Microscopic and biochemical monitoring of endosomal trafficking and extracellular vesicle secretion in an endogenous in vivo model

Extracellular vesicle (EV) secretion enables cell–cell communication in multicellular organisms. During development, EV secretion and the specific loading of signalling factors in EVs contributes to organ development and tissue differentiation. Here, we present an in vivo model to study EV secretion using the fat body and the haemolymph of the fruit fly, Drosophila melanogaster. The system makes use of tissue-specific EV labelling and is amenable to genetic modification by RNAi. This allows the unique combination of microscopic visualisation of EVs in different organs and quantitative biochemical purification to study how EVs are generated within the cells and which factors regulate their secretion in vivo. Characterisation of the system revealed that secretion of EVs from the fat body is mainly regulated by Rab11 and Rab35, highlighting the importance of recycling Rab GTPase family members for EV secretion. We furthermore discovered a so far unknown function of Rab14 along with the kinesin Klp98A in EV biogenesis and secretion

Windei, the Drosophila Homolog of mAM/MCAF1, Is an Essential Cofactor of the H3K9 Methyl Transferase dSETDB1/Eggless in Germ Line Development

The epigenetic regulation of gene expression by the covalent modification of histones is a fundamental mechanism required for the proper differentiation of germ line cells during development. Trimethylation of histone 3 lysine 9 (H3K9me3) leads to chromatin silencing and the formation of heterochromatin by recruitment of heterochromatin protein 1 (HP1). dSETDB1/Eggless (Egg), the ortholog of the human methyltransferase SETDB1, is the only essential H3K9 methyltransferase in Drosophila and is required for H3K9 trimethylation in the female germ line. Here we show that Windei (Wde), the Drosophila homolog of mouse mAM and human MCAF1, is an essential cofactor of Egg required for its nuclear localization and function in female germ line cells. By deletion analysis combined with coimmunoprecipitation, we have identified the protein regions in Wde and Egg that are necessary and sufficient for the interaction between the two proteins. We furthermore identified a region of Egg that gets covalently modified by SUMOylation, which may facilitate the formation of higher order chromatin-modifying complexes. Together with Egg, Wde localizes to euchromatin, is enriched on chromosome 4, and binds to the Painting of fourth (POF) protein. Our data provide the first genetic and phenotypic analysis of a mAM/MCAF1 homolog in a model organism and demonstrate its essential function in the survival of germ line cells

Recommendations for the introduction of metagenomic high-throughput sequencing in clinical virology, part I: wet lab procedure

Metagenomic high-throughput sequencing (mHTS) is a hypothesis-free, universal pathogen detection technique for determination of the DNA/RNA sequences in a variety of sample types and infectious syndromes. mHTS is still in its early stages of translating into clinical application. To support the development, implementation and standardization of mHTS procedures for virus diagnostics, the European Society for Clinical Virology (ESCV) Network on Next-Generation Sequencing (ENNGS) has been established. The aim of ENNGS is to bring together professionals involved in mHTS for viral diagnostics to share methodologies and experiences, and to develop application recommendations. This manuscript aims to provide practical recommendations for the wet lab procedures necessary for implementation of mHTS for virus diagnostics and to give recommendations for development and validation of laboratory methods, including mHTS quality assurance, control and quality assessment protocols.Molecular basis of virus replication, viral pathogenesis and antiviral strategie

Effect of different anaesthetic regimes on the oculocardiac reflex during paediatric strabismus surgery

The oculocardiac reflex (OCR) is induced by mechanical stimulation and therefore is frequently encountered during strabismus surgery. This study was designed to determine how various anaesthetic regimes modulate the haemodynamic effects of the OCR during paediatric strabismus surgery. Thirty-nine patients (4-14 years, ASA I) were randomized to one of four anaesthetic regimes: group P: propofol (12 mg.kg(-1).h(-1)) and alfentanil (0.04 mg.kg(-1).h(-1)); group S: sevoflurane 1-1.2 MAC in 30% O-2/70% N2O; group K: ketamine racemate (10-12 mg.kg(-1).h(-1)) and midazolam (0.3-0.6 mg.kg(-1).h(-1); group H: halothane 1-1.2 MAC in 30% O-2/70% N2O. Electrocardiogram (ECG), beat-to-beat heart rate (HR) and blood pressure (BP) changes were measured during and after a standardized traction was applied to an external eye muscle (4-6 Newton, 90 s). OCR was defined as a 10% change in HR induced by traction. OCR occurred in 77% of patients. Whereas virtually all patients in the P, H and S groups developed OCR, only 22% developed it in group K. Median HR change in group P (-37 bpm) was significantly greater (P < 0.05) than in group H (-17 bpm) or group K (-7 bpm). Median BP change in group K (+10 mmHg) was significantly different (P < 0.05) from group H (-5 mmHg), group S (-3 mmHg) and group P (-8 mmHg). Atrioventricular rhythm disorders were significantly more frequent in group P compared with group K (P < 0.02). Respiration-induced sinus dysrhythmia was significantly less frequent (P < 0.001) in group K (0%), compared with group P (100%), group H (56%) and group S (55%). Of the anaesthetic techniques studied, ketamine anaesthesia is associated with the least haemodynamic changes induced by OCR during strabismus surgery in paediatric patients

Reliability of the volatile agent consumption display in the Draeger Primus™ anesthesia machine

Knowledge of the consumed amount of volatile anesthetic (VA) expressed in liquid agent is necessary to enable agent sparing dosing measures and for billing purposes. The widespread Draeger Primus™ anesthesia machine displays in its logbook the amount of consumed VA at the end of each anesthesia, but the reliability of this parameter is yet unknown. The objective was to evaluate the precision and reliability of the inbuilt VA consumption display in Draeger Primus™ anesthesia machines as compared with the gold standard of weighing the vaporizer before and after anesthesia. In this prospective laboratory investigation we compared the VA consumption displayed by the Draeger Primus™ anesthesia machine with measured vaporizer weight differences before and after 10 sevoflurane and 10 desflurane anesthesias. We assessed the average difference and spread of values between the predicted (displayed) and measured (control) values for VA consumption. The displayed sevoflurane consumption overestimated the measured values by 4.3 ± 5.4 ml (7.6 %). The displayed desflurane consumption underestimated the measured values by -3.5 ± 6.3 ml (6.2 %). Nine from 10 sevoflurane pairs of values and all desflurane pairs of values were within ±1.96 SD. The displayed VA consumption calculations for sevoflurane and desflurane in the Draeger Primus™ are sufficiently reliable to estimate the pharmacoeconomic impact of VA delivery during inhalational anesthesia