplasmic LDs. Taken together, these results reveal a role for mitochondria in dietary lipid transport in enterocytes, which might be relevant for understanding the intestinal defects observed in patients with mitochondrial disorders5.
Subject(s)
Dietary Fats , Enterocytes , Lipid Metabolism , Mitochondria , Animals , Mice , Aspartate-tRNA Ligase/metabolism , Chylomicrons/metabolism , Dietary Fats/metabolism , Electron Transport Complex II/metabolism , Endoplasmic Reticulum/metabolism , Enterocytes/metabolism , Enterocytes/pathology , Epithelial Cells/metabolism , Golgi Apparatus/metabolism , Intestines , Lipid Droplets/metabolism , Mitochondria/metabolism , Mitochondria/pathologyABSTRACT
Lung carcinoma is still one of the most common forms of cancer in both sexes in Germany and the most common cause of cancer-related death. However, we are in the midst of a revolution in the treatment of lung cancer. Above all, the new immune and target therapies as well as the possible combinations of the individual therapy components have expanded the spectrum of drug therapy for lung cancer in recent years.Great progress has also been made in other fields. Be it in the context of early detection, where low-dose CT screening will soon be established for patients at risk, or in the context of molecular diagnostics with the identification of a large number of targets that can be specifically treated with tyrosine kinase inhibitors.Lung carcinoma therapy is moving on the direction of personalized tumor therapy that is specially tailored to each individual patient. Many studies on new drugs or new markers are ongoing. It is all the more important to keep track of the large number of newly approved substances and possibilities. Every patient should therefore, if possible, be treated in a lung cancer center with a therapy decision made in an interdisciplinary tumor board.The updated S3 guideline "Prevention, diagnostics, therapy and aftercare of lung cancer" was published in November 2022 as part of the guideline program oncology of the Working Group of Scientific Medical Societies, the German Cancer Society and the German Cancer Aid, financed by the German Cancer Aid. The full guideline is available at https://www.leitlinienprogramm-onkologie.de/leitlinien/lungenkrebs/ or in the guideline program app.There are already new treatment options that have not yet been taken into account in the recommendations. In order to take account of the dynamics of medical progress, the S3 guideline is going to be continued as a living guideline with annually updates.
Subject(s)
Carcinoma , Lung Neoplasms , Humans , Early Detection of Cancer , Lung Neoplasms/diagnosis , Lung Neoplasms/therapy , Medical Oncology , LungABSTRACT
In preparing space and microgravity experiments, the utilization of ground-based facilities is common for initial experiments and feasibility studies. One approach to simulating microgravity conditions on Earth is to employ a random positioning machine (RPM) as a rotary bioreactor. Combined with a suitable low-mass model system, such as cell cultures, these devices simulating microgravity have been shown to produce results similar to those obtained in a space experiment under real microgravity conditions. One of these effects observed under real and simulated microgravity is the formation of spheroids from 2D adherent cancer cell cultures. Since real microgravity cannot be generated in a laboratory on Earth, we aimed to determine which forces lead to the detachment of individual FTC-133 thyroid cancer cells and the formation of tumor spheroids during culture with exposure to random positioning modes. To this end, we subdivided the RPM motion into different static and dynamic orientations of cell culture flasks. We focused on the molecular activation of the mechanosignaling pathways previously associated with spheroid formation in microgravity. Our results suggest that RPM-induced spheroid formation is a two-step process. First, the cells need to be detached, induced by the cell culture flask's rotation and the subsequent fluid flow, as well as the presence of air bubbles. Once the cells are detached and in suspension, random positioning prevents sedimentation, allowing 3D aggregates to form. In a comparative shear stress experiment using defined fluid flow paradigms, transcriptional responses were triggered comparable to exposure of FTC-133 cells to the RPM. In summary, the RPM serves as a simulator of microgravity by randomizing the impact of Earth's gravity vector especially for suspension (i.e., detached) cells. Simultaneously, it simulates physiological shear forces on the adherent cell layer. The RPM thus offers a unique combination of environmental conditions for in vitro cancer research.
Subject(s)
Thyroid Neoplasms , Weightlessness , Humans , Cell Culture Techniques/methods , Thyroid Neoplasms/metabolismABSTRACT
The current S3 Lung Cancer Guidelines are edited with fundamental changes to the previous edition based on the dynamic influx of information to this field:The recommendations include de novo a mandatory case presentation for all patients with lung cancer in a multidisciplinary tumor board before initiation of treatment, furthermore CT-Screening for asymptomatic patients at risk (after federal approval), recommendations for incidental lung nodule management , molecular testing of all NSCLC independent of subtypes, EGFR-mutations in resectable early stage lung cancer in relapsed or recurrent disease, adjuvant TKI-therapy in the presence of common EGFR-mutations, adjuvant consolidation treatment with checkpoint inhibitors in resected lung cancer with PD-L1 ≥â50%, obligatory evaluation of PD-L1-status, consolidation treatment with checkpoint inhibition after radiochemotherapy in patients with PD-L1-pos. tumor, adjuvant consolidation treatment with checkpoint inhibition in patients withPD-L1 ≥â50% stage IIIA and treatment options in PD-L1 ≥â50% tumors independent of PD-L1status and targeted therapy and treatment option immune chemotherapy in first line SCLC patients.Based on the current dynamic status of information in this field and the turnaround time required to implement new options, a transformation to a "living guideline" was proposed.
Subject(s)
Carcinoma, Non-Small-Cell Lung , Lung Neoplasms , Humans , Lung Neoplasms/diagnosis , Lung Neoplasms/prevention & control , B7-H1 Antigen/genetics , B7-H1 Antigen/therapeutic use , Follow-Up Studies , ErbB Receptors/genetics , Carcinoma, Non-Small-Cell Lung/pathologyABSTRACT
Skeletal muscle research is transitioning toward 3D tissue engineered in vitro models reproducing muscle's native architecture and supporting measurement of functionality. Human induced pluripotent stem cells (hiPSCs) offer high yields of cells for differentiation. It has been difficult to differentiate high-quality, pure 3D muscle tissues from hiPSCs that show contractile properties comparable to primary myoblast-derived tissues. Here, we present a transgene-free method for the generation of purified, expandable myogenic progenitors (MPs) from hiPSCs grown under feeder-free conditions. We defined a protocol with optimal hydrogel and medium conditions that allowed production of highly contractile 3D tissue engineered skeletal muscles with forces similar to primary myoblast-derived tissues. Gene expression and proteomic analysis between hiPSC-derived and primary myoblast-derived 3D tissues revealed a similar expression profile of proteins involved in myogenic differentiation and sarcomere function. The protocol should be generally applicable for the study of personalized human skeletal muscle tissue in health and disease.
Subject(s)
Induced Pluripotent Stem Cells , Humans , Proteomics , Cells, Cultured , Muscle, Skeletal , Tissue Engineering/methods , Myoblasts/metabolism , Cell Differentiation/geneticsABSTRACT
Collagen XII, belonging to the fibril-associated collagens, is a homotrimeric secreted extracellular matrix (ECM) protein encoded by the COL12A1 gene. Mutations in the human COL12A1 gene cause an Ehlers-Danlos/myopathy overlap syndrome leading to skeletal abnormalities and muscle weakness. Here, we studied the role of collagen XII in joint pathophysiology by analyzing collagen XII deficient mice and human patients. We found that collagen XII is widely expressed across multiple connective tissue of the developing joint. Lack of collagen XII in mice destabilizes tendons and the femoral trochlear groove to induce patellar subluxation in the patellofemoral joint. These changes are associated with an ECM damage response in tendon and secondary quadriceps muscle degeneration. Moreover, patellar subluxation was also identified as a clinical feature of human patients with collagen XII deficiency. The results provide an explanation for joint hyperlaxity in mice and human patients with collagen XII deficiency.
ABSTRACT
The lunar dust problem was first formulated in 1969 with NASA's first successful mission to land a human being on the surface of the Moon. Subsequent Apollo missions failed to keep the dust at bay, so exposure to the dust was unavoidable. In 1972, Harrison Schmitt suffered a brief sneezing attack, red eyes, an itchy throat, and congested sinuses in response to lunar dust. Some additional Apollo astronauts also reported allergy-like symptoms after tracking dust into the lunar module. Immediately following the Apollo missions, research into the toxic effects of lunar dust on the respiratory system gained a lot of interest. Moreover, researchers believed other organ systems might be at risk, including the skin and cornea. Secondary effects could translocate to the cardiovascular system, the immune system, and the brain. With current intentions to return humans to the moon and establish a semi-permanent presence on or near the moon's surface, integrated, end-to-end dust mitigation strategies are needed to enable sustainable lunar presence and architecture. The characteristics and formation of Martian dust are different from lunar dust, but advances in the research of lunar dust toxicity, mitigation, and protection strategies can prove strategic for future operations on Mars.
ABSTRACT
OBJECTIVE: Norepinephrine stimulates the adipose tissue thermogenic program through a ß-adrenergic receptor (ßAR)-cyclic adenosine monophosphate (cAMP)-protein kinase A (PKA) signaling cascade. We discovered that a noncanonical activation of the mechanistic target of rapamycin complex 1 (mTORC1) by PKA is required for the ßAR-stimulation of adipose tissue browning. However, the downstream events triggered by PKA-phosphorylated mTORC1 activation that drive this thermogenic response are not well understood. METHODS: We used a proteomic approach of Stable Isotope Labeling by/with Amino acids in Cell culture (SILAC) to characterize the global protein phosphorylation profile in brown adipocytes treated with the ßAR agonist. We identified salt-inducible kinase 3 (SIK3) as a candidate mTORC1 substrate and further tested the effect of SIK3 deficiency or SIK inhibition on the thermogenic gene expression program in brown adipocytes and in mouse adipose tissue. RESULTS: SIK3 interacts with RAPTOR, the defining component of the mTORC1 complex, and is phosphorylated at Ser884 in a rapamycin-sensitive manner. Pharmacological SIK inhibition by a pan-SIK inhibitor (HG-9-91-01) in brown adipocytes increases basal Ucp1 gene expression and restores its expression upon blockade of either mTORC1 or PKA. Short-hairpin RNA (shRNA) knockdown of Sik3 augments, while overexpression of SIK3 suppresses, Ucp1 gene expression in brown adipocytes. The regulatory PKA phosphorylation domain of SIK3 is essential for its inhibition. CRISPR-mediated Sik3 deletion in brown adipocytes increases type IIa histone deacetylase (HDAC) activity and enhances the expression of genes involved in thermogenesis such as Ucp1, Pgc1α, and mitochondrial OXPHOS complex protein. We further show that HDAC4 interacts with PGC1α after ßAR stimulation and reduces lysine acetylation in PGC1α. Finally, a SIK inhibitor well-tolerated in vivo (YKL-05-099) can stimulate the expression of thermogenesis-related genes and browning of mouse subcutaneous adipose tissue. CONCLUSIONS: Taken together, our data reveal that SIK3, with the possible contribution of other SIKs, functions as a phosphorylation switch for ß-adrenergic activation to drive the adipose tissue thermogenic program and indicates that more work to understand the role of the SIKs is warranted. Our findings also suggest that maneuvers targeting SIKs could be beneficial for obesity and related cardiometabolic disease.