Progress feedback in children and adolescents with internalizing and externalizing symptoms in routine care (OPTIE study): study protocol of a randomized parallel-group trial.

BACKGROUND: Progress feedback provides therapists with progress notes on a regular basis through the continuous assessment of participants throughout their treatment (e.g., symptoms, therapeutic alliance). While for adults the evidence base has increased over the years, progress feedback in the therapy of children and adolescents has not been sufficiently investigated. This manuscript describes the trial protocol of the OPTIE study: a randomized trial that tests the efficacy of a progress feedback system in children and adolescents under conditions of routine care. METHODS: The study is based on a randomized parallel-group trial with two treatment groups (routine, feedback) at an outpatient unit of a university hospital. The target sample size is 439 families consisting of children and adolescents aged 6 to17 years old with internalizing and/or externalizing symptoms. Both the patients and the therapists are independently assigned to the treatment groups by stratified block randomization. In both treatment groups patients receive routine care behavioral therapy for a study-related 12 months; additionally, in the feedback group, a progress feedback system with three components is applied (monitoring, report, and supervision). For three informants (caregiver, child [≥ 11 years], therapist) surveys are conducted every 6 weeks (e.g., symptoms, goals, motivation). For both treatment groups, comparison data is collected at baseline and at six and 12 months after the beginning of the intervention (pre, inter, post), and includes five informants (blinded clinician, therapist, caregiver, child [≥ 11 years], teacher). DISCUSSION: The OPTIE study will contribute to the evidence base of progress feedback in children and adolescents and has the potential to uncover treatments' effects in the small to medium range. Noteworthy features are the inclusion of children younger than 10 years old and the consideration of a blinded clinician rating. TRIAL REGISTRATION: German Clinical Trials Register (DRKS) DRKS00016737 ( https://www.drks.de/DRKS00016737 ). Registered 17 September, 2019.

Chemsex and Mental Health of Men Who Have Sex With Men in Germany.

Background: Chemsex is defined as using certain substances immediately before or during sexual activities to facilitate, prolong and/or intensify sexual experience, mainly by some communities of men who have sex with men (MSM). Four substances are typically associated with chemsex: methamphetamine, mephedrone, GHB/GBL, and ketamine. While there is a lot of evidence for increased prevalence of HIV, sexually transmitted infections and other sexual health measures among MSM, who engage in chemsex, there has been little research on mental health aspects. This study aims to describe aspects of mental health among a sample of German men who have sex with men (MSM) who engage in chemsex and to describe potentially adverse consequences of chemsex behavior. Method: This paper refers to a subset of participants from the German Chemsex Survey, an MSM-community recruited, self-completed online survey with a self-selected convenience sample. The survey comprised 420 different items considering recreational substance use, substance use in sexual settings, mental health, sexual transmitted infections, adverse consequences of chemsex behavior, and experiences of non-consensual sex acts. A group of participants who used methamphetamine, mephedrone, GHB/GBL, and/or ketamine in a sexual setting in the last 12 months (n = 280, chemsex group) was analyzed regarding symptoms of depression (PHQ-9), general anxiety disorder (GAD-7), somatization (PHQ-15), and PTSD (Primary Care PTSD Screen). Group comparisons were conducted between the chemsex group and men who did not use substances in a sexual context (n = 177, non-chemsex group). Mean scores of mental health measures were compared, as well as scores above a cut-off that indicates clinically relevant symptoms. Logistical regression was utilized to determine whether mental health measures can predict adverse consequences of engagement in chemsex behaviors. Results: A total of 1,583 men started the survey; 1,050 participants provided information on substance use. Twenty-seven percent of participants (n = 280) reported that they used methamphetamine, mephedrone, GHB/GBL and/or ketamine in a sexual setting in the last 12 months. The chemsex group showed significantly higher mean scores for depression, anxiety, and somatization than the non-chemsex group, but effect sizes were low. Even though mean scores were heightened, they were still far below the cut-off for clinically relevant symptoms. The chemsex group reported significantly higher incidences of non-consensual sex acts compared with the non-chemsex group. Some men in the chemsex-group experienced potentially adverse consequences, such as loss of control regarding time and money spent for chemsex activities or amount of substances used at one occasion (49.6%), negative impacts on social functioning (33.6%), psychotic symptoms (13.2%), and physically aggressive behavior toward others (2.9%). Clinically relevant symptoms did not predict a higher likelihood for adverse consequences. Discussion: Mean scores for depression, anxiety, and somatization were significantly higher in the chemsex-group, but effect sizes were low. Both groups reported poorer mental health compared to men in the German general population. Mental health measures did not contribute to predict potentially adverse consequences of chemsex behavior.

Cutting off ciliary protein import: intraflagellar transport after dendritic femtosecond-laser ablation.

Primary cilia, organelles protruding from the surface of eukaryotic cells, act as cellular antennae to detect and transmit signals from the extracellular environment. They are built and maintained by continuous cycles of intraflagellar transport (IFT), where ciliary proteins are transported between the ciliary base and tip. These proteins originate from the cell body because cilia lack protein synthesis machinery. How input from the cell body affects IFT and ciliary function is not well understood. Here, we use femtosecond-laser ablation to perturb the dendritic input of proteins to chemosensory cilia in living Caenorhabditis elegans. Using fluorescence microscopy, we visualize and quantify the real-time response of ciliary proteins to dendritic ablation. We find that the response occurs in three distinct stages. First, IFT dynein is activated within seconds, redistributing IFT components toward the ciliary base; second, the ciliary axoneme shortens and motors slow down; and third, motors leave the cilium. Depletion of ATP by adding azide also results in IFT slowdown and IFT components leaving the cilium, but not in activation of retrograde IFT. These results indicate that laser ablation triggers a specific mechanism important for IFT regulation that allows the cilium to rapidly adapt to changes in the outside environment.

Single-Molecule Turnarounds of Intraflagellar Transport at the C. elegans Ciliary Tip.

Cilia are microtubule-based sensing hubs that rely on intraflagellar transport (IFT) for their development, maintenance, and function. Kinesin-2 motors transport IFT trains, consisting of IFT proteins and cargo, from ciliary base to tip. There, trains turn around and are transported back by IFT dynein. The mechanism of tip turnaround has remained elusive. Here, we employ single-molecule fluorescence microscopy of IFT components in the tips of phasmid cilia of living C. elegans. Analysis of the trajectories reveals that while motor proteins and IFT-A particle component CHE-11 mostly turn around immediately, the IFT-B particle component OSM-6 pauses for several seconds. Our data indicate that IFT trains disassemble into at least IFT-A, IFT-B, IFT-dynein, and OSM-3 complexes at the tip, where OSM-6 is temporarily retained or undergoes modification, prior to train reassembly and retrograde transport. The single-molecule approach used here is a valuable tool to study how directional switches occur in microtubule-based transport processes.

Interplay between Ciliary Ultrastructure and IFT-Train Dynamics Revealed by Single-Molecule Super-resolution Imaging.

Cilia are built and maintained by intraflagellar transport (IFT), driving IFT trains back and forth along the ciliary axoneme. How IFT brings about the intricate ciliary structure and how this structure affects IFT are not well understood. We identify, using single-molecule super-resolution imaging of IFT components in living C. elegans, ciliary subdomains, enabling correlation of IFT-train dynamics to ciliary ultra-structure. In the transition zone, IFT dynamics are impaired, resulting in frequent pauses. At the ciliary base and tip, IFT trains show intriguing turnaround dynamics. Surprisingly, deletion of IFT motor kinesin-II not only affects IFT-train dynamics but also alters ciliary structure. Super-resolution imaging in these mutant animals suggests that the arrangement of IFT trains with respect to the axonemal microtubules is different than in wild-type animals. Our results reveal a complex, mutual interplay between ciliary ultrastructure and IFT-train dynamics, highlighting the importance of physical cues in the control of IFT dynamics.

Single-Cell Acoustic Force Spectroscopy: Resolving Kinetics and Strength of T Cell Adhesion to Fibronectin.

Assessing the strength and kinetics of molecular interactions of cells with the extracellular matrix is fundamental to understand cell adhesion processes. Given the relevance of these processes, there is a strong need for physical methods to quantitatively assess the mechanism of cell adhesion at the single-cell level, allowing discrimination of cells with different behaviors. Here we introduce single-cell acoustic force spectroscopy (scAFS), an approach that makes use of acoustic waves to exert controlled forces, up to 1 nN, to hundreds of individual cells in parallel. We demonstrate the potential of scAFS by measuring adhesion forces and kinetics of CD4+ T lymphocytes (CD4) to fibronectin. We determined that CD4 adhesion is accelerated by interleukin-7, their main regulatory cytokine, whereas CD4 binding strength remains the same. Activation of these cells likely increases their chance to bind to the vessel wall in the blood flow to infiltrate inflamed tissues and locally coordinate the immune response.

Single-Molecule Imaging of Escherichia coli Transmembrane Proteins.

Single-molecule imaging in living cells can provide unique information about biological processes. Bacteria offer some particular challenges for single-molecule imaging due to their small size, only slightly larger than the diffraction limit of visible light. Here, we describe how reliable and reproducible single-molecule data can be obtained for a transmembrane protein in the Gram-negative bacterium Escherichia coli by using live-cell fluorescence microscopy. Fluorescent labeling of a protein by genetic fusion, cell culturing, sample preparation, imaging, and data analysis are discussed.

Ensemble and single-molecule dynamics of IFT dynein in Caenorhabditis elegans cilia.

Cytoplasmic dyneins drive microtubule-based, minus-end directed transport in eukaryotic cells. Whereas cytoplasmic dynein 1 has been widely studied, IFT dynein has received far less attention. Here, we use fluorescence microscopy of labelled motors in living Caenorhabditis elegans to investigate IFT-dynein motility at the ensemble and single-molecule level. We find that while the kinesin composition of motor ensembles varies along the track, the amount of dynein remains relatively constant. Remarkably, this does not result in directionality changes of cargo along the track, as has been reported for other opposite-polarity, tug-of-war motility systems. At the single-molecule level, IFT-dynein trajectories reveal unexpected dynamics, including diffusion at the base, and pausing and directional switches along the cilium. Stochastic simulations show that the ensemble IFT-dynein distribution depends upon the probability of single-motor directional switches. Our results provide quantitative insight into IFT-dynein dynamics in vivo, shedding light on the complex functioning of dynein motors in general.

MreB-Dependent Organization of the E. coli Cytoplasmic Membrane Controls Membrane Protein Diffusion.

The functional organization of prokaryotic cell membranes, which is essential for many cellular processes, has been challenging to analyze due to the small size and nonflat geometry of bacterial cells. Here, we use single-molecule fluorescence microscopy and three-dimensional quantitative analyses in live Escherichia coli to demonstrate that its cytoplasmic membrane contains microdomains with distinct physical properties. We show that the stability of these microdomains depends on the integrity of the MreB cytoskeletal network underneath the membrane. We explore how the interplay between cytoskeleton and membrane affects trans-membrane protein (TMP) diffusion and reveal that the mobility of the TMPs tested is subdiffusive, most likely caused by confinement of TMP mobility by the submembranous MreB network. Our findings demonstrate that the dynamic architecture of prokaryotic cell membranes is controlled by the MreB cytoskeleton and regulates the mobility of TMPs.

Functional differentiation of cooperating kinesin-2 motors orchestrates cargo import and transport in C. elegans cilia.

Intracellular transport depends on cooperation between distinct motor proteins. Two anterograde intraflagellar transport (IFT) motors, heterotrimeric kinesin-II and homodimeric OSM-3, cooperate to move cargo along Caenorhabditis elegans cilia. Here, using quantitative fluorescence microscopy, with single-molecule sensitivity, of IFT in living strains containing single-copy transgenes encoding fluorescent IFT proteins, we show that kinesin-II transports IFT trains through the ciliary base and transition zone to a 'handover zone' on the proximal axoneme. There, OSM-3 gradually replaces kinesin-II, yielding velocity profiles inconsistent with in vitro motility assays, and then drives transport to the ciliary tip. Dissociated kinesin-II motors undergo rapid turnaround and recycling to the ciliary base, whereas OSM-3 is recycled mainly to the handover zone. This reveals a functional differentiation in which the slower, less processive kinesin-II imports IFT trains into the cilium and OSM-3 drives their long-range transport, thereby optimizing cargo delivery.

morFeus: a web-based program to detect remotely conserved orthologs using symmetrical best hits and orthology network scoring.

BACKGROUND: Searching the orthologs of a given protein or DNA sequence is one of the most important and most commonly used Bioinformatics methods in Biology. Programs like BLAST or the orthology search engine Inparanoid can be used to find orthologs when the similarity between two sequences is sufficiently high. They however fail when the level of conservation is low. The detection of remotely conserved proteins oftentimes involves sophisticated manual intervention that is difficult to automate. RESULTS: Here, we introduce morFeus, a search program to find remotely conserved orthologs. Based on relaxed sequence similarity searches, morFeus selects sequences based on the similarity of their alignments to the query, tests for orthology by iterative reciprocal BLAST searches and calculates a network score for the resulting network of orthologs that is a measure of orthology independent of the E-value. Detecting remotely conserved orthologs of a protein using morFeus thus requires no manual intervention. We demonstrate the performance of morFeus by comparing it to state-of-the-art orthology resources and methods. We provide an example of remotely conserved orthologs, which were experimentally shown to be functionally equivalent in the respective organisms and therefore meet the criteria of the orthology-function conjecture. CONCLUSIONS: Based on our results, we conclude that morFeus is a powerful and specific search method for detecting remotely conserved orthologs. morFeus is freely available at http://bio.biochem.mpg.de/morfeus/. Its source code is available from Sourceforge.net (https://sourceforge.net/p/morfeus/).

Imaging and quantification of trans-membrane protein diffusion in living bacteria.

The cytoplasmic membrane forms the barrier between any cell's interior and the outside world. It contains many proteins that enable essential processes such as the transmission of signals, the uptake of nutrients, and cell division. In the case of prokaryotes, which do not contain intracellular membranes, the cytoplasmic membrane also contains proteins for respiration and protein folding. Mutual interactions and specific localization of these proteins depend on two-dimensional diffusion driven by thermal fluctuations. The experimental investigation of membrane-protein diffusion in bacteria is challenging due to their small size, only a few times larger than the resolution of an optical microscope. Here, we review fluorescence microscopy-based methods to study diffusion of membrane proteins in living bacteria. The main focus is on data-analysis tools to extract diffusion coefficients from single-particle tracking data obtained by single-molecule fluorescence microscopy. We introduce a novel approach, IPODD (inverse projection of displacement distributions), to obtain diffusion coefficients from the usually obtained 2-D projected diffusion trajectories of the highly 3-D curved bacterial membrane. This method provides, in contrast to traditional mean-squared-displacement methods, correct diffusion coefficients and allows unravelling of heterogeneously diffusing populations.

Slow cortical potentials capture decision processes during temporal discounting.

Various neuroimaging studies have detected brain regions involved in discounting the value of temporally delayed rewards. This study used slow cortical potentials (SCPs) to elaborate the time course of cognitive processing during temporal discounting. Depending on their strength of discounting, subjects were categorised as low and high impulsive. Low impulsives, but not high impulsives, showed faster reaction times for making decisions when the delayed reward was of high amount than when it was of low amount. Both low impulsives and high impulsives chose the delayed reward more often when its amount was high than when it was low, but this behavior was more pronounced for low impulsives. Moreover, only low impulsives showed more negative SCPs for low than for high amounts. All three measures indicated that only low impulsives experienced extended conflict for delayed low amounts than for high amounts. Additionally, the SCPs of low impulsives were more sensitive to the delay of the delayed reward than those of high impulsives, extending seconds after the response. This indicates that they continued evaluating their choices even after the decision. Altogether, the present study demonstrated that SCPs are sensitive to decision-related resource allocation during inter-temporal decision-making. Resource allocation depended both on the choice situation and on impulsivity. Furthermore, the time course of SCPs suggested that decision-related processes occurred both prior to and after the response.

Forces driving epithelial spreading in zebrafish gastrulation.

Contractile actomyosin rings drive various fundamental morphogenetic processes ranging from cytokinesis to wound healing. Actomyosin rings are generally thought to function by circumferential contraction. Here, we show that the spreading of the enveloping cell layer (EVL) over the yolk cell during zebrafish gastrulation is driven by a contractile actomyosin ring. In contrast to previous suggestions, we find that this ring functions not only by circumferential contraction but also by a flow-friction mechanism. This generates a pulling force through resistance against retrograde actomyosin flow. EVL spreading proceeds normally in situations where circumferential contraction is unproductive, indicating that the flow-friction mechanism is sufficient. Thus, actomyosin rings can function in epithelial morphogenesis through a combination of cable-constriction and flow-friction mechanisms.