Electrospray Mass Spectrometry to Study Combinatorial iClick Reactions and Multiplexed Kinetics of [Ru(N3)(N∧N)(terpy)]PF6 with Alkynes of Different Steric and Electronic Demand.

In a combinatorial approach, a family of ruthenium(II) azido complexes [Ru(N3)(N∧N)(terpy)]PF6 with terpy = 2,2':6',2″-terpyridine and N∧N as a bidentate chelator derived from 2,2'-biypridine and its 4,4'-disubstituted derivatives, 2,2'-bipyrimidine, and 1,10-phenanthroline were reacted with different internal and terminal alkynes to give access to a total of 7 × 7 = 49 triazolato complexes in a room-temperature catalyst-free iClick reaction. The reactants were mixed in a repurposed high-performance liquid chromatography (HPLC) autosampler, and the reaction progress was monitored by direct injection into an electrospray mass spectrometer. The ratio of the peak intensities of [Ru(N3)(N∧N)(terpy)]+ and [Ru(triazolato)(N∧N)(terpy)]+ was converted to a colored heat map for facile visual inspection of the conversion ratio. By automated multiple injections of the reaction mixture in fixed time intervals and plotting peak intensities over reaction time, pseudo-first-order rate constants were easily determined. Finally, nonoverlapping isotope patterns of the azido starting materials and triazolato products enabled multiplexed parallel determination of rate constants for four different ruthenium(II) azido complexes from a single sample vial, thereby reducing experiment time by 75%.

Time-resolved proteomics profiling of the ciliary Hedgehog response.

The primary cilium is a signaling compartment that interprets Hedgehog signals through changes of its protein, lipid, and second messenger compositions. Here, we combine proximity labeling of cilia with quantitative mass spectrometry to unbiasedly profile the time-dependent alterations of the ciliary proteome in response to Hedgehog. This approach correctly identifies the three factors known to undergo Hedgehog-regulated ciliary redistribution and reveals two such additional proteins. First, we find that a regulatory subunit of the cAMP-dependent protein kinase (PKA) rapidly exits cilia together with the G protein-coupled receptor GPR161 in response to Hedgehog, and we propose that the GPR161/PKA module senses and amplifies cAMP signals to modulate ciliary PKA activity. Second, we identify the phosphatase Paladin as a cell type-specific regulator of Hedgehog signaling that enters primary cilia upon pathway activation. The broad applicability of quantitative ciliary proteome profiling promises a rapid characterization of ciliopathies and their underlying signaling malfunctions.

Trauma-focused psychodynamic therapy and STAIR Narrative Therapy of post-traumatic stress disorder related to childhood maltreatment: trial protocol of a multicentre randomised controlled trial assessing psychological, neurobiological and health economic outcomes (ENHANCE).

INTRODUCTION: Success rates of psychotherapy in post-traumatic stress disorder related to childhood maltreatment (PTSD-CM) are limited. METHODS AND ANALYSIS: Observer-blind multicentre randomised clinical trial (A-1) of 4-year duration comparing enhanced methods of STAIR Narrative Therapy (SNT) and of trauma-focused psychodynamic therapy (TF-PDT) each of up to 24 sessions with each other and a minimal attention waiting list in PTSD-CM. Primary outcome is severity of PTSD (Clinician-Administered PTSD Scale for DSM-5 total) assessed by masked raters. For SNT and TF-PDT, both superiority and non-inferiority will be tested. Intention-to-treat analysis (primary) and per-protocol analysis (secondary). Assessments at baseline, after 10 sessions, post-therapy/waiting period and at 6 and 12 months of follow-up. Adult patients of all sexes between 18 and 65 years with PTSD-CM will be included. Continuing stable medication is permitted. To be excluded: psychotic disorders, risk of suicide, ongoing abuse, acute substance related disorder, borderline personality disorder, dissociative identity disorder, organic mental disorder, severe medical conditions and concurrent psychotherapy. To be assessed for eligibility: n=600 patients, to be e randomly allocated to the study conditions: n=328. Data management, randomisation and monitoring will be performed by an independent European Clinical Research Infrastructure Network (ECRIN)-certified data coordinating centre for clinical trials (KKS Marburg). Report of AEs to a data monitoring and safety board. Complementing study A-1, four inter-related add-on projects, including subsamples of the treatment study A-1, will examine (1) treatment integrity (adherence and competence) and moderators and mediators of outcome (B-1); (2) biological parameters (B-2, eg, DNA damage, reactive oxygen species and telomere shortening); (3) structural and functional neural changes by neuroimaging (B-3) and (4) cost-effectiveness of the treatments (B-4, costs and utilities). ETHICS AND DISSEMINATION: Approval by the institutional review board of the University of Giessen (AZ 168/19). Following the Consolidated Standards of Reporting Trials statement for non-pharmacological trials, results will be reported in peer-reviewed scientific journals and disseminated to patient organisations and media. TRIAL REGISTRATION NUMBER: DRKS 00021142.