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Background Coronavirus disease 2019 (COVID-19)-related acute respiratory distress syndrome requiring veno-venous extracorporeal membrane oxygenation (vv-ECMO) is related with poor outcome, especially in Germany. We aimed to analyze whether changes in vv-ECMO therapy during the pandemic were observed and lead to changes in the outcome of vv-ECMO patients. Methods All patients undergoing vv-ECMO support for COVID-19 between 2020 and 2021 in a single center (n = 75) were retrospectively analyzed. Weaning from vv-ECMO and in-hospital mortality were defined as primary and peri-interventional adverse events as secondary endpoints of the study. Results During the study period, four infective waves were observed in Germany. Patients were assigned correspondingly to four study groups: ECMO implantation between March 2020 and September 2020: first wave (n = 11); October 2020 to February 2021: second wave (n = 23); March 2021 to July 2021: third wave (n = 25); and August 2021 to December 2021: fourth wave (n = 20). Preferred cannulation technique changed within the second wave from femoro-femoral to femoro-jugular access (p Conclusion Preference for femoro-jugular cannulation and awake ECMO combined with preexisting expertise and patient selection are considered to be associated with increased duration of ECMO support and numerically improved ECMO weaning and in-hospital mortality.

Background & AimsExtracellular vesicles (EVs) play an important role in intercellular communication, serving as vehicles for the exchange of biological materials and being involved in the regulation of physiological processes. EVs and their associated cargoes are considered a promising source of disease-associated biomarkers. The purpose of this study was to establish an easy-to-use, reproducible, and scalable workflow to efficiently analyze EVs in the context of liver disease.MethodsAn optimized workflow was established for the pre-analytical processing and isolation of EVs from plasma and serum. Nanoparticle Tracking Analysis (NTA) was used to characterize circulating EVs in the serum of patients with nonalcoholic fatty liver disease (NAFLD), autoimmune liver disease (AIH), and animal models with impaired liver function. EVs were separated from soluble proteins by an optimized, polyethylene glycol (PEG)-based enrichment protocol. Enriched EVs were either labeled and functionally characterized by monitoring cellular uptake or lysed for biomarker identification.ResultsCirculating EVs in the serum of patients with NAFLD or AIH and in different animal models have been characterized by NTA. Here we show that both the quantity and size of EVs in the serum of patients/animal models are significantly different from those of healthy individuals. We show that isolated EVs are functional, and their uptake by acceptor cells can be quantified after fluorescence labelling. Enriched EVs were directly used to analyze RNA biomarkers. Several microRNAs, including miR-15b, -16, -21, -122 and -223, were found to be significantly up-regulated in EVs isolated from the sera of patients with NAFLD and AIH. We show that EVs transport cytokines, and that IL-2, IL-6 and IL-8 were significantly up-regulated in EVs enriched from patients with cholangiocarcinoma (CCA) compared to healthy controls.ConclusionsThe workflow presented here represents an accessible and easy-to-use approach that enables the analysis and enrichment of EVs from complex biological fluids and their preparation for functional characterization or downstream analysis. In this study, the levels of several miRNAs were found to be significantly increased in EVs isolated from AIH and NAFLD patients compared with healthy controls.HighlightsEVs circulating in crude serum reflect the diseased stage of the donors.Enrichment of EVs with the approach presented here efficiently separates soluble proteins from EVs, providing optimal material for further characterization.Exosomal markers are present in the EVs-enriched fraction.Enriched EVs are intact and are functionally taken up by acceptor cells.Enriched EVs are suitable, and have been used for, biomarkers identification both at RNA and protein level.

Acute kidney injury (AKI) is a major kidney disease with a poor clinical outcome. It is a common complication with an incidence of 10-15% of patients admitted to hospital. This rate even increases for patients who are admitted to the intensive care unit with an incidence of >50%. AKI is characterized by a rapid increase in serum creatinine, decrease in urine output, or both. Associated Symptoms include feeling sick or being sick, diarrhoea, dehydration, decreased urine output-although occasionally urine output remains normal, fluid retention-causing swelling in the legs or ankles, shortness of breath, fatigue and nausea. However, sometimes acute kidney injury causes no signs or symptoms and is detected by lab tests. Therefore, an urgent demand for non-invasive biomarkers for early detection of AKI are highly desirable. This might enable the prevention of the progression from AKI to CKD.In this study, we analysed the secretome of urine of an AKI patient cohort employing a kidney-biomarker cytokine assay. Based on these results we suggest, ADIPOQ, EGF and SERPIN3A as potential biomarkers, which might be able to detect AKI as soon as 24 h post-surgery. For the later stages, common biomarkers for the detection of AKI in both male and female patients we suggest, VEGF, SERPIN3A, TNFSF12, ANPEP, CXCL1, REN, CLU and PLAU. These markers in combination might present a robust strategy to identify the development of AKI as early as 24h or 72h post-surgery. Furthermore, we evaluated the effect of patient and healthy urine on human podocyte cells. We conclude that cytokines in the urine of AKI patients trigger processes which are needed to repair the damaged nephron and activate TP53 and SIRT1 to maintain the balance between proliferation, angiogenesis, and cell cycle arrest. In conclusion, the Renin-Angiotensin pathway seems to have major implications.

Despite significant advances in the diagnosis and treatment of cardiovascular diseases, recent calls have emphasized the unmet need to improve precision-based approaches in cardiovascular disease. Although some studies provide preliminary evidence of the diagnostic and prognostic potential of circulating coding and non-coding RNAs, the complex RNA biology and lack of standardization have hampered the translation of these markers into clinical practice. In this position paper of the CardioRNA COST action CA17129, we provide recommendations to standardize the RNA development process in order to catalyse efforts to investigate novel RNAs for clinical use. We list the unmet clinical needs in cardiovascular disease, such as the identification of high-risk patients with ischaemic heart disease or heart failure who require more intensive therapies. The advantages and pitfalls of the different sample types, including RNAs from plasma, extracellular vesicles, and whole blood, are discussed in the sample matrix, together with their respective analytical methods. The effect of patient demographics and highly prevalent comorbidities, such as metabolic disorders, on the expression of the candidate RNA is presented and should be reported in biomarker studies. We discuss the statistical and regulatory aspects to translate a candidate RNA from a research use only assay to an in-vitro diagnostic test for clinical use. Optimal planning of this development track is required, with input from the researcher, statistician, industry, and regulatory partners., peer-reviewed

The global outbreak of SARS-CoV-2/COVID-19 provided the stage to accumulate an enormous biomedical data set and an opportunity as well as a challenge to test new concepts and strategies to combat the pandemic. New research and molecular medical protocols may be deployed in different scientific fields, e.g., glycobiology, nanopharmacology, or nanomedicine. We correlated clinical biomedical data derived from patients in intensive care units with structural biology and biophysical data from NMR and/or CAMM (computer-aided molecular modeling). Consequently, new diagnostic and therapeutic approaches against SARS-CoV-2 were evaluated. Specifically, we tested the suitability of incretin mimetics with one or two pH-sensitive amino acid residues as potential drugs to prevent or cure long-COVID symptoms. Blood pH values in correlation with temperature alterations in patient bodies were of clinical importance. The effects of biophysical parameters such as temperature and pH value variation in relation to physical-chemical membrane properties (e.g., glycosylation state, affinity of certain amino acid sequences to sialic acids as well as other carbohydrate residues and lipid structures) provided helpful hints in identifying a potential Achilles heel against long COVID. In silico CAMM methods and in vitro NMR experiments (including 31P NMR measurements) were applied to analyze the structural behavior of incretin mimetics and SARS-CoV fusion peptides interacting with dodecylphosphocholine (DPC) micelles. These supramolecular complexes were analyzed under physiological conditions by 1H and 31P NMR techniques. We were able to observe characteristic interaction states of incretin mimetics, SARS-CoV fusion peptides and DPC membranes. Novel interaction profiles (indicated, e.g., by 31P NMR signal splitting) were detected. Furthermore, we evaluated GM1 gangliosides and sialic acid-coated silica nanoparticles in complex with DPC micelles in order to create a simple virus host cell membrane model. This is a first step in exploring the structure–function relationship between the SARS-CoV-2 spike protein and incretin mimetics with conserved pH-sensitive histidine residues in their carbohydrate recognition domains as found in galectins. The applied methods were effective in identifying peptide sequences as well as certain carbohydrate moieties with the potential to protect the blood–brain barrier (BBB). These clinically relevant observations on low blood pH values in fatal COVID-19 cases open routes for new therapeutic approaches, especially against long-COVID symptoms.

ZusammenfassungEine akute Nierenschädigung tritt bei 25% aller Krankenhaus- und bei 50% der Intensivpatienten auf. Im Stadium 3 der akuten Nierenschädigung sterben auch bei optimaler Therapie etwa 40 – 50% der betroffenen Patienten. Die Langzeitprognose hängt ganz wesentlich von der Erholung der Nierenfunktion ab. Eine frühe Diagnostik und die konsequente Durchführung von Interventionen zur Prophylaxe und zur schnellen Wiederherstellung der Nierenfunktion sind essenziell 1.

Background A profound inflammation-mediated lung injury with long-term acute respiratory distress and high mortality is one of the major complications of critical COVID-19. Immunoglobulin M (IgM)-enriched immunoglobulins seem especially capable of mitigating the inflicted inflammatory harm. However, the efficacy of intravenous IgM-enriched preparations in critically ill patients with COVID-19 is largely unclear. Methods In this retrospective multicentric cohort study, 316 patients with laboratory-confirmed critical COVID-19 were treated in ten German and Austrian ICUs between May 2020 and April 2021. The primary outcome was 30-day mortality. Analysis was performed by Cox regression models. Covariate adjustment was performed by propensity score weighting using machine learning-based SuperLearner to overcome the selection bias due to missing randomization. In addition, a subgroup analysis focusing on different treatment regimens and patient characteristics was performed. Results Of the 316 ICU patients, 146 received IgM-enriched immunoglobulins and 170 cases did not, which served as controls. There was no survival difference between the two groups in terms of mortality at 30 days in the overall cohort (HRadj: 0.83; 95% CI: 0.55 to 1.25; p = 0.374). An improved 30-day survival in patients without mechanical ventilation at the time of the immunoglobulin treatment did not reach statistical significance (HRadj: 0.23; 95% CI: 0.05 to 1.08; p = 0.063). Also, no statistically significant difference was observed in the subgroup when a daily dose of ≥ 15 g and a duration of ≥ 3 days of IgM-enriched immunoglobulins were applied (HRadj: 0.65; 95% CI: 0.41 to 1.03; p = 0.068). Conclusions Although we cannot prove a statistically reliable effect of intravenous IgM-enriched immunoglobulins, the confidence intervals may suggest a clinically relevant effect in certain subgroups. Here, an early administration (i.e. in critically ill but not yet mechanically ventilated COVID-19 patients) and a dose of ≥ 15 g for at least 3 days may confer beneficial effects without concerning safety issues. However, these findings need to be validated in upcoming randomized clinical trials. Trial registrationDRKS00025794, German Clinical Trials Register, https://www.drks.de. Registered 6 July 2021.

Background: Alterations of the expression of microRNAs (miRNAs) in chronic pain models seem to play a crucial role in the development of neuropathic pain, with microRNA-1 (miR-1) being of particular interest. Recently, we were able to show that decreased miR-1 levels were associated with increased expression of brain-derived neurotrophic factor (BDNF) and Connexin 43 (Cx43). We hypothesized that miR-1 mimetic nucleotides could alleviate neuropathic pain caused by chronic constriction injury in rats. Methods: MiR-1 mimetic nucleotides were evaluated for effectiveness, functionality, and intracellular stability by transfecting human glioblastoma cells (U-87 MG). In vivo transfection with miR-1 mimics and corresponding scrambled miRNAs serving as control was performed by repetitive injection (days 0, 3, and 7) into the sciatic nerve following chronic constriction injury (CCI) in rats. Quantitative PCR was used to measure miR-1 content. Cx43 expression was determined by Western blot analysis. Effects on neuropathic pain were assessed by detecting paw withdrawal thresholds using an automated filament application. Results: Transfection of miR-1 mimics was confirmed in U-87 MG cells, with miR-1 content being increased significantly after 48 h and after 96 h (p

COVID-19 patients who may require invasive therapeutic procedures such as extracorporeal membrane oxygenation (ECMO) have high symptom burden and in-hospital mortality. In addition, awake patients on ECMO are new in the intensive care unit (ICU) setting. Inpatient specialist palliative care (sPC) provides support such as symptom control on a physical, psychosocial and spiritual level. The field of sPC in COVID-19 patients is still new and important to investigate. We aim to analyze sPC of COVID-19 patients in the ICU with regard to patient characteristics and symptoms from a palliative care perspective. We conducted a retrospective analysis (03/2020–04/2021) and identified 51 ICU patients receiving sPC. The statistical analysis included descriptive statistics and comparisons of symptoms. The first sPC contact of patients (mean age 69.5 years, 62.7% male) was around 14 days after COVID-19 confirmation, and 43% were treated with ECMO therapy. The baseline symptom burden was high with a focus on weakness (100%), tiredness (98%), dyspnea (96%) and family burden (92%). The symptom intensity significantly decreased during the time period of sPC and COVID-19 treatment (t(99) = 3.119, p = 0.003, d = 0.437). These results help intensivists and sPC clinicians to identify symptoms and the need for sPC in COVID-19 patients. However, studies with prospective and controlled designs need to follow.

BACKGROUND Ischemic preconditioning (IPC) and remote ischemic preconditioning (RIPC) protect myocardial tissue against subsequent ischemia and reperfusion injury (IRI) and have a high potential to improve patient outcome. The mediators and mechanisms of protection through IPC and RIPC remain largely unknown, but micro-RNAs (miRNAs) are promising candidates. METHODS Systematic review of Medline and Embase databases for biomedical scientific literature. RESULTS A total of 26 relevant publications (21 full-text original articles and 5 conference abstracts) were identified, 8 describing cell culture experiments, 14 animal experiments, and 4 randomized clinical trials in humans. Most commonly reported miRNAs with differential expression between preconditioned and control groups include miR-1, miR-21, and miR-144. Experimental designs and procedures differ widely, thereby limiting the potential to compare results between studies. Two of the four RCTs did not find any differentially expressed miRNAs. CONCLUSIONS Results from RCTs should feed back into basic research and focused studies confirming or rejecting hypotheses generated by these RCTs are needed.

Background: Cardioprotective interventions-such as pharmacological postconditioning- A re a promising strategy to reduce deleterious consequences of ischemia and reperfusion injury (I/RI) in the heart, especially as timing and onset of myocardial infarction are unpredictable. Pharmacological postconditioning by treatment with dexmedetomidine (Dex), an 2-adrenoreceptor agonist, during reperfusion protects hearts from I/RI, independently of time point and duration of application during the reperfusion phase. The mitochondrial ATP-sensitive K +(mKATP) and mitochondrial large-conductance calcium-sensitive potassium channel (mBKCa) play a pivotal role in mediating this cardioprotective effect. Therefore, we investigated whether Dex-induced cardioprotection during early or late reperfusion is mediated variously by these mitochondrial K +-channels. METHODS: Hearts of male Wistar rats were randomized into 8 groups and underwent a protocol of 15 minutes adaption, 33 minutes ischemia, and 60 minutes reperfusion in an in vitro Langendorff-system. A 10-minute treatment phase was started directly (first subgroup, early reperfusion) or 30 minutes (second subgroup, late reperfusion) after the onset of reperfusion. Control (Con) hearts received vehicle only. In the first subgroup, hearts were treated with 3 nM Dex, 100 M mKATP-channel blocker 5-hydroxydecanoate (5HD) or 1 M mBKCa-channel blocker Paxilline (Pax) alone or with respective combinations (5HD + Dex, Pax + Dex). Hearts of the second subgroup received Dex alone (Dex30') or in combination with the respective blockers (5HD + Dex30', Pax + Dex30'). Infarct size was determined with triphenyltetrazoliumchloride staining. Hemodynamic variables were recorded during the whole experiment. RESULTS: During early reperfusion (first subgroup), the infarct size reducing effect of Dex (Con: 57% ± 9%, Dex: 31% ± 7%; P

Objective: The affection of both the peripheral (PNS) and central nervous system (CNS) by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been assumed to play a direct role in the respiratory failure of patients with Corona virus disease 2019 (COVID-19) through affection of medullary cardiorespiratory centers resulting in neurological complications and sequelae. Methods: We used a multimodal electrophysiological approach combined with neuropsychological investigations to study functional alteration of both the PNS and CNS in four patients with severe COVID-19. Results: We found electrophysiological evidence for affection of both the PNS and CNS, and particularly affection of brain stem function. Furthermore, our neuropsychological investigations provide evidence of marked impairment of cognition independent of delirium, and outlasting the duration of acute infection with SARS-CoV-2. Conclusion: This case series provides first direct electrophysiological evidence for functional brain stem involvement in COVID-19 patients without evident morphological changes supporting the notion of the brain stem contributing to respiratory failure and thus promoting severe courses of the disease. Moreover, sustained neuropsychological sequelae in these patients may be of particular psychosocial and possibly also economic relevance for society.

Sepsis and septic shock are characterized by a release of cytokines into the circulation. These mediators contribute to the detrimental hemodynamic and metabolic effects in the early phase of septic shock. Recently, a new polystyrene-based hemoadsorption device was introduced into clinical practice (CytoSorb®). The adsorber binds a variety of molecules including cytokines and removes them from the circulation. Studies in septic patients have shown an improved clinical course following hemoadsorption but no increased survival. We hypothesize that not only cytokines but also antibiotics may be removed which potentially may negate any beneficial effect of the adsorber. To test this hypothesis, we performed polystyrene-based hemoadsorption in three patients in septic shock and analysed glycopeptide elimination by measuring serum levels pre- and post-adsorber. We administered both teicoplanin and vancomycin via a 60-min infusion and vancomycin via continuous infusion, additionally. When applied as 60 min infusion, vancomycin and teicoplanin were removed immediately by the adsorber. However, the adsorptive capacity of the device was saturable. Serum levels of vancomycin, but not teicoplanin, decreased to subtherapeutic levels. With continuous infusion of vancomycin, removal was less and serum levels remained in the therapeutic range. In conclusion, we show effective glycopeptide adsorption using a polystyrene-based hemoadsorber in septic patients. The dose of these antibiotics should be adjusted appropriately and early therapeutic drug monitoring is highly recommended.

Continuous renal replacement therapy is a standard treatment in critically ill patients with acute kidney injury. All CRRT techniques provide a high low-molecular weight clearance but even with hemofiltration, clearance of middle molecules is low. We investigated whether a new super high-flux hemofilter provides effective and sustained middle molecule clearance during citrate-anticoagulated continuous venovenous hemodialysis for up to 72 h. We included 14 critically ill patients with AKI-KDIGO-III in a prospective observational trial. We measured/calculated blood and urine concentrations, clearances and sieving coefficients of eight molecules with molecular weights from 60 to 66 kDa, hemodynamic parameters and SAPS-II scores. All filters were patent at 72 h. Clearance and sieving coefficients of small solutes were high and sustained over time, those for larger solutes decreased over 72 h but remained high enough to decrease blood concentrations of solutes up to 25 kDa. Albumin serum levels remained unaffected. Catecholamine doses and SAPS-II scores decreased significantly. This new hemofilter may improve blood purification in critically ill patients with AKI.

Today, up to 20% of all intensive care unit patients require renal replacement therapy (RRT), and continuous renal replacement therapies (CRRT) are the preferred technique. In CRRT, effective anticoagulation of the extracorporeal circuit is mandatory to prevent clotting of the circuit or filter and to maintain filter performance. At present, a variety of systemic and regional anticoagulation modes for CRRT are available. Worldwide, unfractionated heparin is the most widely used anticoagulant. All systemic techniques are associated with significant adverse effects. Most important are bleeding complications and heparin-induced thrombocytopenia (HIT-II). Regional citrate anticoagulation (RCA) is a safe and effective technique. Compared to systemic anticoagulation, RCA prolongs filter running times, reduces bleeding complications, allows effective control of acid-base status, and reduces adverse events like HIT-II. In this review, we will discuss systemic and regional anticoagulation techniques for CRRT including anticoagulation for patients with HIT-II. Today, RCA can be recommended as the therapy of choice for the majority of critically ill patients requiring CRRT.

Systemic administration of the local anaesthetic lidocaine is antinociceptive in both acute and chronic pain states, especially in acute postoperative and chronic neuropathic pain. These effects cannot be explained by its voltage-gated sodium channel blocking properties alone, but the responsible mechanisms are still elusive. This narrative review focuses on available experimental evidence of the molecular mechanisms by which systemic lidocaine exerts its clinically documented analgesic effects. These include effects on the peripheral nervous system and CNS, where lidocaine acts via silencing ectopic discharges, suppression of inflammatory processes, and modulation of inhibitory and excitatory neurotransmission. We highlight promising objectives for future research to further unravel these antinociceptive mechanisms, which subsequently may facilitate the development of new analgesic strategies and therapies for acute and chronic pain.

Healthcare personnel are at risk to aquire the corona virus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We evaluated the prevalence of SARS-CoV-2 antibodies and positive nasopharyngeal reverse transcriptase polymerase-chain reaction (RT-PCR) tests in German intensive care and emergency physicians. Physicians attending intensive care and emergency medicine training courses between June 16th and July 2nd 2020 answered a questionnaire and were screened for SARS-CoV-2 antibodies via automated electrochemiluminiscence immunoassay. We recruited 516 physicans from all parts of Germany, 445/516 (86%) worked in high risk areas, and 379/516 (73%) had treated patients with COVID-19. The overall positive rate was 18/516 (3.5%), 16/18 (89%) had antibodies against SARS-COV-2, another 2 reported previous positive RT-PCR results although antibody testing was negative. Of those positive, 7/18 (39%) were unaware of their infection. A stay abroad was stated by 173/498 (35%), mostly in Europe. 87/516 (17%) reported a febrile respiratory infection after January 1st 2020 which was related to SARS-CoV-2 in 4/87 (4.6%). Contact to COVID-19 positive relatives at home was stated by 22/502 (4.4%). This was the only significant risk factor for Covid-19 infection (Fisher´s exact test, p = 0.0005). N95 masks and eye protection devices were available for 87% and 73%, respectively. A total of 254/502 (51%) had been vaccinated against seasonal influenza. The overall SARS-CoV-2 infection rate of german physicians from intensive care and emergency medicine was low compared to reports from other countries and settings. This finding may be explained by the fact that the German health care system was not overwhelmed by the first wave of the SARS-CoV-2 pandemic.

The delivery of an effective dialysis dose in continuous renal replacement therapy (CRRT) depends on adequate anticoagulation of the extracorporeal circuit. In most patients, either systemic heparin anticoagulation (SHA) or regional citrate anticoagulation (RCA) is used. This review will outline the basics and rationale of RCA and summarize data on safety and efficacy of both techniques.The basic principle of RCA is to reduce the level of ionized calcium in the extracorporeal circuit via infusion of citrate. This way, effective anticoagulation restricted to the extracorporeal circuit is achieved. SHA and RCA were compared in a variety of studies. RCA significantly prolonged filter lifetime, reduced bleeding complications and provided excellent control of uremia and acid-base status. RCA was also safe in the majority of patients with impaired liver function, whereas caution must be exerted in those with severe multiorgan failure and persistent hyperlactatemia.RCA per se is safe and effective for anticoagulation of CRRT. Compared to SHA, efficacy of anticoagulation is improved and adverse effects are reduced. RCA can be recommended as the anticoagulation mode of choice for CRRT in most ICU patients.

Acute kidney injury (AKI) is an important health issue concerning ∼50% of patients treated in intensive care units. AKI mainly occurs after sepsis, acute ischemia, nephrotoxicity, or hypoxia and leads to severe damage of the kidney and to an increased risk of mortality. The diagnosis of AKI is currently based on creatinine urea levels and diuresis. Yet, novel markers may improve the accuracy of this diagnosis at an early stage of the disease, thereby allowing early prevention and therapy, ultimately leading to a reduction in the need for renal replacement therapy and decreased mortality. Non-protein-coding RNAs or noncoding RNAs are central players in development and disease. They are important regulatory molecules that allow a fine-tuning of gene expression and protein synthesis. This regulation is necessary to maintain homeostasis, and its dysregulation is often associated with disease development. Noncoding RNAs are present in the kidney and in body fluids and their expression is modulated during AKI. This review article assembles the current knowledge of the role of noncoding RNAs, including microRNAs, long noncoding RNAs and circular RNAs, in the pathogenesis of AKI. Their potential as biomarkers and therapeutic targets as well as the challenges to translate research findings to clinical application are discussed. Although microRNAs have entered clinical testing, preclinical and clinical trials are needed before long noncoding RNAs and circular RNAs may be considered as useful biomarkers or therapeutic targets of AKI.

Remote ischemic preconditioning (RIPC) seems to be a promising cardioprotective strategy with contradictive clinical data suggesting the anesthetic regimen influencing the favorable impact of RIPC. This study aimed to investigate whether cardio protection by RIPC is abolished by anesthetic regimens. Male Wistar rats were randomized to 6 groups. Anesthesia was either maintained by pentobarbital (Pento) alone or a combination of sevoflurane (Sevo) and remifentanil or propofol (Prop) and remifentanil in combination with and without RIPC. RIPC reduced infarct size in Pento- and Sevo-anesthetized rats (Pento-RIPC: 30% ± 9% versus Pento-control [Con]: 65% ± 6%, P < .001; Sevo-RIPC: 31% ± 6% versus Sevo-Con: 61% ± 8%, P < .001), but RIPC did not initiate cardio protection in Prop-anesthetized animals (Prop-RIPC: 59% ± 6% versus Prop-Con: 59% ± 8%, P = 1.000). Cardio protection by RIPC is abolished by Prop.

Continuous renal replacement therapy is a standard treatment in critically ill patients with acute kidney injury. All CRRT techniques provide a high low-molecular weight clearance but even with hemofiltration, clearance of middle molecules is low. We investigated whether a new super high-flux hemofilter provides effective and sustained middle molecule clearance during citrate-anticoagulated continuous venovenous hemodialysis for up to 72 h. We included 14 critically ill patients with AKI-KDIGO-III in a prospective observational trial. We measured/calculated blood and urine concentrations, clearances and sieving coefficients of eight molecules with molecular weights from 60 to 66 kDa, hemodynamic parameters and SAPS-II scores. All filters were patent at 72 h. Clearance and sieving coefficients of small solutes were high and sustained over time, those for larger solutes decreased over 72 h but remained high enough to decrease blood concentrations of solutes up to 25 kDa. Albumin serum levels remained unaffected. Catecholamine doses and SAPS-II scores decreased significantly. This new hemofilter may improve blood purification in critically ill patients with AKI.

Background Lidocaine exerts antinociceptive effects when applied systemically. The mechanisms are not fully understood but glycinergic mechanisms might be involved. The synaptic glycine concentration is controlled by glycine transporters. Whereas neurons express two types of glycine transporters, astrocytes specifically express glycine transporter 1 (GlyT1). This study focuses on effects of lidocaine and its major metabolites on GlyT1 function. Methods The effects of lidocaine and its metabolites monoethylglycinexylidide (MEGX), glycinexylidide, and N-ethylglycine on GlyT1 function were investigated in uptake experiments with [¹⁴C]-labeled glycine in primary rat astrocytes. Furthermore, the effect of lidocaine and its metabolites on glycine-induced currents were investigated in GlyT1-expressing Xenopus laevis oocytes. Results Lidocaine reduced glycine uptake only at toxic concentrations. The metabolites MEGX, glycinexylidide, and N-ethylglycine, however, significantly reduced glycine uptake (P < 0.05). Inhibition of glycine uptake by a combination of lidocaine with its metabolites at a clinically relevant concentration was diminished with increasing extracellular glycine concentrations. Detailed analysis revealed that MEGX inhibits GlyT1 function (P < 0.05), whereas N-ethylglycine was identified as an alternative GlyT1 substrate (EC₅₀ = 55 μM). Conclusions Although lidocaine does not function directly on GlyT1, its metabolites MEGX and N-ethylglycine [corrected] were shown to inhibit GlyT1-mediated glycine uptake by at least two different mechanisms. Whereas N-ethylglycine [corrected] was demonstrated to be an alternative GlyT1 substrate, MEGX was shown to inhibit GlyT1 activity in both primary astrocytes and in GlyT1-expressing Xenopuslaevis oocytes at clinically relevant concentrations. These findings provide new insights into the possible mechanisms for the antinociceptive effect of systemic lidocaine.

Neuropathic pain is accompanied by significant alterations of gene expression patterns in the somatosensory nervous system. The spinal cord is particularly prone to neuroplastic changes. Since the expression of microRNAs (miRNAs) has been linked to numerous pathophysiological processes, a contribution of miRNAs to the maladaptive plasticity of the spinal cord in neuropathic pain is possible. Aim of the present study therefore was to characterize the specific expression pattern of miRNAs in the rat spinal cord. Furthermore, we evaluated the time-dependent changes in expression patterns of spinal miRNAs in the chronic constriction injury (CCI) model of neuropathic pain in rats. Results from miRNA microarrays revealed a distinct expression pattern of miRNAs in the rat spinal cord. MiRNAs-494, -720, -690 and -668 showed the highest signal intensities. Members of the let-7 family as well as miR-124 belong to the group of the most highly expressed miRNAs. Induction of neuropathic pain by CCI did not lead to relevant differences in spinal miRNA expression levels compared to sham-operated animals at any studied time point. Therefore, modulation of miRNAs does not seem to contribute significantly to the changes in gene expression that cause neural plasticity in the spinal cord in this model of chronic neuropathic pain.

Ca(2+) and Ca(2+)-dependent signals are essential for sperm maturation and fertilization. In mouse sperm the plasma membrane Ca(2+)-ATPase (PMCA) isoform 4 plays a crucial role in Ca(2+) transport. The two major splice variants of PMCA4 are PMCA4a and PMCA4b. PMCA4a differs from PMCA4b in the mechanism of calmodulin binding and activation. PMCA4a shows a much higher basal activity and is more effective than PMCA4b in returning Ca(2+) to resting levels. Knock-out mice carrying a PMCA4-null mutation are infertile because their sperm cannot achieve a hyperactivated state of motility. As sperm reach functional maturity during their transit through the epididymis, the expression of PMCA4a and 4b was assessed in bull testis and epididymis. Quantitative PCR revealed that PMCA4b is the major splice variant in testis, caput, and corpus epididymidis. In contrast, PMCA4a is the major splice variant in cauda epididymidis, whereas sperm are transcriptionally silent. Immunohistochemical staining using a new antibody against bovine PMCA4a located the PMCA4a to the apical membrane of the epithelium of cauda epididymidis, whereas testis, caput, and corpus epididymidis were negative. Western blotting of testis, epididymis, and sperm isolated from caput and cauda epididymidis showed a much higher level of PMCA4a in cauda epididymidis and sperm from cauda epididymidis compared with testis membranes and sperm from caput epididymidis. These findings suggest that PMCA4a is transferred to bovine sperm membranes in cauda epididymidis. This isoform switch may facilitate a higher calcium turnover in sperm necessary to traverse the female genital tract.

The plasma membrane Ca(2+) -ATPase (PMCA) is the main restorer of Ca(2+) balance in sperm. Particularly, PMCA isoform 4 has an essential function in sperm fertility by its participation in gaining sperm hypermotility. PMCA activity is influenced by its lipid environment. Sperm membranes exhibit lipid raft microdomains or detergent-resistant membrane domains, enriched in sphingolipids and cholesterol, forming functional specialized areas. Lipid and protein composition of lipid rafts alters during the capacitation process, which is characterized by a cholesterol efflux. In this study, the localization of PMCA4 in lipid membrane fractions of the sperm plasma membrane was investigated. We identified PMCA4 in both the detergent-resistant membrane (DRM) and in the detergent-soluble (DS) fraction of caput and cauda sperm, respectively. Capacitation did not influence PMCA4 localization. In immunocytochemical studies PMCA4 was co-localized with the lipid raft/DRM marker caveolin in the mid piece of caput and cauda sperm. Functional studies with seminal vesicle major protein PDC-109 showed that the Ca(2+) -ATPase activity in DS fractions of cauda sperm and capacitated cauda sperm was stronger enhanced than in the DRMs. In both fractions the effect was statistically significant. In contrast, in lipid overlay experiments PDC-109 interacted stronger with the lipids extracted from DRMs than with lipids extracted from DS. Our results indicate a possible functional compartmentalization of PMCA in bull sperm membranes and point to a presumptive, yet unknown interaction partner of Ca(2+) -ATPase and PDC-109, mediating the PDC-109 action from DRMs to the DS fraction of sperm plasma membrane.

It has recently been shown in mice that the plasma membrane Ca(2+)-ATPase isoform 4 (PMCA4) is essential for sperm fertilization capacity. We analyzed whether sperm PMCA4 is formed in the rat during spermatogenesis or is synthesized in the epididymis and transferred onto sperm during sperm maturation. We could show that PMCA4 is conserved in sperm from testis to epididymis. In testis, PMCA4 mRNA was restricted to spermatogonia and early spermatocytes, while the PMCA4 protein was detected in spermatogonia, late spermatocytes, spermatids and in epididymal sperm. In epididymis PMCA4 mRNA was localized in basolateral plasma membranes of epithelial cells of the caput, corpus and cauda epididymidis. In contrast, the protein was only detectable in the epithelial cells of the caput, indicating that PMCA4 mRNA is only translated into protein in caput epithelium. In the epididymal corpus and cauda, PMCA4 mRNA and protein, respectively, was localized and in peritubular cells. Furthermore, we detected an identical distribution of PMCA4a and b splice variants in rat testis, epididymal corpus and cauda. In the caput epididymidis, where PMCA4 is located in the epithelium splice variant 4b was more prominent. Further experiments have to clarify the functional importance of the differences in the PMCA4 distribution.

Calcium (Ca(2+)) signals, produced by the opening of plasma membrane entry channels, regulate a number of functions in spermatozoa such as capacitation and motility. The mechanisms of Ca(2+) removal from the sperm, required to restore resting [Ca(2+)](i), include plasma membrane Ca(2+)-dependent ATPase (PMCA) isoenzymes as well as a plasma membrane Na(+)-Ca(2+) exchanger. We have recently shown that bovine sperm PMCA is stimulated by PDC-109, a secretory protein of bovine seminal vesicles. To demonstrate the subcellular localization and regulation of bovine sperm PMCA, we have performed cell fractionation, enzyme activity determination and Western blotting studies of PMCA in spermatozoa removed from the cauda epididymidis of bull. Fractionation of sperm heads and tails resulted in a distinct association of ATPase activity with the tail membrane fraction. In vitro stimulation studies with PDC-109 using intact and fractionated sperm showed an increase in enzyme activity up to 105% in sperm tail membranes. Furthermore, thapsigargin inhibition did not alter the stimulatory effect of PDC-109 on ATPase activity, indicating that no sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA), but only PMCA isoenzymes are involved in this effect. Western blotting studies using a polyvalent PMCA antibody showed the exclusive presence of a 135 kDa band in the tail plasma membrane fraction. To elucidate whether or not the stimulatory effect was a direct one or indirectly mediated through PKA and PKC activation, PKA and PKC inhibitors, respectively, were used in the Ca(2+)-ATPase activity assays, which was followed by PDC-109 stimulation. The stimulatory effect of PDC-109 on PMCA was still observed under these conditions, while no phosphotyrosine proteins could be detected by Western blotting in sperm extracts following PDC-109 treatment. Co-immunoprecipitation studies, PDC-109 affinity chromatography as well as overlay blots failed to show a strong association of both PMCA and PDC-109, pointing to an indirect, perhaps phospholipid-mediated effect.

The liver enzymes aspartate aminotransferase (AST), alanine aminotransferase (ALT) and lactate dehydrogenase (LDH) are commonly used but not specific markers to quantify hepatic injury. In this in vivo study it was determined whether hepatic expression and serum levels of the microRNAs (miRNA) miR-122, -21 and -223 are altered and correlated with the release of liver enzymes after warm hepatic ischaemia and reperfusion (IR). Male Wistar rats were subjected to either 45 min of partial (70%) hepatic ischaemia and 240 min of reperfusion (n=7) or sham operation (n=5). Expression levels of miR-122, -21 and -223 were analysed in serum and liver tissue by quantitative polymerase chain reaction and tested for correlation with serum activities of AST, ALT and LDH. The relative expression levels of circulating miR-122 increased after IR and correlated with the serum activity of AST, ALT and LDH. Neither increased serum level of miR-21 nor elevated relative hepatic expression of miR-223 correlated with the serum activity of liver enzymes. The hepatic expression of miR-122 was unaffected by IR. The correlation between circulating miR-122 expression levels and liver enzyme activity qualifies miR-122 as a potential biomarker of warm hepatic IR injury.