Characterization of obesity-related diseases and inflammation using single cell immunophenotyping in two different diet-induced obesity models.

BACKGROUND: Obesity is a growing problem worldwide and a major risk factor for many chronic diseases. The accumulation of adipose tissue leads to the release of significant amounts of pro-inflammatory cytokines and adipokines, resulting in a low-grade systemic inflammation. However, the mechanisms behind the development of obesity-related diseases are not fully understood. Therefore, our study aimed to investigate the pathological changes and inflammatory processes at systemic level and in individual organs in two different diet-induced mouse obesity models. METHODS: Male C57BL6/J mice were fed by high-fat diet (HFD), high-fat/high-fructose diet (HFD + FR) or normal chow for 21 weeks starting at 3 months of age (n = 15 animals/group). Insulin resistance was tested by oral glucose tolerance test. Pathological changes were investigated on hematoxylin-eosin-stained liver and brown adipose tissue sections. The gene expression levels of adipokines and cytokines were analyzed by qPCR in adipose tissues, whereas serum protein concentrations were determined by multiplex immunoassays. Immunophenotyping of isolated blood, bone marrow and spleen cells was performed by single-cell mass cytometry. RESULTS: Weight gain, glucose intolerance and hepatic steatosis were more severe in the HFD + FR group than in the control and HFD groups. This was accompanied by a higher level of systemic inflammation, as indicated by increased expression of pro-inflammatory genes in visceral white adipose tissue and by a higher serum TNFα level. In addition, immunophenotyping revealed the increase of the surface expressions of CD44 and CD69 on various cell types, such as CD8+ and CD4 + T-cells, B-cells and macrophages, in animals with obesity. CONCLUSIONS: The combination of HFD with fructose supplementation promotes more properly the symptoms of metabolic syndrome. Therefore, the combined high-fat/high-fructose nutrition can be a more suitable model of the Western diet. However, despite these differences, both models showed immunophenotypic changes that may be associated with increased risk of obesity-related cancer.

The Use of the Symani Surgical System® in Emergency Hand Trauma Care.

Background: The use of robotic systems for microsurgery has gained popularity in recent years. Despite its drawbacks, such as increased learning time and lack of haptic feedback, robot-assisted microsurgery is beneficial for emergency care due to its reduced risk of tremor and fatigue. The Symani Surgical System® is 1 example of this advanced technology. The device offers a range of possibilities in the field of microsurgery by combining precision and dexterity, revolutionizing microsurgical procedures. This article explores the applications of the Symani in microsurgical procedures in emergency hand trauma care, highlighting its advantages and limitations. Material and Methods: We present the results of 62 anastomoses of blood vessels under .8 mm diameter after hand trauma. 31 anastomoses were conducted using the Symani Surgical System®, and the other 31 were done as a control group in hand-sewn technique. Study Sample: The patient characteristics, including sex, age, and risk factors, were matched. Results: We found no significant differences in the anastomosis surgery length when performed with the Symani (arterial 17.3 ± 1.9 min; venous 11.5 ± 1.3 min) vs the hand-sewn technique (arterial 16.1 ± 1.4 min; venous 10.2 ± 1.8 min). Additionally, the learning curve consistently decreased over time, with the 10th surgery taking 30% (arterial) less time. Conclusion: Our study indicates that robot-assisted microsurgery can help surgeons maintain a relaxed and focused state while producing results comparable to hand-sutured procedures in emergency care.

Organization of Hannover Skin Bank: Sterile culture and procurement protocols for viable cryopreserved allogeneic skin grafts of living donors.

Preserved allogeneic donor skin still represents one of the gold standard therapies in temporary wound coverage in severely burned patients or chronic wounds. Allogeneic skin grafts are currently commercially available as cryo- or glycerol-preserved allografts through skin tissue banks all over the world. Most of the skin tissue banks rely on human cadaveric skin donations. Due to the chronic shortage of human allogeneic transplants, such as skin, and increasing costs in the procurement of allografts from other skin tissue banks, Hannover Medical School has been building up its own skin tissue bank based on allogeneic skin grafts from living donors who underwent surgical treatment (i.e., body-contouring procedures, such as abdominioplasties). This article presents procedures and protocols for the procurement and processing of allogeneic skin grafts according to national legislation and European regulations and guidelines. Beside protocols, initial microbiological data regarding the sterility of the harvested grafts are presented. The results currently form the basis for further investigations as well as clinical applications. In summary, a microbiological testing and acceptance procedure is presented that ensures adequate patient safety and skin viability.

Netrin G1 Is a Novel Target Antigen in Primary Membranous Nephropathy.

BACKGROUND: Primary membranous nephropathy (MN) is caused by circulating autoantibodies binding to antigens on the podocyte surface. PLA2R1 is the main target antigen in 70%-80% of cases, but the pathogenesis is unresolved in 10%-15% of patients. METHODS: We used native western blotting to identify IgG4 autoantibodies, which bind an antigen endogenously expressed on podocyte membranes, in the serum of the index patient with MN. These IgG4 autoantibodies were used to immunoprecipitate the target antigen, and mass spectrometry was used to identify Netrin G1 (NTNG1). Using native western blot and ELISA, NTNG1 autoantibodies were analyzed in cohorts of 888 patients with MN or other glomerular diseases. RESULTS: NTNG1 was identified as a novel target antigen in MN. It is a membrane protein expressed in healthy podocytes. Immunohistochemistry confirmed granular NTNG1 positivity in subepithelial glomerular immune deposits. In prospective and retrospective MN cohorts, we identified three patients with NTNG1-associated MN who showed IgG4-dominant circulating NTNG1 autoantibodies, enhanced NTNG1 expression in the kidney, and glomerular IgG4 deposits. No NTNG1 autoantibodies were identified in 561 PLA2R1 autoantibodies-positive patients, 27 THSD7A autoantibodies-positive patients, and 77 patients with other glomerular diseases. In two patients with available follow-up of 2 and 4 years, both NTNG1 autoantibodies and proteinuria persisted. CONCLUSIONS: NTNG1 expands the repertoire of target antigens in patients with MN. The clinical role of NTNG1 autoantibodies remains to be defined.

Remodeling of Liver and Plasma Lipidomes in Mice Lacking Cyclophilin D.

In recent years, several studies aimed to investigate the metabolic effects of non-functioning or absent cyclophilin D (CypD), a crucial regulatory component of mitochondrial permeability transition pores. It has been reported that the lack of CypD affects glucose and lipid metabolism. However, the findings are controversial regarding the metabolic pathways involved, and most reports describe the effect of a high-fat diet on metabolism. We performed a lipidomic analysis of plasma and liver samples of CypD-/- and wild-type (WT) mice to reveal the lipid-specific alterations resulting from the absence of CypD. In the CypD-/- mice compared to the WT animals, we found a significant change in 52% and 47% of the measured 225 and 201 lipid species in liver and plasma samples, respectively. The higher total lipid content detected in these tissues was not accompanied by abdominal fat accumulation assessed by nuclear magnetic resonance imaging. We also documented characteristic changes in the lipid composition of the liver and plasma as a result of CypD ablation with the relative increase in polyunsaturated membrane lipid species. In addition, we did not observe remarkable differences in the lipid distribution of hepatocytes using histochemistry, but we found characteristic changes in the hepatocyte ultrastructure in CypD-/- animals using electron microscopy. Our results highlight the possible long-term effects of CypD inhibition as a novel therapeutic consideration for various diseases.

The Germline Marker Piwi Expressed in the Skin Layer of the Polychaete Perinereis wilsoni After Injury.

Nereidid polychaete Perinereis wilsoni is a homonomous metameric worm with a complete septum between each segment. Each segment has germ cells localized in the distal area of the parapodia. Perinereis wilsoni is also known to have high abilities of tissue regeneration; however, it is still unclear whether germ cells can regenerate in the healing tissue. To address this, we surgically operated the parapodia of an adult worm to remove germ cells from the segments and observed the germ cell regeneration using the germ cell genetic marker Pw-piwi. At day 20 post-surgical operation of the parapodia in one side of the segment, we found that Pw-piwi was expressed in the regenerating parapodia. We surgically operated the parapodia on both sides of the segment to remove the germ cells completely and it gave a similar result. However, before the expression of this gene marker in the regenerating parapodia, we observed that Pw-piwi was expressed in cells in the skin layer of the worm just after surgical operations. These Pw-piwi-positive cells were not observed in the un-operated worm. Our observations showed that germ cells of Perinereis wilsoni can regenerate even after the complete removal of germ cells from the defined habitat. The Pw-piwipositive cells that appeared in the skin layer after the disappearance of germ cells may be involved in the regeneration of new germ cells.

Lipids and Trehalose Actively Cooperate in Heat Stress Management of Schizosaccharomyces pombe.

Homeostatic maintenance of the physicochemical properties of cellular membranes is essential for life. In yeast, trehalose accumulation and lipid remodeling enable rapid adaptation to perturbations, but their crosstalk was not investigated. Here we report about the first in-depth, mass spectrometry-based lipidomic analysis on heat-stressed Schizosaccharomyces pombe mutants which are unable to synthesize (tps1Δ) or degrade (ntp1Δ) trehalose. Our experiments provide data about the role of trehalose as a membrane protectant in heat stress. We show that under conditions of trehalose deficiency, heat stress induced a comprehensive, distinctively high-degree lipidome reshaping in which structural, signaling and storage lipids acted in concert. In the absence of trehalose, membrane lipid remodeling was more pronounced and increased with increasing stress dose. It could be characterized by decreasing unsaturation and increasing acyl chain length, and required de novo synthesis of stearic acid (18:0) and very long-chain fatty acids to serve membrane rigidification. In addition, we detected enhanced and sustained signaling lipid generation to ensure transient cell cycle arrest as well as more intense triglyceride synthesis to accommodate membrane lipid-derived oleic acid (18:1) and newly synthesized but unused fatty acids. We also demonstrate that these changes were able to partially substitute for the missing role of trehalose and conferred measurable stress tolerance to fission yeast cells.

Male and Female Animals Respond Differently to High-Fat Diet and Regular Exercise Training in a Mouse Model of Hyperlipidemia.

Inappropriate nutrition and a sedentary lifestyle can lead to obesity, one of the most common risk factors for several chronic diseases. Although regular physical exercise is an efficient approach to improve cardiometabolic health, the exact cellular processes are still not fully understood. We aimed to analyze the morphological, gene expression, and lipidomic patterns in the liver and adipose tissues in response to regular exercise. Healthy (wild type on a normal diet) and hyperlipidemic, high-fat diet-fed (HFD-fed) apolipoprotein B-100 (APOB-100)-overexpressing mice were trained by treadmill running for 7 months. The serum concentrations of triglyceride and tumor necrosis factor α (TNFα), as well as the level of lipid accumulation in the liver, were significantly higher in HFD-fed APOB-100 males compared to females. However, regular exercise almost completely abolished lipid accumulation in the liver of hyperlipidemic animals. The expression level of the thermogenesis marker, uncoupling protein-1 (Ucp1), was significantly higher in the subcutaneous white adipose tissue of healthy females, as well as in the brown adipose tissue of HFD-fed APOB-100 females, compared to males. Lipidomic analyses revealed that hyperlipidemia essentially remodeled the lipidome of brown adipose tissue, affecting both the membrane and storage lipid fractions, which was partially restored by exercise in both sexes. Our results revealed more severe metabolic disturbances in HFD-fed APOB-100 males compared to females. However, exercise efficiently reduced the body weight, serum triglyceride levels, expression of pro-inflammatory factors, and hepatic lipid accumulation in our model.

Germ Cell Development in Male Perinereis nuntia and Gamete Spawning Mechanisms in Males and Females.

Perinereis nuntia is a fully segmented worm with complete intersegmental septa. A previous study of females revealed that germ cells of this animal originate in the tail end segment, called the pygidium. Germ cells were duplicated in the pygidium, transferred to a newly generated segment, and then settled in the parapodia. Within each segment, the settled germ cells proliferated in the parapodia and then migrated into a body cavity area to begin meiotic development. Currently, there is not much information about differences between male and female germ cell development. Therefore, we conducted monthly in situ hybridization analyses using the germ cell marker Pn-piwi and histological examinations. Germ cells detected by Pn-piwi initially settled in the distal areas of the parapodia on both sides of each segment, then formed a large germ cell cluster in each parapodium, and finally, small germ cell clusters were formed by the separation of the large clusters. The small clusters migrated to the deeper body cavity area during growth by segment addition. Until the female germ cells began vitellogenesis, the sex of germ cells could not be identified by morphological observation. Thus, male and female P. nuntia may have the same mechanism of germ cell provision to all segments. At the time of spawning, sperm were released from nephridiopores at the 2nd through 15th segments from the pygidium, while eggs were released through ruptures in the skin of 2-3 segments between the 10th and 30th segments from the tail.

[Reduction of needlestick injuries by 48 % in 1 year : Effects of improvement of the safety concept according to the European Union Council directive 2010/32/EU at a large regional hospital]. / Reduktion von Nadelstichverletzungen um 48 % in einem Jahr : Auswirkungen einer Verbesserung des Sicherheitskonzeptes nach der EU-Direktive 2010/32/EU an einem großen regionalen Krankenhaus.

BACKGROUND: Needlestick injuries (NSI) are potentially infectious injuries from sharp or pointed medical instruments and through contact with blood on mucous membranes or nonintact skin. Although the European Union (EU) Council directive 2010/32/EU on the prevention of NSI was implemented in EU countries in 2013, information on the effectiveness of the measures is limited. OBJECTIVE: The aim of this study was to evaluate the effectiveness of a safety concept according to the EU Council Directive 2010/32/EU on prevention of NSI. MATERIAL AND METHODS: In 2016 the NSI safety concept at a large regional hospital was improved according to 2010/32/EU, specifically by an update of blood screening profiles and standard operating procedures (SOP), better dissemination of information to employees and complete conversion to safety cannulas and scalpels. The medical records of all NSIs from 2015-2017 were retrospectively anonymized and evaluated and a cost analysis was performed. RESULTS: The number of NSIs in 2017 was significantly reduced by 48.4% as compared to 2016 and NSIs with scalpels were completely prevented. The proportion of employees with NSIs who were adequately immunized against hepatitis B was significantly increased to 84.1% in 2017. Furthermore, identification of the index patient was significantly increased to 82.5% in 2017. The cost of avoiding NSIs increased by a total of 24.1% in 2017 as compared to 2015 before introduction of the safety concept. CONCLUSION: Implementation of the EU Council directive 2010/32/EU, resulted in an almost 50% reduction in NSIs over 1 year, including the complete prevention of NSIs due to scalpels. In addition, the anamnestic presence of immunization against hepatitis B and index patient identification were significantly increased.

Epidemiologic and Molecular Investigation of a MRSA Outbreak Caused by a Contaminated Bathtub for Carbon Dioxide Hydrotherapy and Review of the Literature.

METHODS: We conducted an outbreak investigation and performed a molecular typing of the outbreak strains with pulsed-field gel electrophoresis (PFGE). In addition, we reviewed PubMed and the Outbreak Database for MRSA outbreaks related to hydrotherapy or other bathing activities. RESULTS: Four patients acquired nosocomial MRSA during the 4-week outbreak period. Environmental sampling revealed the presence of MRSA in the bathtub used for hydrotherapy. The environmental and the patients' isolates showed an indistinguishable restriction pattern in the PFGE. Subsequent discontinuation of bathing stopped the outbreak. The literature search found 9 MRSA outbreak reports related to bathing activities or hydrotherapy. CONCLUSION: The epidemiologic outbreak investigation together with the molecular findings suggests monoclonal spread of MRSA due to surface contamination of the bathtub. After enhancing the disinfection and cleaning process accompanied by staff training with respect to hand hygiene, no further cases occurred. Standardized and best practice cleaning and disinfection protocols are crucial, especially in critical facilities such as hydrotherapy units. Regular environmental sampling is helpful to monitor these processes and to detect potential contamination.

Reconstructed evolution of insulin receptors in insects reveals duplications in early insects and cockroaches.

Social insects show an extreme degree of phenotypic plasticity. In highly eusocial species, this manifests in the generation of distinct castes with extreme differences in both morphology and life span. The molecular basis of these differences is highly entangled and not fully understood, but several recent studies demonstrated that insulin/insulin-like growth factor signaling (IIS) is one of the key pathways. Here, we investigate the molecular evolution of insect insulin receptors (InRs), which are membrane-bound dimers that enable IIS by relaying extracellular signals to intracellular signaling cascades. Classic models of invertebrate IIS include only one InR gene, but some recent studies on less commonly studied insects have found two InRs, which act in an antagonistic manner to facilitate polyphenism in at least one documented case. We search 22 arthropod genomes and identify several InR copies and their evolutionary origin that were lacking from previous annotations. Phylogenetic analysis shows that the two insect InR genes date back at least 400 million years to a common ancestor of winged insects. Most notably, we also identified the evolutionary origin of a third InR copy that is unique to the clade of Blattodea, just before therein the eusocial termites evolved. One of the InR paralogs consistently shows caste-biased expression in all three termites, which strongly suggests a role in caste differentiation. These results have important ramifications for past and future InR inhibition/InR knockdown experiments in insects and they provide a set of key genes regulating life span and morphology in termite castes.

Nanotubes connecting B lymphocytes: High impact of differentiation-dependent lipid composition on their growth and mechanics.

Nanotubes (NTs) are thin, long membranous structures forming novel, yet poorly known communication pathways between various cell types. Key mechanisms controlling their growth still remained poorly understood. Since NT-forming capacity of immature and mature B cells was found largely different, we investigated how lipid composition and molecular order of the membrane affect NT-formation. Screening B cell lines with various differentiation stages revealed that NT-growth linearly correlates with membrane ganglioside levels, while it shows maximum as a function of cholesterol level. NT-growth of B lymphocytes is promoted by raftophilic phosphatidylcholine and sphingomyelin species, various glycosphingolipids, and docosahexaenoic acid-containing inner leaflet lipids, through supporting membrane curvature, as demonstrated by comparative lipidomic analysis of mature versus immature B cell membranes. Targeted modification of membrane cholesterol and sphingolipid levels altered NT-forming capacity confirming these findings, and also highlighted that the actual lipid raft number may control NT-growth via defining the number of membrane-F-actin coupling sites. Atomic force microscopic mechano-manipulation experiments further proved that mechanical properties (elasticity or bending stiffness) of B cell NTs also depend on the actual membrane lipid composition. Data presented here highlight importance of the lipid side in controlling intercellular, nanotubular, regulatory communications in the immune system.

Oxidized Phospholipids Inhibit the Formation of Cholesterol-Dependent Plasma Membrane Nanoplatforms.

We previously developed a single-molecule microscopy method termed TOCCSL (thinning out clusters while conserving stoichiometry of labeling), which allows for direct imaging of stable nanoscopic platforms with raft-like properties diffusing in the plasma membrane. As a consensus raft marker, we chose monomeric GFP linked via a glycosylphosphatidylinositol (GPI) anchor to the cell membrane (mGFP-GPI). With this probe, we previously observed cholesterol-dependent homo-association to nanoplatforms diffusing in the plasma membrane of live CHO cells. Here, we report the release of this homo-association upon addition of 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine (POVPC) or 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphocholine, two oxidized phospholipids (oxPLs) that are typically present in oxidatively modified low-density lipoprotein. We found a dose-response relationship for mGFP-GPI nanoplatform disintegration upon addition of POVPC, correlating with the signal of the apoptosis marker Annexin V-Cy3. Similar concentrations of lysolipid showed no effect, indicating that the observed phenomena were not linked to properties of the lipid bilayer itself. Inhibition of acid sphingomyelinase by NB-19 before addition of POVPC completely abolished nanoplatform disintegration by oxPLs. In conclusion, we were able to determine how oxidized lipid species disrupt mGFP-GPI nanoplatforms in the plasma membrane. Our results favor an indirect mechanism involving acid sphingomyelinase activity rather than a direct interaction of oxPLs with nanoplatform constituents.

Lipidomic analysis reveals a radiosensitizing role of gamma-linolenic acid in glioma cells.

Previous studies have demonstrated that gamma-linolenic acid (GLA) is effective against glioma cells under both in vitro and in vivo conditions. In the present study we determined how GLA alone or in combination with irradiation alters the fatty acid (FA) and lipid profiles, the lipid droplet (LD) content, the lipid biosynthetic gene expression and the apoptosis of glioma cells. In GLA-treated cells direct correlations were found between the levels of various FAs and the expression of the corresponding FA biosynthetic genes. The total levels of saturated and monosaturated FAs decreased in concert with the down-regulation of FASN and SCD1 gene expression. Similarly, decreased FADS1 gene expression was paralleled by lowered arachidonic acid (20:4 n-6) and eicosapentaenoic acid (20:5 n-3) contents, while the down-regulation of FADS2 expression was accompanied by a diminished docosahexaenoic acid (22:6 n-3) content. Detailed mass spectrometric analyses revealed that individual treatments gave rise to distinct lipidomic fingerprints. Following uptake, GLA was subjected to elongation, resulting in dihomo-gamma-linolenic acid (20:3 n-6, DGLA), which was used for the synthesis of the LD constituent triacylglycerols and cholesteryl esters. Accordingly, an increased number of LDs were observed in response to GLA administration after irradiation. GLA increased the radioresponsiveness of U87 MG cells, as demonstrated by an increase in the number of apoptotic cells determined by FACS analysis. In conclusion, treatment with GLA increased the apoptosis of irradiated glioma cells, and GLA might therefore increase the therapeutic efficacy of irradiation in the treatment of gliomas.

7DHC-induced changes of Kv1.3 operation contributes to modified T cell function in Smith-Lemli-Opitz syndrome.

In vitro manipulation of membrane sterol level affects the regulation of ion channels and consequently certain cellular functions; however, a comprehensive study that confirms the pathophysiological significance of these results is missing. The malfunction of 7-dehydrocholesterol (7DHC) reductase in Smith-Lemli-Opitz syndrome (SLOS) leads to the elevation of the 7-dehydrocholesterol level in the plasma membrane. T lymphocytes were isolated from SLOS patients to assess the effect of the in vivo altered membrane sterol composition on the operation of the voltage-gated Kv1.3 channel and the ion channel-dependent mitogenic responses. We found that the kinetic and equilibrium parameters of Kv1.3 activation changed in SLOS cells. Identical changes in Kv1.3 operation were observed when control/healthy T cells were loaded with 7DHC. Removal of the putative sterol binding sites on Kv1.3 resulted in a phenotype that was not influenced by the elevation in membrane sterol level. Functional assays exhibited impaired activation and proliferation rate of T cells probably partially due to the modified Kv1.3 operation. We concluded that the altered membrane sterol composition hindered the operation of Kv1.3 as well as the ion channel-controlled T cell functions.

Evaporative Water Loss in Superficial to Full Thickness Burns.

INTRODUCTION: Increased evaporative water loss (EWL) in burn patients leads to dehydration and hypothermia. Early clinical studies performed with outdated hygrometers suggested a 17 to 75 times increased EWL in burns with contradicting results for the different burn depths.Our study proposals were: (1) obtain reliable data of the EWL of all burn depths, (2) compare these results with findings from earlier studies, (3) evaluate the usefulness of the EWL in differentiating between superficial and deep partial thickness burns, (4) determine the effect of Biobrane on the EWL of superficial partial thickness burns in vivo, and (5) evaluate the effect of the sterile incision foil Opraflex on the EWL in split skin graft donor sites. METHODS: We measured the EWL of all burn depths in 28 patients under stable and recorded conditions regarding room temperature and humidity with a modern digital evaporimeter (Tewameter TM 300). For the first time in vivo, we also determined the effect of Biobrane on the EWL of burns and evaluated the EWL in split skin graft donor sites covered with Opraflex. RESULTS: The EWL in all burn depths was significantly increased (P < 0.001) compared with unburned skin. There was no significant difference (P > 0.05) in the EWL of superficial compared with deep partial thickness burns, whereas full thickness burns had a significantly lower EWL (P < 0.05) compared with superficial and deep partial thickness burns. Biobrane significantly reduced the EWL (P < 0.05) of superficial partial thickness burns. The EWL of Opraflex covered skin graft donor sites was significantly reduced compared with uncovered donor sites (P < 0.05). CONCLUSIONS: Our data suggest that the actual EWL in burns is approximately 3 times higher in full thickness burns and approximately 4 times higher in superficial and deep partial thickness burns compared with normal skin and therefore much lower than suggested previously.Because there was no significant difference in the EWL of superficial compared with deep partial thickness burns, the EWL cannot be used to differentiate between these burn depths.Biosynthetic wound dressings can significantly reduce the EWL of superficial partial thickness burns and sterile incision foil protects split skin graft donor sites from an increased EWL.

Plasma membranes as heat stress sensors: from lipid-controlled molecular switches to therapeutic applications.

The classic heat shock (stress) response (HSR) was originally attributed to protein denaturation. However, heat shock protein (Hsp) induction occurs in many circumstances where no protein denaturation is observed. Recently considerable evidence has been accumulated to the favor of the "Membrane Sensor Hypothesis" which predicts that the level of Hsps can be changed as a result of alterations to the plasma membrane. This is especially pertinent to mild heat shock, such as occurs in fever. In this condition the sensitivity of many transient receptor potential (TRP) channels is particularly notable. Small temperature stresses can modulate TRP gating significantly and this is influenced by lipids. In addition, stress hormones often modify plasma membrane structure and function and thus initiate a cascade of events, which may affect HSR. The major transactivator heat shock factor-1 integrates the signals originating from the plasma membrane and orchestrates the expression of individual heat shock genes. We describe how these observations can be tested at the molecular level, for example, with the use of membrane perturbers and through computational calculations. An important fact which now starts to be addressed is that membranes are not homogeneous nor do all cells react identically. Lipidomics and cell profiling are beginning to address the above two points. Finally, we observe that a deregulated HSR is found in a large number of important diseases where more detailed knowledge of the molecular mechanisms involved may offer timely opportunities for clinical interventions and new, innovative drug treatments. This article is part of a Special Issue entitled: Membrane Structure and Function: Relevance in the Cell's Physiology, Pathology and Therapy.

Mild Hyperthermia-Induced Thermogenesis in the Endoplasmic Reticulum Defines Stress Response Mechanisms.

Previous studies reported that a mild, non-protein-denaturing, fever-like temperature increase induced the unfolded protein response (UPR) in mammalian cells. Our dSTORM super-resolution microscopy experiments revealed that the master regulator of the UPR, the IRE1 (inositol-requiring enzyme 1) protein, is clustered as a result of UPR activation in a human osteosarcoma cell line (U2OS) upon mild heat stress. Using ER thermo yellow, a temperature-sensitive fluorescent probe targeted to the endoplasmic reticulum (ER), we detected significant intracellular thermogenesis in mouse embryonic fibroblast (MEF) cells. Temperatures reached at least 8 °C higher than the external environment (40 °C), resulting in exceptionally high ER temperatures similar to those previously described for mitochondria. Mild heat-induced thermogenesis in the ER of MEF cells was likely due to the uncoupling of the Ca2+/ATPase (SERCA) pump. The high ER temperatures initiated a pronounced cytosolic heat-shock response in MEF cells, which was significantly lower in U2OS cells in which both the ER thermogenesis and SERCA pump uncoupling were absent. Our results suggest that depending on intrinsic cellular properties, mild hyperthermia-induced intracellular thermogenesis defines the cellular response mechanism and determines the outcome of hyperthermic stress.

Reconstruction of an Extensive Segmental Radial Shaft Bone Defect by Vascularized 3D-Printed Graft Cage.

We report here a 46-year-old male patient with a 14 cm segmental bone defect of the radial shaft after third degree open infected fracture caused by a shrapnel injury. The patient underwent fixed-angle plate osteosynthesis and bone reconstruction of the radial shaft by a vascularized 3D-printed graft cage, including plastic coverage with a latissimus dorsi flap and an additional central vascular pedicle. Bony reconstruction of segmental defects still represents a major challenge in musculo-skeletal surgery. Thereby, 3D-printed scaffolds or graft cages display a new treatment option for bone restoration. As missing vascularization sets the limits for the treatment of large-volume bone defects by 3D-printed scaffolds, in the present case, we firstly describe the reconstruction of an extensive radial shaft bone defect by using a graft cage with additional vascularization.