Histone recognition and large-scale structural analysis of the human bromodomain family.

Bromodomains (BRDs) are protein interaction modules that specifically recognize ε-N-lysine acetylation motifs, a key event in the reading process of epigenetic marks. The 61 BRDs in the human genome cluster into eight families based on structure/sequence similarity. Here, we present 29 high-resolution crystal structures, covering all BRD families. Comprehensive crossfamily structural analysis identifies conserved and family-specific structural features that are necessary for specific acetylation-dependent substrate recognition. Screening of more than 30 representative BRDs against systematic histone-peptide arrays identifies new BRD substrates and reveals a strong influence of flanking posttranslational modifications, such as acetylation and phosphorylation, suggesting that BRDs recognize combinations of marks rather than singly acetylated sequences. We further uncovered a structural mechanism for the simultaneous binding and recognition of diverse diacetyl-containing peptides by BRD4. These data provide a foundation for structure-based drug design of specific inhibitors for this emerging target family.

Chemical proteomics reveals the target landscape of 1,000 kinase inhibitors.

Medicinal chemistry has discovered thousands of potent protein and lipid kinase inhibitors. These may be developed into therapeutic drugs or chemical probes to study kinase biology. Because of polypharmacology, a large part of the human kinome currently lacks selective chemical probes. To discover such probes, we profiled 1,183 compounds from drug discovery projects in lysates of cancer cell lines using Kinobeads. The resulting 500,000 compound-target interactions are available in ProteomicsDB and we exemplify how this molecular resource may be used. For instance, the data revealed several hundred reasonably selective compounds for 72 kinases. Cellular assays validated GSK986310C as a candidate SYK (spleen tyrosine kinase) probe and X-ray crystallography uncovered the structural basis for the observed selectivity of the CK2 inhibitor GW869516X. Compounds targeting PKN3 were discovered and phosphoproteomics identified substrates that indicate target engagement in cells. We anticipate that this molecular resource will aid research in drug discovery and chemical biology.

The Chemical Probes Portal: an expert review-based public resource to empower chemical probe assessment, selection and use.

We describe the Chemical Probes Portal (https://www.chemicalprobes.org/), an expert review-based public resource to empower chemical probe assessment, selection and use. Chemical probes are high-quality small-molecule reagents, often inhibitors, that are important for exploring protein function and biological mechanisms, and for validating targets for drug discovery. The publication, dissemination and use of chemical probes provide an important means to accelerate the functional annotation of proteins, the study of proteins in cell biology, physiology, and disease pathology, and to inform and enable subsequent pioneering drug discovery and development efforts. However, the widespread use of small-molecule compounds that are claimed as chemical probes but are lacking sufficient quality, especially being inadequately selective for the desired target or even broadly promiscuous in behaviour, has resulted in many erroneous conclusions in the biomedical literature. The Chemical Probes Portal was established as a public resource to aid the selection and best-practice use of chemical probes in basic and translational biomedical research. We describe the background, principles and content of the Portal and its technical development, as well as examples of its applications and use. The Chemical Probes Portal is a community resource and we therefore describe how researchers can be involved in its content and development.

Wnt/ß-catenin and NFκB signaling synergize to trigger growth factor-free regeneration of adult primary human hepatocytes.

BACKGROUND AND AIMS: The liver has a remarkable capacity to regenerate, which is sustained by the ability of hepatocytes to act as facultative stem cells that, while normally quiescent, re-enter the cell cycle after injury. Growth factor signaling is indispensable in rodents, whereas Wnt/ß-catenin is not required for effective tissue repair. However, the molecular networks that control human liver regeneration remain unclear. METHODS: Organotypic 3D spheroid cultures of primary human or murine hepatocytes were used to identify the signaling network underlying cell cycle re-entry. Furthermore, we performed chemogenomic screening of a library enriched for epigenetic regulators and modulators of immune function to determine the importance of epigenomic control for human hepatocyte regeneration. RESULTS: Our results showed that, unlike in rodents, activation of Wnt/ß-catenin signaling is the major mitogenic cue for adult primary human hepatocytes. Furthermore, we identified TGFß inhibition and inflammatory signaling through NF-κB as essential steps for the quiescent-to-regenerative switch that allows Wnt/ß-catenin-induced proliferation of human cells. In contrast, growth factors, but not Wnt/ß-catenin signaling, triggered hyperplasia in murine hepatocytes. High-throughput screening in a human model confirmed the relevance of NFκB and revealed the critical roles of polycomb repressive complex 2, as well as of the bromodomain families I, II, and IV. CONCLUSIONS: This study revealed a network of NFκB, TGFß, and Wnt/ß-catenin that controls human hepatocyte regeneration in the absence of exogenous growth factors, identified novel regulators of hepatocyte proliferation, and highlighted the potential of organotypic culture systems for chemogenomic interrogation of complex physiological processes.

A national surveillance system for continuous monitoring of blood transfusion safety: German haemovigilance data.

BACKGROUND AND OBJECTIVES: Haemovigilance (HV) systems aim to improve transfusion outcomes in patients and donor safety. An important question for blood regulators is how to ensure an effective HV system. MATERIALS AND METHODS: We retrospectively analysed the HV reports submitted to Paul-Ehrlich-Institut over the last two decades. RESULTS: Between 2011 and 2020, 50.86 million units of blood components were used, and 8931 suspected serious donor and recipient adverse reactions (SARs), 874 serious adverse events (SAEs) and 12,073 donor look-backs were reported. Following implementation of specific risk-minimization measures (RMMs) between 2000 and 2010, SAR reporting rates decreased for transfusion-transmitted viral infections (TTVIs), transfusion-related acute lung injury (TRALI) and transfusion-transmitted bacterial infections (TTBIs), while increasing for other serious adverse transfusion reactions. Within this decade, the overall blood component use decreased. CONCLUSION: Long-term data collection forms the basis to establish trends and changes in reporting and to evaluate the effect of RMM. Standardized criteria for reaction types, seriousness and imputability assessments and availability of a denominator are important elements. Central data collection and independent assessment allow for monitoring HV data in a nationwide context over time. Stakeholder involvement and transparent feedback on the benefit of RMM will help to achieve the objectives of HV.

Large-scale structural analysis of the classical human protein tyrosine phosphatome.

Protein tyrosine phosphatases (PTPs) play a critical role in regulating cellular functions by selectively dephosphorylating their substrates. Here we present 22 human PTP crystal structures that, together with prior structural knowledge, enable a comprehensive analysis of the classical PTP family. Despite their largely conserved fold, surface properties of PTPs are strikingly diverse. A potential secondary substrate-binding pocket is frequently found in phosphatases, and this has implications for both substrate recognition and development of selective inhibitors. Structural comparison identified four diverse catalytic loop (WPD) conformations and suggested a mechanism for loop closure. Enzymatic assays revealed vast differences in PTP catalytic activity and identified PTPD1, PTPD2, and HDPTP as catalytically inert protein phosphatases. We propose a "head-to-toe" dimerization model for RPTPgamma/zeta that is distinct from the "inhibitory wedge" model and that provides a molecular basis for inhibitory regulation. This phosphatome resource gives an expanded insight into intrafamily PTP diversity, catalytic activity, substrate recognition, and autoregulatory self-association.

Mechanical Thrombectomy Versus Best Medical Treatment in the Late Time Window in Non-DEFUSE-Non-DAWN Patients: A Multicenter Cohort Study.

BACKGROUND: We assessed the efficacy and safety of mechanical thrombectomy (MT) in adult stroke patients with anterior circulation large vessel occlusion presenting in the late time window not fulfilling the DEFUSE-3 (Thrombectomy for Stroke at 6 to 16 Hours With Selection by Perfusion Imaging trial) and DAWN (Thrombectomy 6 to 24 Hours After Stroke With a Mismatch Between Deficit and Infarct trial) inclusion criteria. METHODS: Cohort study of adults with anterior circulation large vessel occlusion admitted between 6 and 24 hours after last-seen-well at 5 participating Swiss stroke centers between 2014 and 2021. Mismatch was assessed by computer tomography or magnetic resonance imaging perfusion with automated software (RAPID or OLEA). We excluded patients meeting DEFUSE-3 and DAWN inclusion criteria and compared those who underwent MT with those receiving best medical treatment alone by inverse probability of treatment weighting using the propensity score. The primary efficacy end point was a favorable functional outcome at 90 days, defined as a modified Rankin Scale score shift toward lower categories. The primary safety end point was symptomatic intracranial hemorrhage within 7 days of stroke onset; the secondary was all-cause mortality within 90 days. RESULTS: Among 278 patients with anterior circulation large vessel occlusion presenting in the late time window, 190 (68%) did not meet the DEFUSE-3 and DAWN inclusion criteria and thus were included in the analyses. Of those, 102 (54%) received MT. In the inverse probability of treatment weighting analysis, patients in the MT group had higher odds of favorable outcomes compared with the best medical treatment alone group (modified Rankin Scale shift: acOR, 1.46 [1.02-2.10]; P=0.04) and lower odds of all-cause mortality within 90 days (aOR, 0.59 [0.37-0.93]; P=0.02). There were no significant differences in symptomatic intracranial hemorrhage (MT versus best medical treatment alone: 5% versus 2%, P=0.63). CONCLUSIONS: Two out of 3 patients with anterior circulation large vessel occlusion presenting in the late time window did not meet the DEFUSE-3 and DAWN inclusion criteria. In these patients, MT was associated with higher odds of favorable functional outcomes without increased rates of symptomatic intracranial hemorrhage. These findings support the enrollment of patients into ongoing randomized trials on MT in the late window with more permissive inclusion criteria.

PREDICT-juvenile-stroke: PRospective evaluation of a prediction score determining individual clinical outcome three months after ischemic stroke in young adults - a study protocol.

BACKGROUND: Although of high individual and socioeconomic relevance, a reliable prediction model for the prognosis of juvenile stroke (18-55 years) is missing. Therefore, the study presented in this protocol aims to prospectively validate the discriminatory power of a prediction score for the 3 months functional outcome after juvenile stroke or transient ischemic attack (TIA) that has been derived from an independent retrospective study using standard clinical workup data. METHODS: PREDICT-Juvenile-Stroke is a multi-centre (n = 4) prospective observational cohort study collecting standard clinical workup data and data on treatment success at 3 months after acute ischemic stroke or TIA that aims to validate a new prediction score for juvenile stroke. The prediction score has been developed upon single center retrospective analysis of 340 juvenile stroke patients. The score determines the patient's individual probability for treatment success defined by a modified Rankin Scale (mRS) 0-2 or return to pre-stroke baseline mRS 3 months after stroke or TIA. This probability will be compared to the observed clinical outcome at 3 months using the area under the receiver operating characteristic curve. The primary endpoint is to validate the clinical potential of the new prediction score for a favourable outcome 3 months after juvenile stroke or TIA. Secondary outcomes are to determine to what extent predictive factors in juvenile stroke or TIA patients differ from those in older patients and to determine the predictive accuracy of the juvenile stroke prediction score on other clinical and paraclinical endpoints. A minimum of 430 juvenile patients (< 55 years) with acute ischemic stroke or TIA, and the same number of older patients will be enrolled for the prospective validation study. DISCUSSION: The juvenile stroke prediction score has the potential to enable personalisation of counselling, provision of appropriate information regarding the prognosis and identification of patients who benefit from specific treatments. TRIAL REGISTRATION: The study has been registered at https://drks.de on March 31, 2022 ( DRKS00024407 ).

PROTAC degraders as chemical probes for studying target biology and target validation.

Small molecule degraders such as PROTACs (PROteolysis TArgeting Chimeras) have emerged as new promising pharmacological modalities and the first PROTAC drug candidates are now studied clinically. The catalytic properties of PROTACs, acting as chemical degraders of a protein of interest (POI), represent an attractive new strategy for drug development. The development and characterization of PROTACs requires an array of additional assay systems that track the degradation pathway leading ultimately to degradation of the POI, identifying critical steps for PROTAC optimization. In addition to their exciting translational potential, PROTACs represent versatile chemical tools that considerably expanded our chemical biology toolbox and significantly enlarged the proteome that can be modulated by small molecules. Similar to conventional chemical probes, PROTACs used as chemical probes in target validation require comprehensive characterization. As a consequence, PROTAC-specific quality criteria should be defined by the chemical biology community. These criteria need to comprise additional or alternative parameters compared to those for conventional occupancy-driven chemical probes, such as the maximum level of target degradation (Dmax), confirmation of a proteasome dependent degradation mechanism and, importantly, also kinetic parameters of POI degradation. The kinetic aspects are particularly relevant for PROTACs that harbor covalent binding moieties. Here, we review recent progress in the development of assay systems for PROTAC characterization and suggest a set of criteria for PROTACs as high quality chemical probes.

Analysis of Transfusion-Transmitted Bacterial Infections according to German Hemovigilance Data (2011-2020).

Introduction: Following the first assessment of the effects of safety measures taken against transfusion-transmitted bacterial infections (TTBI), the Paul-Ehrlich-Institut (PEI) decided to newly analyze risk minimization measures (RMM) using German hemovigilance data from 2011 to 2020, focusing on blood components, recipients, and bacterial strains. Materials and Methods: The PEI assessed the imputability of all reported serious adverse reactions (SAR) relying mainly on microbiological test results. Reporting rates (RR) of suspected, confirmed, and fatal confirmed TTBI were calculated and compared to the previous reporting 10-year period (2001-2010) using Poisson regression to estimate RR ratios (RRR). Furthermore, details on blood component age, patients' medical history, and bacterial pathogenicity were collected. Results: With respect to the previous 10-year period, the number of suspected TTBI increased (n = 403), while fewer cases were confirmed (n = 40); the number of deaths remained more or less unchanged (n = 8). The RR for suspected TTBI were 7.9, 18.7, and 1.6 cases per million units transfused for red blood cells (RBC), platelet concentrates (PC), and fresh frozen plasma (FFP), respectively. RRR showed a statistically significant 2.5-fold increase in the RR for suspected TTBI after RBC administration from 2001-2010 to the period under review (p < 0.0001). The RR for confirmed TTBI were 0.4, 5.0, and 0.0 cases per million units transfused for RBC, PC, and FFP, respectively. Compared to the period 2001-2010, there was a statistically significant decrease in the RR of confirmed TTBI by half for PC (p = 0.0052). The RR for confirmed PC-caused TTBI with fatal outcome was 1.4 cases per million units transfused. Regardless of type of blood product transfused and outcome of SAR, the majority of TTBI occurred after administration of a product at the end of shelf life (40.0%) and to recipients of advanced age (median age: 68.5 years) and/or with severe immunosuppression (72.5%) due to decreased myelopoiesis (62.5%). 72.5% of the involved bacteria had a middle/high human pathogenicity. Conclusion: Despite a significant decrease in confirmed TTBI following PC transfusion in Germany after implementation of RMM, the current manufacture of blood products can still not prevent TTBI with fatal outcomes. As demonstrated in various countries, RMM like bacterial screening or pathogen reduction may measurably improve the safety of blood transfusion.

Covalent Modification of Bromodomain Proteins by Peptides Containing a DNA Damage-Induced, Histone Post-Translational Modification.

An electrophilic 5-methylene-2-pyrrolone modification (KMP ) is produced at lysine residues of histone proteins in nucleosome core particles upon reaction with a commonly formed DNA lesion (C4-AP). The nonenzymatic KMP modification is also generated in the histones of HeLa cells treated with the antitumor agent, bleomycin that oxidizes DNA and forms C4-AP. This nonenzymatic covalent histone modification has the same charge as the N-acetyllysine (KAc ) modification but is more electrophilic. In this study we show that KMP -containing histone peptides are recognized by, and covalently modify bromodomain proteins that are KAc readers. Distinct selectivity preferences for covalent bromodomain modification are observed following incubation with KMP -containing peptides of different sequence. MS/MS analysis of 3 covalently modified bromodomain proteins confirmed that Cys adduction was selective. The modified Cys was not always proximal to the KAc binding site, indicating that KMP -containing peptide interaction with bromodomain protein is distinct from the former. Analysis of protein adduction yields as a function of bromodomain pH at which the protein charge is zero (pI) or cysteine solvent accessible surface area are also consistent with non-promiscuous interaction between the proteins and electrophilic peptides. These data suggest that intracellular formation of KMP could affect cellular function and viability by modifying proteins that regulate genetic expression.

C81-evoked inhibition of the TNFR1-NFκB pathway during inflammatory processes for stabilization of the impaired vascular endothelial barrier for leukocytes.

Chronic inflammation-related diseases are characterized by persistent leukocyte infiltration into the underlying tissue. The vascular endothelium plays a major role in this pathophysiological condition. Only few therapeutic strategies focus on the vascular endothelium as a major target for an anti-inflammatory approach. In this study, we present the natural compound-derived carbazole derivative C81 as chemical modulator interfering with leukocyte-endothelial cell interactions. An in vivo assay employing intravital microscopy to monitor leukocyte trafficking after C81 treatment in postcapillary venules of a murine cremaster muscle was performed. Moreover, in vitro assays using HUVECs and monocytes were implemented. The impact of C81 on cell adhesion molecules and the NFκB signaling cascade was analyzed in vitro in endothelial cells. Effects of C81 on protein translation were determined by incorporation of a puromycin analog-based approach and polysome profiling. We found that C81 significantly reduced TNF-activated leukocyte trafficking in postcapillary venules. Similar results were obtained in vitro when C81 reduced leukocyte-endothelial cell interactions by down-regulating cell adhesion molecules. Focusing on the NFκB signaling cascade, we found that C81 reduced the activation on multiple levels of the cascade through promoted IκBα recovery by attenuation of IκBα ubiquitination and through reduced protein levels of TNFR1 caused by protein translation inhibition. We suggest that C81 might represent a promising lead compound for interfering with inflammation-related processes in endothelial cells by down-regulation of IκBα ubiquitination on the one hand and inhibition of translation on the other hand without exerting cytotoxic effects.

Conformational plasticity of the ULK3 kinase domain.

The human protein kinase ULK3 regulates the timing of membrane abscission, thus being involved in exosome budding and cytokinesis. Herein, we present the first high-resolution structures of the ULK3 kinase domain. Its unique features are explored against the background of other ULK kinases. An inhibitor fingerprint indicates that ULK3 is highly druggable and capable of adopting a wide range of conformations. In accordance with this, we describe a conformational switch between the active and an inactive ULK3 conformation, controlled by the properties of the attached small-molecule binder. Finally, we discuss a potential substrate-recognition mechanism of the full-length ULK3 protein.

A Screening of Epigenetic Therapeutic Targets for Non-Small Cell Lung Cancer Reveals PADI4 and KDM6B as Promising Candidates.

Despite advances in diagnostic and therapeutic approaches for lung cancer, new therapies targeting metastasis by the specific regulation of cancer genes are needed. In this study, we screened a small library of epigenetic inhibitors in non-small-cell lung cancer (NSCLC) cell lines and evaluated 38 epigenetic targets for their potential role in metastatic NSCLC. The potential candidates were ranked by a streamlined approach using in silico and in vitro experiments based on publicly available databases and evaluated by real-time qPCR target gene expression, cell viability and invasion assays, and transcriptomic analysis. The survival rate of patients with lung adenocarcinoma is inversely correlated with the gene expression of eight epigenetic targets, and a systematic review of the literature confirmed that four of them have already been identified as targets for the treatment of NSCLC. Using nontoxic doses of the remaining inhibitors, KDM6B and PADI4 were identified as potential targets affecting the invasion and migration of metastatic lung cancer cell lines. Transcriptomic analysis of KDM6B and PADI4 treated cells showed altered expression of important genes related to the metastatic process. In conclusion, we showed that KDM6B and PADI4 are promising targets for inhibiting the metastasis of lung adenocarcinoma cancer cells.

Discovery of 3-Amino-1H-pyrazole-Based Kinase Inhibitors to Illuminate the Understudied PCTAIRE Family.

The PCTAIRE subfamily belongs to the CDK (cyclin-dependent kinase) family and represents an understudied class of kinases of the dark kinome. They exhibit a highly conserved binding pocket and are activated by cyclin Y binding. CDK16 is targeted to the plasma membrane after binding to N-myristoylated cyclin Y and is highly expressed in post-mitotic tissues, such as the brain and testis. Dysregulation is associated with several diseases, including breast, prostate, and cervical cancer. Here, we used the N-(1H-pyrazol-3-yl)pyrimidin-4-amine moiety from the promiscuous inhibitor 1 to target CDK16, by varying different residues. Further optimization steps led to 43d, which exhibited high cellular potency for CDK16 (EC50 = 33 nM) and the other members of the PCTAIRE and PFTAIRE family with 20-120 nM and 50-180 nM, respectively. A DSF screen against a representative panel of approximately 100 kinases exhibited a selective inhibition over the other kinases. In a viability assessment, 43d decreased the cell count in a dose-dependent manner. A FUCCI cell cycle assay revealed a G2/M phase cell cycle arrest at all tested concentrations for 43d, caused by inhibition of CDK16.

Image-Based Annotation of Chemogenomic Libraries for Phenotypic Screening.

Phenotypical screening is a widely used approach in drug discovery for the identification of small molecules with cellular activities. However, functional annotation of identified hits often poses a challenge. The development of small molecules with narrow or exclusive target selectivity such as chemical probes and chemogenomic (CG) libraries, greatly diminishes this challenge, but non-specific effects caused by compound toxicity or interference with basic cellular functions still pose a problem to associate phenotypic readouts with molecular targets. Hence, each compound should ideally be comprehensively characterized regarding its effects on general cell functions. Here, we report an optimized live-cell multiplexed assay that classifies cells based on nuclear morphology, presenting an excellent indicator for cellular responses such as early apoptosis and necrosis. This basic readout in combination with the detection of other general cell damaging activities of small molecules such as changes in cytoskeletal morphology, cell cycle and mitochondrial health provides a comprehensive time-dependent characterization of the effect of small molecules on cellular health in a single experiment. The developed high-content assay offers multi-dimensional comprehensive characterization that can be used to delineate generic effects regarding cell functions and cell viability, allowing an assessment of compound suitability for subsequent detailed phenotypic and mechanistic studies.

One-Minute Multi-contrast Echo Planar Brain MRI in Ischemic Stroke: A Retrospective Observational Study of Diagnostic Performance.

BACKGROUND: Fast multi-contrast echo planar MRI (EPIMix) has comparable diagnostic performance to standard MRI for detecting brain pathology but its performance in detecting acute cerebral infarctions has not been determined. PURPOSE: To assess the diagnostic performance of EPIMix for the detection of acute cerebral infarctions. STUDY TYPE: Retrospective observational cohort. POPULATION: One hundred and seventy-two consecutive patients with a clinical suspicion of non-hyperacute ischemic stroke (January 2018 to December 2019). FIELD STRENGTH AND SEQUENCE: 1.5 T or 3 T. EPIMix ((echo-planar based: diffusion weighted (DWI), T2*-weighted, T2-weighted, T2- and T1-fluid attenuated inversion recovery (FLAIR) images) vs. standard MRI: echo-planar DWI, echo-planar T2*-weighted or susceptibility weighted, turbo spin-echo T2-weighted, T2- and T1-FLAIR turbo spin-echo sequences. ASSESSMENT: Three neuroradiologists rated EPIMix and standard MRI on two separate occasions. Incongruent assessments were resolved in consensus with the fourth reader. The ratings included the diagnostic category (acute infarct, normal, and other pathology). Congruent diagnoses together with consensus diagnoses served as the reference standard. STATISTICAL TESTS: The diagnostic performance of EPIMix and standard MRI against the reference standard was calculated by the area under the receiver operating characteristic curve (AUC) and compared by DeLong's test. Sensitivity and specificity were determined. Inter-rater agreements were evaluated by Fleiss's kappa. RESULTS: Of 172 patients (61 ± 16 years, 103 men), acute infarcts were present in 80/172 (47%), normal findings in 60/172 (35%), and other pathology in 32/172 (19%). Across readers, the AUCs were .94-.95 for EPIMix and .95-.99 for standard MRI, with overlapping 95% CI (P = .02-.18). Inter-rater agreement for EPIMix was 0.90 and for standard MRI was 0.93. The sensitivity for EPIMix and standard MRI was 88-91% and 91-98%, respectively, while the specificity was 98-100% and 98-99%, both with overlapping 95% CI. CONCLUSION: Multi-contrast echo planar MRI showed a high but marginally lower diagnostic performance compared to standard MRI for the detection and characterization of acute brain infarct. LEVEL OF EVIDENCE: 3 TECHNICAL EFFICACY: Stage 2.

Therapists' emotional state after sessions in which suicidality is addressed: need for improved management of suicidal tendencies in patients with borderline personality pathology.

INTRODUCTION: Patient suicidality is a frequently experienced topic for psychotherapists. Especially adolescents with borderline personality pathology (BPP) often exhibit suicidal tendencies. Previous research which examined therapists' countertransference towards suicidal patients suggested that therapists are negatively affected and distressed by them. We hypothesize that this emotional response of the therapists is related to specific sessions in which suicidality came up as a topic. Accordingly, the objective of this study consists in examining therapists' emotional state on a session level of analysis. METHODS: The sample consisted of N = 21 adolescents (age 13-19 years) with BPD or subthreshold BPD. Therapists' emotional states were measured in n = 418 sessions using the Session Evaluation Questionnaire. Principal component analysis was used to reduce dimensionality of the therapist response. The emotional states were compared depending on whether suicidality has been addressed in the session (SS) or not (NSS). RESULTS: Two components could be identified. Firstly, therapists were more aroused, excited, afraid, angry and uncertain after SS than after NSS. Secondly, therapists were more aroused, excited, definite and pleased after SS than after NSS. DISCUSSION: Suicidality does not always have to be a burden for therapists: Both a "distress" and an "eustress" component occur in this context from which the latter is supposed to help clinicians master a difficult situation. Since countertransference feelings are often not fully conscious, it is necessary to do research on therapists' emotional states after sessions in which suicidality is addressed. This is crucial to both prevent the therapeutic process from being endangered and preserve clinicians' mental health. Clinical implications and limitations are discussed.

Suppressor mutations reveal an NtrC-like response regulator, NmpR, for modulation of Type-IV Pili-dependent motility in Myxococcus xanthus.

Two-component signaling systems (TCS) regulate bacterial responses to environmental signals through the process of protein phosphorylation. Specifically, sensor histidine kinases (SK) recognize signals and propagate the response via phosphorylation of a cognate response regulator (RR) that functions to initiate transcription of specific genes. Signaling within a single TCS is remarkably specific and cross-talk between TCS is limited. However, regulation of the flow of information through complex signaling networks that include closely related TCS remains largely unknown. Additionally, many bacteria utilize multi-component signaling networks which provide additional genetic and biochemical interactions that must be regulated for signaling fidelity, input and output specificity, and phosphorylation kinetics. Here we describe the characterization of an NtrC-like RR that participates in regulation of Type-IV pilus-dependent motility of Myxococcus xanthus and is thus named NmpR, NtrC Modulator of Pili Regulator. A complex multi-component signaling system including NmpR was revealed by suppressor mutations that restored motility to cells lacking PilR, an evolutionarily conserved RR required for expression of pilA encoding the major Type-IV pilus monomer found in many bacterial species. The system contains at least four signaling proteins: a SK with a protoglobin sensor domain (NmpU), a hybrid SK (NmpS), a phospho-sink protein (NmpT), and an NtrC-like RR (NmpR). We demonstrate that ΔpilR bypass suppressor mutations affect regulation of the NmpRSTU multi-component system, such that NmpR activation is capable of restoring expression of pilA in the absence of PilR. Our findings indicate that pilus gene expression in M. xanthus is regulated by an extended network of TCS which interact to refine control of pilus function.

The Kinase Chemogenomic Set (KCGS): An Open Science Resource for Kinase Vulnerability Identification.

We describe the assembly and annotation of a chemogenomic set of protein kinase inhibitors as an open science resource for studying kinase biology. The set only includes inhibitors that show potent kinase inhibition and a narrow spectrum of activity when screened across a large panel of kinase biochemical assays. Currently, the set contains 187 inhibitors that cover 215 human kinases. The kinase chemogenomic set (KCGS), current Version 1.0, is the most highly annotated set of selective kinase inhibitors available to researchers for use in cell-based screens.