Benefits and harms of perioperative high fraction inspired oxygen for surgical site infection prevention: a protocol for a systematic review and meta-analysis of individual patient data of randomised controlled trials.

INTRODUCTION The use of high fraction of inspired oxygen (FiO2) intraoperatively for the prevention of surgical site infection (SSI) remains controversial. Promising results of early randomised controlled trials (RCT) have been replicated with varying success and subsequent meta-analysis are equivocal. Recent advancements in perioperative care, including the increased use of laparoscopic surgery and pneumoperitoneum and shifts in fluid and temperature management, can affect peripheral oxygen delivery and may explain the inconsistency in reproducibility. However, the published data provides insufficient detail on the participant level to test these hypotheses. The purpose of this individual participant data meta-analysis is to assess the described benefits and harms of intraoperative high FiO2compared with regular (0.21-0.40) FiO2 and its potential effect modifiers. METHODS AND ANALYSIS Two reviewers will search medical databases and online trial registries, including MEDLINE, Embase, CENTRAL, CINAHL, ClinicalTrials.gov and WHO regional databases, for randomised and quasi-RCT comparing the effect of intraoperative high FiO2 (0.60-1.00) to regular FiO2 (0.21-0.40) on SSI within 90 days after surgery in adult patients. Secondary outcome will be all-cause mortality within the longest available follow-up. Investigators of the identified trials will be invited to collaborate. Data will be analysed with the one-step approach using the generalised linear mixed model framework and the statistical model appropriate for the type of outcome being analysed (logistic and cox regression, respectively), with a random treatment effect term to account for the clustering of patients within studies. The bias will be assessed using the Cochrane risk-of-bias tool for randomised trials V.2 and the certainty of evidence using Grading of Recommendations, Assessment, Development and Evaluation methodology. Prespecified subgroup analyses include use of mechanical ventilation, nitrous oxide, preoperative antibiotic prophylaxis, temperature (2.5 hour). ETHICS AND DISSEMINATION Ethics approval is not required. Investigators will deidentify individual participant data before it is shared. The results will be submitted to a peer-review journal. PROSPERO REGISTRATION NUMBER CRD42018090261

Plasticity in D1-Like Receptor Expression Is Associated with Different Components of Cognitive Processes

Dopamine D1-like receptors consist of D1 (D1A) and D5 (D1B) receptors and play a key role in working memory. However, their possibly differential contribution to working memory is unclear. We combined a working memory training protocol with a stepwise increase of cognitive subcomponents and real-time RT-PCR analysis of dopamine receptor expression in pigeons to identify molecular changes that accompany training of isolated cognitive subfunctions. In birds, the D1-like receptor family is extended and consists of the D1A, D1B, and D1D receptors. Our data show that D1B receptor plasticity follows a training that includes active mental maintenance of information, whereas D1A and D1D receptor plasticity in addition accompanies learning of stimulus-response associations. Plasticity of D1-like receptors plays no role for processes like response selection and stimulus discrimination. None of the tasks altered D2 receptor expression. Our study shows that different cognitive components of working memory training have distinguishable effects on D1-like receptor expression

Die Rolle der Gerüstproteine an der Aktiven Zone in der Verteilung und Freisetzungswahrscheinlichkeit synaptischer Vesikel

At neuronal cell-cell-contacts called synapses, transmitter is released from the synaptic vesicles (SVs) into the synaptic cleft at specialized areas of the plasma membrane, so-called Active Zones (AZs). Here, upon arrival of an action potential, Ca2+ flows into the cell through voltage-gated Ca2+ channels and triggers SV fusion; the Ca2+ sensor is located on the SV membrane. At the postsynapse, the transmitter binds to membrane-residing receptors and causes a change in membrane potential. At the AZ, few SVs are in direct contact with the cell membrane (docked SV) which partially serve as an immediately release- ready pool for efficient and reliable synaptic transmission upon stimulation. Due to a limitation in the number of Ca2+ channels, the change in presynaptic Ca2+ concentration is locally restricted and forms a so-called nanodomain. A fast and direct SV fusion upon Ca2+ influx is accomplished by SV placement in a sufficiently small distance to the Ca2+ source to obtain tight coupling. In my thesis, I studied the role of AZ components in SV distribution and the functional conse-quences of SV rearrangements at the neuromuscular junction (NMJ) of Drosophila melanogaster larvae. I focused my work in particular on the priming factor Unc13; additionally, the roles of the AZ components BRP (a large scaffold protein), Phosphatidylinositol-4 kinase PI4KIIIα (which influences lipid composition at the AZ), and Rabconnectin3B and Spinophilin (regulatory proteins) in synaptic transmission were analyzed. To unveil aspects of SV docking and priming, I characterized mutant larvae mainly in electron microscopy (EM) and electrophysiological recordings, partly with Ca2+ buffer treatment. In these investigations, I focused specifically on SV placement, vesicle release probabilities, and the Ca2+ dependence of release. Two Unc13 isoforms are expressed in Drosophila, namely Unc13A and Unc13B. In this work it was found that the two Unc13 variants are localized in an isoform-specific pattern at the AZ. Unc13A is located in ~60 nm distance to the AZ center. I could show that Unc13A is the essential isoform for synaptic transmission and responsible for tight coupling of SVs to the Ca2+ channels. Unc13B is located in ~120 nm distance to the Ca2+ channels, and was not found to be especially relevant for signal transduction at the NMJ. Furthermore, the Unc13 N-term was found to be crucial in restricting the protein localization to the aforementioned sites at the AZ. Thus, Unc13 is the only identified protein of the core fusion machinery (additionally consisting of SNAREs and Unc18) which is not distributed over the whole neuronal membrane, but whose specific localization determines the sites of release. Via the BRP C-term, SVs tether to the T-Bar, an electron-dense structure at the AZs of Drosophila. Upon loss of the last 17 amino acids of BRP, the T-Bar is devoid of SVs, and the brp allele is thus called brpnude. I could show that the altered SV distribution in brpnude leads to a decreased SV replenishment rate at the release sites. Furthermore, I established in vivo Ca2+ imaging to investigate the participation of single AZs in spontaneous and evoked release at the NMJ. Here, I could show a profound heterogeneous distribution between individual AZs concerning the probability to participate in evoked and/or spontaneous release. Additionally it was found that evoked SV fusion is not only dependent on the BRP level the AZ, where with increasing BRP levels the participation in evoked release was proportionally elevated, but that evoked release is furthermore affected by the presence of Spinophilin. In addition, the lipid composition at the plasma membrane alters SV release probability. In this work, I could show that the AZ component PI4KIIIα altered synaptic transmission properties. In summary, I discovered that SV release probability is highly influenced by the spatial arrange-ment of SVs at the AZ, which in turn is altered by a number of AZ proteins I characterized and describe in this work. The location of docked SVs is determined by Unc13, which in turn defines the sites of SV fusion. An altered distribution of T-Bar associated SVs, as observed in brpnude, leads to a diminished rate of SV recruitment rate to the fusion sites at the AZ. Furthermore, the composition of the protein matrix and the plasma membrane at the AZ influences synaptic transmission in general, and alters the probability to participate in evoked release already at the level of individual AZs in particular.Neurone haben besondere Zell-Zell-Kontakte namens Synapsen, wo synaptische Vesikel (SV) an spezialisierten Bereichen der Plasmamembran, sog. Aktiven Zonen (AZ), fusionieren und ihren Inhalt in den synaptischen Spalt freisetzen. Bei Eintreffen eines Aktionspotentials an der Präsynapse strömt durch spannungsgesteuerte Ca2+-Kanäle Ca2+ ein und löst innerhalb von Millisekunden die Transmitterauschüttung aus. Der Transmitter bindet an Rezeptoren auf der postsynaptischen Zelle und verursacht dort wiederum eine Veränderung des Membranpotentials. Die SV sind teilweise schon direkt an der AZ angelagert (diese SV sind "gedockt"). Bei Ca2+ Einstrom können gedockte, fusionskompetente SV unverzüglich mit der Plasmamembran fusionieren. Der Ca2+ Sensor befindet sich auf den SV. Durch die begrenzte Anzahl an Ca2+ Kanälen bildet sich nur ein lokaler Ca2+ Konzentrationsgradient mit kleiner Reichweite aus, die sog. Ca2+ Nanodomäne. Eine schnelle und unvermittelte SV-Fusion ist gewährleistet, wenn die SV sich in ausreichend kleiner Distanz zur Ca2+ Quelle befinden, man spricht auch von enger Kopplung. In meiner Doktorarbeit habe ich die Funktion einzelner AZ-Proteine bei der Verteilung der SV an der Synapse untersucht. In diesem Zusammenhang wurde an der Modellsynapse der neuromuskulären Endplatte (neuromuscular junction, NMJ) in Drosophila melanogaster Larven die Auswirkung von veränderter Vesikelanordung auf die Freisetzungswahrscheinlichkeit von synaptischen Vesikeln ermittelt. Meine Arbeit fokussierte sich dabei vor allem auf den Primingfaktor Unc13; außerdem wurde die Rolle des Gerüstproteins BRP, der Phosphatidylinositol-4 Kinase PI4KIIIα, die die Lipidzusammensetzung der Plasmamembran verändert, und den Regulatorproteinen Rabconnectin3B und Spinophilin in der synaptischen Transmission untersucht. Dafür wurden transgene Larven hauptsächlich elektronenmikroskopisch analysiert und elektrophysiologischen Messungen unterzogen, teilweise unter Anwendung von Ca2+ Puffern. Hierbei konzentrierte ich mich im Speziellen auf die Analyse der Platzierung der SV relativ zur Ca2+ Quelle, ihre Freisetzungswahrscheinlichkeit und die Ca2+-Abhängigkeit der Freisetzung. In Drosophila sind zwei Isoformen von Unc13 bekannt, Unc13A und Unc13B. In dieser Arbeit wurde festgestellt, dass die beiden Varianten an isoformspezifischen Positionen an der AZ verteilt sind. Unc13A, das in ca. 60 nm Entfernung zum Zentrum der AZ lokalisiert ist, wurde als die essentielle Isoform für synaptische Transmission identifiziert. Sie ist verantwortlich für eine enge Kopplung der SV zu Ca2+ Kanälen und gewährleistet schnelle und zuverlässige Transmitterausschüttung. Unc13B befindet sich in ca. 120 nm Entfernung zum AZ-Zentrum und hat nur eine untergeordnete Rolle in der Signalübertragung an der NMJ. Darüber hinaus wurde der N-Term von Unc13 als notwendige Lokalisierungseinheit identifiziert, die die Lage des Protein an die o.g. Stellen an der AZ begrenzt. Dadurch wurde Unc13 als einziges Mitglied der Kern-Fusionsmaschinerie (neben den SNARE-Proteinen und Unc18) ermittelt, das nicht über die gesamte neuronale Membran verteilt ist, sondern dessen Lokalisierung die physikalischen Fusionsnischen in AZ determiniert. Über den BRP C-Term binden SV an den T-Bar, die elektronendichte Struktur, die an der präsy-naptischen Membran angelagert ist. Bei Verlust der letzten 17 Aminosäuren des C-Terms ist der T-Bar frei von SV, das entsprechende Allel heißt brpnude. Es konnte gezeigt werden, dass in brpnude die veränderte Verteilung der T-Bar-assoziierten SV ihre Rekrutierung zur AZ beeinträchtigt. Weiterhin habe ich in vivo Ca2+ imaging im Labor etabliert, um die Beteiligung einzelner AZ in spontaner und evozierter Vesikelfusion zu untersuchen. Dabei wurde festgestellt, dass die Teilnahme an einem oder beiden Modi (spontan oder evoziert) unter den AZ einer NMJ hochgradig heterogen verteilt ist. Außerdem konnte gezeigt werden, dass die Vesikelfusion nicht nur vom BRP-Level an der AZ abhängt, denn je mehr BRP vorhanden ist, desto größer ist die Wahrscheinlichkeit der AZ an evozierter SV-Fusion beteiligt zu sein; weiterhin wurde gezeigt, dass auch Spinophilin für evozierte Vesikelfusion relevant ist. Doch nicht nur die Anwesenheit bestimmter Proteine an der AZ ist relevant für die synaptische Transmission, auch die Lipidkomposition der Plasmamembran wirkt sich auf die Fusionswahr-scheinlichkeit aus. Es konnte in dieser Arbeit gezeigt werden, dass auch die an der AZ befindliche PI4KIIIα die synaptische Transmission beeinflusst. Zusammenfassend konnte ich zeigen, dass Verteilung der SV an der AZ maßgebliche Bedeutung für ihre Freisetzungswahrscheinlichkeit hat. Ich habe mehrere Komponenten der Proteinmatrix der AZ identifiziert und charakterisiert, die die SV Verteilung beeinflussen. Der Ort gedockter SV wird von Unc13 festgelegt; Unc13 determiniert somit die Orte der SV-Fusion und hat damit einen direkten und maßgeblichen Einfluss auf die Freisetzungswahrscheinlichkeit von SV. Ist die Verteilung der T-Bar- assoziierten SV verändert wie in brpnude, ist die Rekrutierung der SV zu den Fusionsstellen eingeschränkt. Darüber hinaus bestimmen sowohl die Komposition der Proteinmatrix als auch Lipide der Plasmamembran an der Aktiven Zone ganz generell die Wahrscheinlichkeit der evozierten und spontanen Transmitterausschüttung schon innerhalb einer einzelnen NMJ (wie in Fall von Spinophilin gezeigt wurde)

Local Anesthetics, Procaine, Lidocaine, and Mepivacaine Show Vasodilatation but No Type 1 Allergy: A Double-Blind, Placebo-Controlled Study

Background. Therapy with local anesthetics (LAs), also known as neural therapy, is used in integrative medicine because of its anti-inflammatory properties. Ester-linked LAs are often avoided because of their alleged high allergenicity. Little data supports this assumption and hence the importance of our investigation on type-1 allergies against ester- and amide-linked LAs. We performed a prospective, double-blinded, placebo-controlled observational study. Methods. 177 patients received 340 intracutaneous injections with 1% procaine, 0.5% lidocaine, 1% mepivacaine, or saline solution. Every patient received two different tests on each forearm. Reactions were monitored for 15 minutes. Results. No type-1 allergy was observed. The mean erythema diameter of the wheals after 10 minutes was procaine 8.0±6.4 mm, mepivacaine 7.6±6.3 mm, lidocaine 4.4±4.8 mm, and NaCl 3.7±3.2 mm. The wheal diameter of all substances showed a crescendo-decrescendo phenomenon. The procaine and mepivacaine wheals were significantly larger than those of lidocaine and NaCl. No general signs of hypersensitivity were observed. Diameter and intensity were independent of the injection site, order of injection, age, gender, and body mass index. Conclusion. This study shows no higher type-1 allergenicity of the ester-linked LA procaine, compared to the amide-linked LAs lidocaine and mepivacaine, and supports its use in therapy and short-track surgery

Presynaptic spinophilin tunes neurexin signalling to control active zone architecture and function

Assembly and maturation of synapses at the Drosophila neuromuscular junction (NMJ) depend on trans-synaptic neurexin/neuroligin signalling, which is promoted by the scaffolding protein Syd-1 binding to neurexin. Here we report that the scaffold protein spinophilin binds to the C-terminal portion of neurexin and is needed to limit neurexin/neuroligin signalling by acting antagonistic to Syd-1. Loss of presynaptic spinophilin results in the formation of excess, but atypically small active zones. Neuroligin-1/neurexin-1/Syd-1 levels are increased at spinophilin mutant NMJs, and removal of single copies of the neurexin-1, Syd-1 or neuroligin-1 genes suppresses the spinophilin-active zone phenotype. Evoked transmission is strongly reduced at spinophilin terminals, owing to a severely reduced release probability at individual active zones. We conclude that presynaptic spinophilin fine-tunes neurexin/neuroligin signalling to control active zone number and functionality, thereby optimizing them for action potential-induced exocytosis

Schematic illustration of the different paradigms for the animal groups in cognitive training.

<p>(A) Control group without training in an operant task. (B) S-R task. During training with colored operant keys, each trial started with the presentation of either a green or a red stimulus on one of the three keys. After 15 correct pecks the REWARD phase started with 3 s food access. This was followed by an intertrial interval (ITI) before the next trial started. (C) SMTS task. Training in the simultaneous matching-to-sample task always started with the presentation of either a green or red stimulus as the SAMPLE on the central key. 15 pecks onto this directly started the CHOICE period, where the pigeons had to peck the lateral key that matched the color of the sample. During this phase all keys were simultaneously illuminated. No maintaining of information was required. A single correct peck started the REWARD phase with 3 s food access. This was followed by an ITI before the next trial started. (D) DMTS task. During training of the delayed matching-to-sample task each trial started with the presentation of either a green or red stimulus as the SAMPLE on the central key. 15 pecks onto this started a 4 s DELAY period during which the animals had to memorize the sample color. Then, the lateral keys lit and started the CHOICE period, where the pigeons had to peck the lateral key that matched the color of the sample. A single correct peck started the REWARD phase with 3 s food access. This was followed by an ITI before the next trial started.</p

Differences of D1-like mRNA levels in the NCL (A) and the anterior forebrain (aFB; B) between the trained groups.

<p>In the NCL and in the aFB, D1A receptor expression levels decreased in the S-R and in the SMTS groups, and increased to control levels after training in the DMTS group. D1B receptor expression increased in both areas in the DMTS group. D1D receptor expression levels decreased in the S-R and the SMTS groups in both areas, and increased to control levels in the NCL while increasing above control levels in the aFB. Thus, a rigid training program that involved a reward-dependent learning of an association between external stimuli and own responses resulted in a down-regulation of the expression of D1A and D1D. D1B expression is only affects after DMTS training. A sole comparison of control and DMTS tasks would have resulted in the wrong conclusion that a DMTS procedure increases D1B expression levels but has no effect on D1A or D1D. All data is presented as mean ± SEM; n = 10 each group. All statistical analyses were only performed on the original data (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036484#pone-0036484-g003" target="_blank">Figure 3</a>). Significant differences between groups are marked with asterisks (*p<0.05; **p<0.01; ***p<0.001).</p

Quantification of dopamine receptor (DAR) mRNA levels in the NCL (A) and the anterior forebrain (aFB; B) of the control and the trained groups.

<p>Expression of different DA receptors at the mRNA level is shown relative to the expression of the housekeeping gene histone H3.3B (mean ± SEM; n = 10 each group). Significant differences between groups are marked with asterisks (*p<0.05; **p<0.01; ***p<0.001).</p

Primers used for real-time RT-PCR.

<p>The primers were used for quantitative RT-PCR. Each primer pair binds specifically the indicated gene without cross-reactions. The obtained fragments were verified by sequence analysis.</p

Comparison of pigeon DA receptor probe sequences to gene sequences in chicken (c) and human (h).

<p>Data is presented as x/y (%), with x the number of identical bases and y the total length of the fragment followed by the percentage value of sequence identity. Similarities to pigeon sequences differ between chicken and human and are generally larger for chicken sequences. For the D1D probe only low correspondences were detected to the D1B/D5 gene, while high correlations were found with the chicken D1D gene. Empty boxes indicate absence of any significant identities.</p