ER stress increases expression of intracellular calcium channel RyR1 to modify Ca2+ homeostasis in pancreatic beta cells.

Pancreatic beta cells maintain glucose homeostasis by secreting pulses of insulin in response to a rise in plasma glucose. Pulsatile insulin secretion occurs as a result of glucose-induced oscillations in beta-cell cytosolic Ca2+. The endoplasmic reticulum (ER) helps regulate beta-cell cytosolic Ca2+, and ER stress can lead to ER Ca2+ reduction, beta-cell dysfunction, and an increased risk of type 2 diabetes. However, the mechanistic effects of ER stress on individual calcium channels are not well understood. To determine the effects of tunicamycin-induced ER stress on ER inositol 1,4,5-triphosphate receptors (IP3Rs) and ryanodine receptors (RyRs) and their involvement in subsequent Ca2+ dysregulation, we treated INS-1 832/13 cells and primary mouse islets with ER stress inducer tunicamycin (TM). We showed TM treatment increased RyR1 mRNA without affecting RyR2 mRNA and decreased both IP3R1 and IP3R3 mRNA. Furthermore, we found stress reduced ER Ca2+ levels, triggered oscillations in cytosolic Ca2+ under subthreshold glucose conditions, and increased apoptosis and that these changes were prevented by cotreatment with the RyR1 inhibitor dantrolene. In addition, we demonstrated silencing RyR1-suppressed TM-induced subthreshold cytosolic Ca2+ oscillations, but silencing RyR2 did not affect these oscillations. In contrast, inhibiting IP3Rs with xestospongin-C failed to suppress the TM-induced cytosolic Ca2+ oscillations and did not protect beta cells from TM-induced apoptosis although xestospongin-C inclusion did prevent ER Ca2+ reduction. Taken together, these results show changes in RyR1 play a critical role in ER stress-induced Ca2+ dysfunction and beta-cell apoptosis.

Pyridylpiperazine-based allosteric inhibitors of RND-type multidrug efflux pumps.

Efflux transporters of the RND family confer resistance to multiple antibiotics in Gram-negative bacteria. Here, we identify and chemically optimize pyridylpiperazine-based compounds that potentiate antibiotic activity in E. coli through inhibition of its primary RND transporter, AcrAB-TolC. Characterisation of resistant E. coli mutants and structural biology analyses indicate that the compounds bind to a unique site on the transmembrane domain of the AcrB L protomer, lined by key catalytic residues involved in proton relay. Molecular dynamics simulations suggest that the inhibitors access this binding pocket from the cytoplasm via a channel exclusively present in the AcrB L protomer. Thus, our work unveils a class of allosteric efflux-pump inhibitors that likely act by preventing the functional catalytic cycle of the RND pump.

Binding of Tetracyclines to Acinetobacter baumannii TetR Involves Two Arginines as Specificity Determinants.

Acinetobacter baumannii is an important nosocomial pathogen that requires thoughtful consideration in the antibiotic prescription strategy due to its multidrug resistant phenotype. Tetracycline antibiotics have recently been re-administered as part of the combination antimicrobial regimens to treat infections caused by A. baumannii. We show that the TetA(G) efflux pump of A. baumannii AYE confers resistance to a variety of tetracyclines including the clinically important antibiotics doxycycline and minocycline, but not to tigecycline. Expression of tetA(G) gene is regulated by the TetR repressor of A. baumannii AYE (AbTetR). Thermal shift binding experiments revealed that AbTetR preferentially binds tetracyclines which carry a O-5H moiety in ring B, whereas tetracyclines with a 7-dimethylamino moiety in ring D are less well-recognized by AbTetR. Confoundingly, tigecycline binds to AbTetR even though it is not transported by TetA(G) efflux pump. Structural analysis of the minocycline-bound AbTetR-Gln116Ala variant suggested that the non-conserved Arg135 interacts with the ring D of minocycline by cation-π interaction, while the invariant Arg104 engages in H-bonding with the O-11H of minocycline. Interestingly, the Arg135Ala variant exhibited a binding preference for tetracyclines with an unmodified ring D. In contrast, the Arg104Ala variant preferred to bind tetracyclines which carry a O-6H moiety in ring C except for tigecycline. We propose that Arg104 and Arg135, which are embedded at the entrance of the AbTetR binding pocket, play important roles in the recognition of tetracyclines, and act as a barrier to prevent the release of tetracycline from its binding pocket upon AbTetR activation. The binding data and crystal structures obtained in this study might provide further insight for the development of new tetracycline antibiotics to evade the specific efflux resistance mechanism deployed by A. baumannii.

Allosteric drug transport mechanism of multidrug transporter AcrB.

Gram-negative bacteria maintain an intrinsic resistance mechanism against entry of noxious compounds by utilizing highly efficient efflux pumps. The E. coli AcrAB-TolC drug efflux pump contains the inner membrane H+/drug antiporter AcrB comprising three functionally interdependent protomers, cycling consecutively through the loose (L), tight (T) and open (O) state during cooperative catalysis. Here, we present 13 X-ray structures of AcrB in intermediate states of the transport cycle. Structure-based mutational analysis combined with drug susceptibility assays indicate that drugs are guided through dedicated transport channels toward the drug binding pockets. A co-structure obtained in the combined presence of erythromycin, linezolid, oxacillin and fusidic acid shows binding of fusidic acid deeply inside the T protomer transmembrane domain. Thiol cross-link substrate protection assays indicate that this transmembrane domain-binding site can also accommodate oxacillin or novobiocin but not erythromycin or linezolid. AcrB-mediated drug transport is suggested to be allosterically modulated in presence of multiple drugs.

Selective intra-carotid blood cooling in acute ischemic stroke: A safety and feasibility study in an ovine stroke model.

Selective therapeutic hypothermia (TH) showed promising preclinical results as a neuroprotective strategy in acute ischemic stroke. We aimed to assess safety and feasibility of an intracarotid cooling catheter conceived for fast and selective brain cooling during endovascular thrombectomy in an ovine stroke model.Transient middle cerebral artery occlusion (MCAO, 3 h) was performed in 20 sheep. In the hypothermia group (n = 10), selective TH was initiated 20 minutes before recanalization, and was maintained for another 3 h. In the normothermia control group (n = 10), a standard 8 French catheter was used instead. Primary endpoints were intranasal cooling performance (feasibility) plus vessel patency assessed by digital subtraction angiography and carotid artery wall integrity (histopathology, both safety). Secondary endpoints were neurological outcome and infarct volumes.Computed tomography perfusion demonstrated MCA territory hypoperfusion during MCAO in both groups. Intranasal temperature decreased by 1.1 °C/3.1 °C after 10/60 minutes in the TH group and 0.3 °C/0.4 °C in the normothermia group (p < 0.001). Carotid artery and branching vessel patency as well as carotid wall integrity was indifferent between groups. Infarct volumes (p = 0.74) and neurological outcome (p = 0.82) were similar in both groups.Selective TH was feasible and safe. However, a larger number of subjects might be required to demonstrate efficacy.

Quality and validity of large animal experiments in stroke: A systematic review.

An important factor for successful translational stroke research is study quality. Low-quality studies are at risk of biased results and effect overestimation, as has been intensely discussed for small animal stroke research. However, little is known about the methodological rigor and quality in large animal stroke models, which are becoming more frequently used in the field. Based on research in two databases, this systematic review surveys and analyses the methodological quality in large animal stroke research. Quality analysis was based on the Stroke Therapy Academic Industry Roundtable and the Animals in Research: Reporting In Vivo Experiments guidelines. Our analysis revealed that large animal models are utilized with similar shortcomings as small animal models. Moreover, translational benefits of large animal models may be limited due to lacking implementation of important quality criteria such as randomization, allocation concealment, and blinded assessment of outcome. On the other hand, an increase of study quality over time and a positive correlation between study quality and journal impact factor were identified. Based on the obtained findings, we derive recommendations for optimal study planning, conducting, and data analysis/reporting when using large animal stroke models to fully benefit from the translational advantages offered by these models.

Corrigendum: Development of a Routinely Applicable Imaging Protocol for Fast and Precise Middle Cerebral Artery Occlusion Assessment and Perfusion Deficit Measure in an Ovine Stroke Model: A Case Study.

[This corrects the article DOI: 10.3389/fneur.2019.01113.].

Binding and Transport of Carboxylated Drugs by the Multidrug Transporter AcrB.

AcrAB(Z)-TolC is the main drug efflux transporter complex in Escherichia coli. The extrusion of various toxic compounds depends on several drug binding sites within the trimeric AcrB transporter. Membrane-localized carboxylated substrates, such as fusidic acid and hydrophobic ß-lactams, access the pump via a groove between the transmembrane helices TM1 and TM2. In this article, the transport route from the initial TM1/TM2 groove binding site toward the deep binding pocket located in the periplasmic part has been addressed via molecular modeling studies followed by functional and structural characterization of several AcrB variants. We propose that membrane-embedded drugs bind initially to the TM1/TM2 groove, are oriented by the AcrB PN2 subdomain, and are subsequently transported via a PN2/PC1 interface pathway directly toward the deep binding pocket. Our work emphasizes the exploitation of multiple transport pathways by AcrB tuned to substrate physicochemical properties related to the polyspecificity of the pump.

Development of a Routinely Applicable Imaging Protocol for Fast and Precise Middle Cerebral Artery Occlusion Assessment and Perfusion Deficit Measure in an Ovine Stroke Model: A Case Study.

Temporary middle cerebral artery occlusion (MCAO) in sheep allows modeling of acute large vessel occlusion stroke and subsequent vessel recanalization. However, rapid and precise imaging-based assessment of vessel occlusion and the resulting perfusion deficit during MCAO still represents an experimental challenge. Here, we tested feasibility and suitability of a strategy for MCAO verification and perfusion deficit assessment. We also compared the extent of the initial perfusion deficit and subsequent lesion size for different MCAO durations. The rete mirabile prevents reliable vascular imaging investigation of middle cerebral artery filling status. Hence, computed tomography perfusion imaging was chosen for indirect confirmation of MCAO. Follow-up infarct size evaluation by diffusion-weighted magnetic resonance imaging revealed fluctuating results, with no apparent relationship of lesion size with MCAO at occlusion times below 4 h, potentially related to the variable collateralization of the MCA territory. This underlines the need for intra-ischemic perfusion assessment and future studies focusing on the correlation between perfusion deficit, MCAO duration, and final infarct volume. Temporary MCAO and intra-ischemic perfusion imaging nevertheless has the potential to be applied for the simulation of novel recanalization therapies, particularly those that aim for a fast reperfusion effect in combination with mechanical thrombectomy in a clinically realistic scenario.

Large animals in neurointerventional research: A systematic review on models, techniques and their application in endovascular procedures for stroke, aneurysms and vascular malformations.

Neuroendovascular procedures have led to breakthroughs in the treatment of ischemic stroke, intracranial aneurysms, and intracranial arteriovenous malformations. Due to these substantial successes, there is continuous development of novel and refined therapeutic approaches. Large animal models feature various conceptual advantages in translational research, which makes them appealing for the development of novel endovascular treatments. However, the availability and role of large animal models have not been systematically described so far. Based on comprehensive research in two databases, this systematic review describes current large animal models in neuroendovascular research including their primary use. It may therefore serve as a compact compendium for researchers entering the field or looking for opportunities to refine study concepts. It also describes particular applications for ischemic stroke and aneurysm therapy, as well as for the treatment of arteriovenous malformations. It focuses on most promising study designs and readout parameters, as well as on important pitfalls in endovascular translational research including ways to circumvent them.

Dose-intensified granulocyte-monocyte apheresis in therapy refractory ulcerative colitis.

Granulocyte-monocyte apheresis (GMA) is an emerging therapeutic option in active course of ulcerative colitis (UC). Appropriate GMA dose, including total number, frequency, and duration of the individual GMA session, is a matter of debate. It was the aim of the present study to evaluate the efficacy of a dose-intensified GMA regimen in patients with moderately to severely active UC. A prospective open-label, single-center study was performed in 10 patients with active UC (Rachmilewitz Clinical Activity Index [CAI] ≥ 8 points; Rachmilewitz Endoscopic Index ≥ 7 points). Patients had failed to improve after treatment with steroids and/or immunomodulators. GMA was performed twice weekly for 2 h to a maximum of 10 sessions. In each GMA session, the adsorber was changed after 1 h of treatment time. Four patients achieved remission with a CAI ≤ 4 points. Three patients had a response with an improvement of CAI of ≥3 points. Three patients showed no benefit from GMA. The quality of life score determined by the inflammatory bowel disease questionnaire-Deutschland increased by 26 points in median. First and second filters had similar efficiency in granulocyte and monocyte adsorption. No major adverse effects were observed. Dose-intensified GMA as reported in this study provided an encouraging short-term response rate of 70% in patients with moderately to severely active UC not responding to standard steroid or immunomodulator therapy. Although all patients relapsed not later than 16 weeks, GMA might be useful to reduce steroid and immunomodulator usage, or to delay surgery in this patient group.

MediaDB: a database of microbial growth conditions in defined media.

Isolating pure microbial cultures and cultivating them in the laboratory on defined media is used to more fully characterize the metabolism and physiology of organisms. However, identifying an appropriate growth medium for a novel isolate remains a challenging task. Even organisms with sequenced and annotated genomes can be difficult to grow, despite our ability to build genome-scale metabolic networks that connect genomic data with metabolic function. The scientific literature is scattered with information about defined growth media used successfully for cultivating a wide variety of organisms, but to date there exists no centralized repository to inform efforts to cultivate less characterized organisms by bridging the gap between genomic data and compound composition for growth media. Here we present MediaDB, a manually curated database of defined media that have been used for cultivating organisms with sequenced genomes, with an emphasis on organisms with metabolic network models. The database is accessible online, can be queried by keyword searches or downloaded in its entirety, and can generate exportable individual media formulation files. The data assembled in MediaDB facilitate comparative studies of organism growth media, serve as a starting point for formulating novel growth media, and contribute to formulating media for in silico investigation of metabolic networks. MediaDB is freely available for public use at https://mediadb.systemsbiology.net.

Small-GTPase-associated signaling by the guanine nucleotide exchange factors CpDock180 and CpCdc24, the GTPase effector CpSte20, and the scaffold protein CpBem1 in Claviceps purpurea.

Monomeric GTPases of the Rho subfamily are important mediators of polar growth and NADPH (Nox) signaling in a variety of organisms. These pathways influence the ability of Claviceps purpurea to infect host plants. GTPase regulators contribute to the nucleotide loading cycle that is essential for proper functionality of the GTPases. Scaffold proteins gather GTPase complexes to facilitate proper function. The guanine nucleotide exchange factors (GEFs) CpCdc24 and CpDock180 activate GTPase signaling by triggering nucleotide exchange of the GTPases. Here we show that CpCdc24 harbors nucleotide exchange activity for both Rac and Cdc42 homologues. The GEFs partly share the cellular distribution of the GTPases and interact with the putative upstream GTPase CpRas1. Interaction studies show the formation of higher-order protein complexes, mediated by the scaffold protein CpBem1. Besides the GTPases and GEFs, these complexes also contain the GTPase effectors CpSte20 and CpCla4, as well as the regulatory protein CpNoxR. Functional characterizations suggest a role of CpCdc24 mainly in polarity, whereas CpDock180 is involved in stress tolerance mechanisms. These findings indicate the dynamic formation of small GTPase complexes and improve the model for GTPase-associated signaling in C. purpurea.

Molecular characterization of the NADPH oxidase complex in the ergot fungus Claviceps purpurea: CpNox2 and CpPls1 are important for a balanced host-pathogen interaction.

Reactive oxygen species producing NADPH oxidase (Nox) complexes are involved in defense reactions in animals and plants while they trigger infection-related processes in pathogenic fungi. Knowledge about the composition and localization of these complexes in fungi is limited; potential components identified thus far include two to three catalytical subunits, a regulatory subunit (NoxR), the GTPase Rac, the scaffold protein Bem1, and a tetraspanin-like membrane protein (Pls1). We showed that, in the biotrophic grass-pathogen Claviceps purpurea, the catalytical subunit CpNox1 is important for infection. Here, we present identification of major Nox complex partners and a functional analysis of CpNox2 and the tetraspanin CpPls1. We show that, as in other fungi, Nox complexes are important for formation of sclerotia; CpRac is, indeed, a complex partner because it interacts with CpNoxR, and CpNox1/2 and CpPls1 are associated with the endoplasmatic reticulum. However, unlike in all other fungi, Δcppls1 is more similar to Δcpnox1 than to Δcpnox2, and CpNox2 is not essential for infection. In contrast, Δcpnox2 shows even more pronounced disease symptoms, indicating that Cpnox2 controls the infection process and moderates damage to the host. These data confirm that fungal Nox complexes have acquired specific functions dependent of the lifestyle of the pathogen.

The quantitative estimation of IT-related risk probabilities.

How well can people estimate IT-related risk? Although estimating risk is a fundamental activity in software management and risk is the basis for many decisions, little is known about how well IT-related risk can be estimated at all. Therefore, we executed a risk estimation experiment with 36 participants. They estimated the probabilities of IT-related risks and we investigated the effect of the following factors on the quality of the risk estimation: the estimator's age, work experience in computing, (self-reported) safety awareness and previous experience with this risk, the absolute value of the risk's probability, and the effect of knowing the estimates of the other participants (see: Delphi method). Our main findings are: risk probabilities are difficult to estimate. Younger and inexperienced estimators were not significantly worse than older and more experienced estimators, but the older and more experienced subjects better used the knowledge gained by knowing the other estimators' results. Persons with higher safety awareness tend to overestimate risk probabilities, but can better estimate ordinal ranks of risk probabilities. Previous own experience with a risk leads to an overestimation of its probability (unlike in other fields like medicine or disasters, where experience with a disease leads to more realistic probability estimates and nonexperience to an underestimation).

Advanced hemodynamic monitoring before and after transjugular intrahepatic portosystemic shunt: implications for selection of patients--a prospective study.

PURPOSE: To investigate immediate and short-term effects of transjugular intrahepatic portosystemic shunt (TIPS) on cardiocirculatory, hepatic, and renal function and characterize predictors for TIPS outcome in terms of organ function after TIPS. MATERIALS AND METHODS: This prospective study was approved by the ethics committee at a university hospital and was conducted in a medical intensive care unit. Informed consent was obtained. Twenty patients with indication for TIPS were enrolled. Monitoring of hemodynamic and hepatic function (transpulmonary thermodilution, indocyanine green plasma disappearance rate [ICG-PDR]) was performed. Biochemical markers of organ function were obtained. Statistical analysis (Wilcoxon test, Spearman correlation, multivariate linear regression analysis, receiver operating characteristic [ROC] analysis) was performed. RESULTS: After TIPS, central venous pressure (median, 11 vs 15 cm H(2)O; P < .001), cardiac index (3.4 vs 3.8 L/min/m(2); P = .001), and global end-diastolic volume index (GEDVI) (726 vs 775 mL/m(2); P = .003) increased significantly. Portosystemic pressure gradient (28 vs 11 cm H(2)O; P < .001) and systemic vascular resistance index (1610 vs 1384 dyn · sec · cm(-5) · m(2); P = .015) decreased significantly. Creatinine (1.1 vs 1.1 mg/dL; P = .008) and blood urea nitrogen (BUN) (27 vs 21 mg/dL; P = .006) decreased significantly. Bilirubin (1.8 vs 2.2 mg/dL; P = .032) and international normalized ratio (1.4 vs 1.5; P = .022) increased significantly. ICG-PDR significantly deteriorated after TIPS (P = .006). Higher baseline creatinine was independently associated with a decrease in creatinine after TIPS (R = 0.816, P < .001). ROC analysis identified baseline BUN (P = .026, area under ROC curve [AUC] = 0.818), cystatin C (P = .033, AUC = 0.805), and creatinine (P = .052, AUC = 0.779) as predictors of a decrease in creatinine of 0.5 mg/dL or greater and/or 25% or greater. An increase in bilirubin of 1 mg/dL or greater 1 week after TIPS was significantly associated with high baseline BUN (P = .007, AUC = 0.893) and high central venous pressure (P = .040, AUC = 0.800). Lower baseline alanine aminotransferase (P = .002, AUC = 1.000) and cardiac power index · GEDVI (P = .005, AUC = 0.960) predicted favorable TIPS outcome (creatinine decrease of ≥ 0.2 mg/dL without model for end-stage liver disease score increase of more than one point). CONCLUSION: Patients with renal insufficiency, compensated hepatocellular function, decreased cardiac preload, and decreased cardiac performance benefit most from TIPS.