Calcium-permeable channelrhodopsins for the photocontrol of calcium signalling.

Channelrhodopsins are light-gated ion channels used to control excitability of designated cells in large networks with high spatiotemporal resolution. While ChRs selective for H+, Na+, K+ and anions have been discovered or engineered, Ca2+-selective ChRs have not been reported to date. Here, we analyse ChRs and mutant derivatives with regard to their Ca2+ permeability and improve their Ca2+ affinity by targeted mutagenesis at the central selectivity filter. The engineered channels, termed CapChR1 and CapChR2 for calcium-permeable channelrhodopsins, exhibit reduced sodium and proton conductance in connection with strongly improved Ca2+ permeation at negative voltage and low extracellular Ca2+ concentrations. In cultured cells and neurons, CapChR2 reliably increases intracellular Ca2+ concentrations. Moreover, CapChR2 can robustly trigger Ca2+ signalling in hippocampal neurons. When expressed together with genetically encoded Ca2+ indicators in Drosophila melanogaster mushroom body output neurons, CapChRs mediate light-evoked Ca2+ entry in brain explants.

Postsynaptic plasticity of cholinergic synapses underlies the induction and expression of appetitive and familiarity memories in Drosophila.

In vertebrates, several forms of memory-relevant synaptic plasticity involve postsynaptic rearrangements of glutamate receptors. In contrast, previous work indicates that Drosophila and other invertebrates store memories using presynaptic plasticity of cholinergic synapses. Here, we provide evidence for postsynaptic plasticity at cholinergic output synapses from the Drosophila mushroom bodies (MBs). We find that the nicotinic acetylcholine receptor (nAChR) subunit α5 is required within specific MB output neurons for appetitive memory induction but is dispensable for aversive memories. In addition, nAChR α2 subunits mediate memory expression and likely function downstream of α5 and the postsynaptic scaffold protein discs large (Dlg). We show that postsynaptic plasticity traces can be induced independently of the presynapse, and that in vivo dynamics of α2 nAChR subunits are changed both in the context of associative and non-associative (familiarity) memory formation, underlying different plasticity rules. Therefore, regardless of neurotransmitter identity, key principles of postsynaptic plasticity support memory storage across phyla.

[Is the discipline associated with self-confidence in handling rational antibiotic prescription? : Results from the MR2 study in German hospitals]. / Ist die Fachrichtung assoziiert mit der Selbstsicherheit im Umgang mit rationaler Antibiotikaverordnung? : Ergebnisse der MR2-Studien an deutschen Kliniken.

BACKGROUND: Besides public awareness and specialist knowledge and training of physicians, their self-confidence plays a key role for clinical decision-making in the respective area. OBJECTIVE: This exploratory study investigated the influence of the discipline on differences in self-confidence in dealing with antibiotics and in the self-rated knowledge. METHODS: In 2015 the multi-institutional reconnaissance of practice with multiresistant bacteria (MR2) questionnaire containing items on antibiotic prescription and multiresistant pathogens was sent out to 1061 physicians working in departments for internal medicine, general surgery, gynecology and obstetrics and urology. In 2017 a similar MR2 survey was sent to 1268 specialist and assistant physicians in anesthesiology in Germany. Besides demographic data 4 items on self-confidence in the use of antibiotic treatment and 11 items concerning self-rated knowledge about rational antibiotic therapy and multiresistant pathogens were included in the present analysis. Logistic regression analysis, the χ2-test and the Kruskal-Wallis test were used for statistical analysis of the influence of the discipline on these items. RESULTS: The response rates were 43% (456 out of 1061) from the non-anesthetists and 56% (705 out of 1268) from the anesthetists. Of the non-anesthetists 44% and 57% of the anesthetists had had no advanced training on antibiotic stewardship during the year before the study. In the overall analysis anesthetists (mean±SD: 2.53±0.54) were significantly less self-confident about antibiotics than colleagues from other departments (internal medicine: 3.10±0.50, general surgery: 2.97±0.44, gynecology and obstetrics: 3.12±0.42 and urology: 3.15±0.44) in the unadjusted (all p<0.001) and adjusted comparison. The analysis of self-rated knowledge about rational antibiotic prescription showed similar results. Senior consultant status and advanced training in infectiology were significantly associated with self-confidence and self-rated knowledge about antibiotics. CONCLUSION: Anesthetists showed significantly less self-confidence in dealing with antibiotics than colleagues from other disciplines. Advanced training on a rational prescription of antibiotics was associated with a greater self-confidence, so that the implementation of compulsory courses on rational antibiotic stewardship in the respective residency curriculum needs to be considered.

Network-Specific Synchronization of Electrical Slow-Wave Oscillations Regulates Sleep Drive in Drosophila.

Slow-wave rhythms characteristic of deep sleep oscillate in the delta band (0.5-4 Hz) and can be found across various brain regions in vertebrates. Across phyla, however, an understanding of the mechanisms underlying oscillations and how these link to behavior remains limited. Here, we discover compound delta oscillations in the sleep-regulating R5 network of Drosophila. We find that the power of these slow-wave oscillations increases with sleep need and is subject to diurnal variation. Optical multi-unit voltage recordings reveal that single R5 neurons get synchronized by activating circadian input pathways. We show that this synchronization depends on NMDA receptor (NMDAR) coincidence detector function, and that an interplay of cholinergic and glutamatergic inputs regulates oscillatory frequency. Genetically targeting the coincidence detector function of NMDARs in R5, and thus the uncovered mechanism underlying synchronization, abolished network-specific compound slow-wave oscillations. It also disrupted sleep and facilitated light-induced wakening, establishing a role for slow-wave oscillations in regulating sleep and sensory gating. We therefore propose that the synchronization-based increase in oscillatory power likely represents an evolutionarily conserved, potentially "optimal," strategy for constructing sleep-regulating sensory gates.

Delay of transfer from the intensive care unit: a prospective observational analysis on economic effects of delayed in-house transfer.

BACKGROUND: Intensive care unit (ICU) capacity is a scant and precious resource in hospitals. Therefore, an optimal occupancy rate as well as detailed occupation planning is of great importance. Most literature deals with admission to the ICU, while only few discuss discharge from the ICU. Specifically, a delay of transfer from the ICU can cause a shortness of beds, jeopardize urgent patient treatment and lead to a decrease in treatment quality as well as economic downsides. This study examined the incidence, costs and reasons for delayed discharge from the ICU and analyzed the influence of the department the patient was admitted to. METHODS: Over the course of 12 months, the discharges of all 1643 patients of two surgical intensive care units of a large academic medical center were analyzed. Delay in minutes and reasons were recorded and translated into financial figures. A univariate logistic regression model was developed to evaluate the impact of length of stay at the ICU, age, gender, subspecialty and specific ICU on the delay of transfer. In a next step, significant factors of the univariate logistic regression were incorporated into a multivariate regression model. RESULTS: In 326 out of 1312 patients ready for discharge (24.8%), the transfer to the floor was delayed. Time of delay for all patients added up to a total of 265,691 min in 1 year. The application of the internal cost allocation, in which 1 min corresponds to 0.75 Euro cents, led to costs of 199,268 Euros (~ $240,000) for the study period. In 91.7% of the cases, the reason for the delay was the lack of an available or appropriate bed on the regular ward. Multivariate regression analysis revealed that the type of department the patient is admitted to poses a significantly influencing factor for delayed discharge from the ICU. CONCLUSION: Delay in discharge from the ICU is a common problem of economic relevance. The main reason is a lack of appropriate floor beds. Patients from certain specific departments are at a higher risk to be discharged with delay. A solution to this problem lies in the focus on the downstream units. A proper use of the scarce resources is to be pursued because of ethical as well as economic reasons in an increasingly aging population.

Right on track? Performance of satellite telemetry in terrestrial wildlife research.

Satellite telemetry is an increasingly utilized technology in wildlife research, and current devices can track individual animal movements at unprecedented spatial and temporal resolutions. However, as we enter the golden age of satellite telemetry, we need an in-depth understanding of the main technological, species-specific and environmental factors that determine the success and failure of satellite tracking devices across species and habitats. Here, we assess the relative influence of such factors on the ability of satellite telemetry units to provide the expected amount and quality of data by analyzing data from over 3,000 devices deployed on 62 terrestrial species in 167 projects worldwide. We evaluate the success rate in obtaining GPS fixes as well as in transferring these fixes to the user and we evaluate failure rates. Average fix success and data transfer rates were high and were generally better predicted by species and unit characteristics, while environmental characteristics influenced the variability of performance. However, 48% of the unit deployments ended prematurely, half of them due to technical failure. Nonetheless, this study shows that the performance of satellite telemetry applications has shown improvements over time, and based on our findings, we provide further recommendations for both users and manufacturers.

Effect of hepatitis B virus on steatosis in hepatitis C virus co-infected subjects: A multi-centre study and systematic review.

It remains unclear whether hepatitis B virus (HBV) infection may modify the severity of viral steatosis in patients coinfected with chronic hepatitis C virus (HCV). We examined the influence of coinfection with HBV on prevalence of steatosis in chronic hepatitis C in a multi-centre cohort of HBV-HCV subjects, and by performing a systematic review and meta-analysis of the literature. We centrally and blindly assessed steatosis prevalence and severity in a cohort of HBV-HCV coinfected subjects compared to HCV and HBV monoinfected controls and we performed a systematic review of studies addressing the prevalence of steatosis in HBV-HCV subjects compared to HCV controls. In the clinical cohort, we included 85 HBV-HCV, 69 HBV and 112 HCV subjects from 16 international centres. There was no significant difference in steatosis prevalence between the HBV-HCV and the HCV groups (33% vs 45%, P = .11). In subgroup analysis, lean HBV-HCV subjects with detectable HBV DNA had less steatosis than lean HCV subjects matched for HCV viremia (15% vs 45%, P = .02). Our literature search identified 5 additional studies included in a systematic review. Overall, prevalence of steatosis > 5% was similar in HBV-HCV infection compared to HCV (pooled odds ratio [OR] 0.91, 95% CI 0.53-1.6) although there was significant heterogeneity (I2 69%, P = .007). In conclusion, although the prevalence of steatosis is similar in HBV-HCV compared to HCV subjects, our analysis suggests that there may be an inhibitory effect of HCV-induced steatogenesis by HBV in certain subgroups of patients.

Genetic profiling using plasma-derived cell-free DNA in therapy-naïve hepatocellular carcinoma patients: a pilot study.

Background: Hepatocellular carcinomas (HCCs) are not routinely biopsied, resulting in a lack of tumor materials for molecular profiling. Here we sought to determine whether plasma-derived cell-free DNA (cfDNA) captures the genetic alterations of HCC in patients who have not undergone systemic therapy. Patients and methods: Frozen biopsies from the primary tumor and plasma were synchronously collected from 30 prospectively recruited, systemic treatment-naïve HCC patients. Deep sequencing of the DNA from the biopsies, plasma-derived cfDNA and matched germline was carried out using a panel targeting 46 coding and non-coding genes frequently altered in HCCs. Results: In 26/30 patients, at least one somatic mutation was detected in biopsy and/or cfDNA. Somatic mutations in HCC-associated genes were present in the cfDNA of 63% (19/30) of the patients and could be detected 'de novo' without prior knowledge of the mutations present in the biopsy in 27% (8/30) of the patients. Mutational load and the variant allele fraction of the mutations detected in the cfDNA positively correlated with tumor size and Edmondson grade. Crucially, among the seven patients in whom the largest tumor was ≥5 cm or was associated with metastasis, at least one mutation was detected 'de novo' in the cfDNA of 86% (6/7) of the cases. In these patients, cfDNA and tumor DNA captured 87% (80/92) and 95% (87/92) of the mutations, suggesting that cfDNA and tumor DNA captured similar proportions of somatic mutations. Conclusion: In patients with high disease burden, the use of cfDNA for genetic profiling when biopsy is unavailable may be feasible. Our results support further investigations into the clinical utility of cfDNA in a larger cohort of patients.

In vivo analysis at the cellular level reveals similar steatosis induction in both hepatitis C virus genotype 1 and 3 infections.

Steatosis is a frequent histological feature of hepatitis C virus (HCV) infection. Cohort studies of patients with chronic hepatitis C identified HCV genotype 3 (HCV GT3) as the prevalent steatotic genotype. Moreover, Huh-7 cells over-expressing HCV GT3 core protein accumulate more triglyceride in larger lipid droplets than cells expressing core proteins of other HCV genotypes. However, little is known about the relationship of steatosis and HCV infection at the cellular level in vivo. In this study, we used highly sensitive multiplex in situ hybridization methodology together with lipid staining to investigate HCV-induced lipid droplet accumulation at the cellular level in liver biopsies. Consistent with previous reports, histological steatosis grades were significantly higher in GT3 compared to GT1 infected livers, but independent of viral load. Using nile red lipid stainings, we observed that the frequency of lipid droplet containing cells was similar in HCV GT1- and HCV GT3-infected livers. Lipid droplet formation preferentially occurred in HCV-infected cells irrespective of the genotype, but was also observed in noninfected cells. These findings demonstrate that the main difference between GT1- and GT3-induced steatosis is the size of lipid droplets, but not the number or relative distribution of lipid droplets in infected vs uninfected hepatocytes.

[Current status of teaching in lung ultrasound : Query of knowledge, utilization, need, and preferred teaching method]. / Zum Stand der Ausbildung im Lungenultraschall : Abfrage von Kenntnissen, Verwendung, Bedarf und bevorzugter Unterrichtsform.

BACKGROUND: Lung ultrasound (LUS) is a point-of-care technique which can quickly identify or rule out pathological findings. To date, it is unclear if knowledge about the use of LUS is readily available. OBJECTIVES: We aimed to identify how much knowledge about the use of LUS is present, if there is a need for teaching in LUS, as well as the preferred teaching method in LUS. MATERIALS AND METHODS: A total of 54 participants from two university departments of anesthesiology were randomized into the groups Online, Classroom, and Control. The Online group was taught by videos, the Classroom group by a traditional lecture with hands-on training, and the Control group was not taught at all. We conducted a pre- and posttest as well as a retention test 4 weeks after the end of the study by means of a survey (comparison with Mann-Whitney U test or t­test, respectively, with p < 0.05 considered to be significant). RESULTS: LUS is used "rarely" or "never", and mainly if there is a suspicion for pleural effusion (41.3%). There is a need for LUS (Online: 21.7%; Classroom: 60.9%; Control: 62.5%, p < 0.05). Hybrid teaching consisting of classroom-based and online-based teaching is preferred by the users (Online: 52.2%; Classroom: 56.5%; Control: 62.5%). At the end of the study, 32.6% of the participants of the intervention groups had used LUS in the diagnosis of a pneumothorax. Of the participants, 93.5% planned to use LUS more often in the future. CONCLUSIONS: LUS is rarely used. There is a considerable need for teaching of LUS. Internet-based teaching and traditional lectures are considered equal. Both teaching methods improve the knowledge about LUS and lead to increased use of LUS in daily practice. The participants prefer hybrid teaching incorporating both teaching methods.

Hepatitis B virus covalently closed circular DNA homeostasis is independent of the lymphotoxin pathway during chronic HBV infection.

Current treatment options for patients with chronic hepatitis B virus (HBV) infection are not curative as they are not effective in eliminating covalently closed circular DNA (cccDNA). cccDNA is a stable template for HBV transcription in the nucleus of hepatocytes and is thought to be one of the main factors responsible for HBV persistence. Recently, activation of the lymphotoxin beta receptor (LTßR) has been shown to trigger degradation of cccDNA through induction of cytidine deaminases of the APOBEC3 family in HBV cell culture model systems. To assess the presence and relevance of such mechanisms in the liver of chronically HBV-infected patients, we compared intrahepatic cccDNA levels with the expression levels of lymphotoxins and some of their target genes (eg APOBEC deaminases) in liver biopsy tissue. Our results confirm elevated gene expression levels of components of the lymphotoxin pathway including lymphotoxin alpha (LTα), lymphotoxin beta (LTß), APOBEC3B (A3B) and APOBEC3G (A3G) in the chronically HBV-infected liver compared to uninfected liver. Furthermore, expression levels of the genes of the APOBEC deaminase family were correlated with those of LTα and LTß gene expression, consistent with lymphotoxin-mediated upregulation of APOBEC gene expression. However, intrahepatic cccDNA and HBV replication levels were not correlated with LTα, LTß and APOBEC gene expression. In conclusion, these results suggest that although the lymphotoxin pathway is activated in the chronically HBV-infected liver, it has no major impact on HBV cccDNA metabolism in chronic HBV infection.

[How Much Blood and What Components does the Patient need Intra- and Perioperatively?]. / Wie viel Blut und welche seiner Bestandteile benötigt der Mensch intra- und perioperativ? - neue wichtige Aspekte für den Allgemein-(Viszeral-)Chirurgen aus der Transfusionsmedizin und Hämostaseologie.

By the optimised availability of less expensive and safe red cell packs and other blood products over the last 20 years, numerous surgical interventions have become possible without any demand for comments on the precise need. However, a number of publications indicates that blood transfusion may also induce disadvantageous effects on the postoperative course by immunomodulation, which requires a rather restrictive indication for transfusion. Furthermore, demographic development leads to a decrease in that portion of the population with the potential for blood donation accompanied simultaneously by an increase of the percentage of older patients with more need of blood products during medical treatment. This makes blood-sparing measures necessary. In addition, costs for red cell packs have increased, in particular, for the generally compatible blood group 0 - an extra amount for rhesus negative blood. The present narrative review highlights, therefore, important news from the clinical transfusion medicine, immunohaematology and haemostaseology and their impact on daily transfusion practice. In this context, "blood management" is considered as one of the very effective blood-sparing measures, which focusses especially i) on the substitution of iron in case of depressed preoperative haemoglobin as well as ii) to elucidate disorders of coagulation by structured medical history and, subsequently, to balance possible need by a specific plan for substitution. Simultaneously, prospective studies are initiated to investigate how far the transfusion trigger of a patient can be lowered down to a still appropriate level. As far as consolidated findings are already available, they are described with regard to the single blood components and taking into account the cross-sectional guidelines of the "Bundesärztekammer" (Federal Physicians Chamber). Finally, initial evidence is provided characterising patient- and blood donor-specific, blood group-dependent features of a reasonable haemotherapy.

Impact of common risk factors of fibrosis progression in chronic hepatitis C.

OBJECTIVE: The natural course of chronic hepatitis C varies widely. To improve the profiling of patients at risk of developing advanced liver disease, we assessed the relative contribution of factors for liver fibrosis progression in hepatitis C. DESIGN: We analysed 1461 patients with chronic hepatitis C with an estimated date of infection and at least one liver biopsy. Risk factors for accelerated fibrosis progression rate (FPR), defined as ≥ 0.13 Metavir fibrosis units per year, were identified by logistic regression. Examined factors included age at infection, sex, route of infection, HCV genotype, body mass index (BMI), significant alcohol drinking (≥ 20 g/day for ≥ 5 years), HIV coinfection and diabetes. In a subgroup of 575 patients, we assessed the impact of single nucleotide polymorphisms previously associated with fibrosis progression in genome-wide association studies. Results were expressed as attributable fraction (AF) of risk for accelerated FPR. RESULTS: Age at infection (AF 28.7%), sex (AF 8.2%), route of infection (AF 16.5%) and HCV genotype (AF 7.9%) contributed to accelerated FPR in the Swiss Hepatitis C Cohort Study, whereas significant alcohol drinking, anti-HIV, diabetes and BMI did not. In genotyped patients, variants at rs9380516 (TULP1), rs738409 (PNPLA3), rs4374383 (MERTK) (AF 19.2%) and rs910049 (major histocompatibility complex region) significantly added to the risk of accelerated FPR. Results were replicated in three additional independent cohorts, and a meta-analysis confirmed the role of age at infection, sex, route of infection, HCV genotype, rs738409, rs4374383 and rs910049 in accelerating FPR. CONCLUSIONS: Most factors accelerating liver fibrosis progression in chronic hepatitis C are unmodifiable.

Hepatitis C virus dysregulates glucose homeostasis by a dual mechanism involving induction of PGC1α and dephosphorylation of FoxO1.

The maintenance of glucose homeostasis is a complex process in which the insulin signalling pathway plays a major role. Disruption of insulin-regulated glucose homeostasis is frequently observed in chronic hepatitis C (CHC) infection and might potentially contribute to type 2 diabetes mellitus (T2DM) development. Presently, the mechanism that links HCV infection to insulin resistance remains unclear. Previously, we have reported that HCV protein expression in HCV transgenic mice (B6HCV) leads to an overexpression of protein phosphatase 2A (PP2A) through an ER stress response. In the present work, we describe an association of FoxO1 hypophosphorylation and upregulation of both PGC-1α and G6Pase to phenotypic hyperglycaemia and insulin resistance in B6HCV mice. In vitro, we observed that PGC1α is concomitantly induced with PP2A. Moreover, we show that the enhanced PP2A expression is sufficient to inhibit insulin-induced FoxO1 phosphorylation via blockade of insulin-mediated Akt activation or/and through direct association and dephosphorylation of pS-FoxO1. Consequently, we found that the gluconeogenic gene glucose-6-phosphatase is upregulated. These observations were confirmed in liver biopsies obtained from CHC patients. In summary, our results show that HCV-mediated upregulation of PP2A catalytic subunit alters signalling pathways that control hepatic glucose homeostasis by inhibiting Akt and dephosphorylation of FoxO1.

Treatment of severe autoimmune blistering skin diseases with combination of protein A immunoadsorption and rituximab: a protocol without initial high dose or pulse steroid medication.

BACKGROUND: Skin blistering diseases due to autoantibodies are typically treated with high dose systemic corticosteroids and other conventional immunosuppressants. However, in severe cases, this treatment may not be sufficient to achieve disease control or contraindicated because of comorbidity. METHODS: We describe 15 patients (pts.) with such diseases: 6 pts. with pemphigus vulgaris, 3 pts. with bullous pemphigoid, 3 pts. with mucous membrane pemphigoid (MMP), one being anti-laminin-332-MMP (AL332-MMP), 2 pts. with pemphigus foliaceus and 1 pt. with epidermolysis bullosa acquisita (EBA). Patients were treated with a combination of protein A immunoadsorption (PAIA, 3-21 treatments) and rituximab (3-6 treatments) in addition to low dose conventional immunosuppression. RESULTS: All patients showed rapid clinical improvement starting within the first 4 weeks and decline of circulating autoantibody levels. Complete/partial remission was 88%/12% in pemphigus and 71%/29% in subepidermal blistering diseases. Overall relapse rate was 13% with an average follow-up of 22 months. In the AL332-MMP pt. the PAIA/rituximab treatment was stopped because of an oesophagus cancer considered as the paraneoplastic cause of the skin disease. CONCLUSION: Combined treatment with PAIA and rituximab showed rapid and long-lasting response, thereby allowing substantial reduction of dosage of concomitant immunosuppressive medication. We hereby confirm data from other investigators that PAIA/rituximab treatment is a promising therapeutical modality for pemphigus, pemphigoids and EBA, characterized by a favourable ratio of beneficial efficacy and minimized long-term adverse effects.

[Vocational rehabilitation after total laryngectomy]. / Berufliche Rehabilitation nach Laryngektomie.

BACKGROUND: Aim of this study was to find out how many patients after a total laryngectomy (TLE) return to work successfully and what factors support vocational rehabilitation. PATIENTS AND METHODS: Laryngectomees (n=231) aged up to 60 years completed questionnaires and structured interviews before TLE (t1), before rehabilitation (t2), at the end of rehabilitation (t3), 1 year after TLE (t4), 2 years after TLE (t5), and 3 years after TLE (t6). RESULTS: Prior to TLE, 38% of all respondents were employed, 34% were unemployed, 23% received disability-related and 3% age-related pension retirement. One year after TLE, 13% were employed, 15% 2 years and 14% 3 years after TLE. Unemployed were 10% (t4), 5% (t5), and 7% (t6) of the patients. For 59% of all respondents it was very important to have a job. Predictors of successful vocational rehabilitation were employment prior to TLE, age <50 years, being self-employed or clerical employee, good physical functioning, good speech intelligibility, high motivation to go back to work, and support from colleagues. CONCLUSION: Only few laryngectomees return to work. However, even before TLE only a third of the patients was employed, another third was unemployed. Most of the patients receive pension retirement after TLE. As return to work is important for many patients, patient consultations should consider possibilities to support vocational rehabilitation before offering to apply for retirement.