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Background: Local adaptation to divergent environmental conditions can promote population genetic differentiation even in the absence of geographic barriers and hence, lead to speciation. Perturbations by catastrophic events, however, can distort such parapatric ecological speciation processes. Here, we asked whether an exceptionally strong flood led to homogenization of gene pools among locally adapted populations of the Atlantic molly (Poecilia mexicana, Poeciliidae) in the Cueva del Azufre system in southern Mexico, where two strong environmental selection factors (darkness within caves and/or presence of toxic H2S in sulfidic springs) drive the diversification of P. mexicana. Nine nuclear microsatellites as well as heritable female life history traits (both as a proxy for quantitative genetics and for trait divergence) were used as markers to compare genetic differentiation, genetic diversity, and especially population mixing (immigration and emigration) before and after the flood. Results: Habitat type (i.e., non-sulfidic surface, sulfidic surface, or sulfidic cave), but not geographic distance was the major predictor of genetic differentiation. Before and after the flood, each habitat type harbored a genetically distinct population. Only a weak signal of individual dislocation among ecologically divergent habitat types was uncovered (with the exception of slightly increased dislocation from the Cueva del Azufre into the sulfidic creek, El Azufre). By contrast, several lines of evidence are indicative of increased flood-induced dislocation within the same habitat type, e.g., between different cave chambers of the Cueva del Azufre. Conclusions: The virtual absence of individual dislocation among ecologically different habitat types indicates strong natural selection against migrants. Thus, our current study exemplifies that ecological speciation in this and other systems, in which extreme environmental factors drive speciation, may be little affected by temporary perturbations, as adaptations to physico-chemical stressors may directly affect the survival probability in divergent habitat types.
Nosological delineation of congenital ocular motor apraxia type Cogan : an observational study
(2016)
Background: The nosological assignment of congenital ocular motor apraxia type Cogan (COMA) is still controversial. While regarded as a distinct entity by some authorities including the Online Mendelian Inheritance in Man catalog of genetic disorders, others consider COMA merely a clinical symptom.
Methods: We performed a retrospective multicenter data collection study with re-evaluation of clinical and neuroimaging data of 21 previously unreported patients (8 female, 13 male, ages ranging from 2 to 24 years) diagnosed as having COMA.
Results: Ocular motor apraxia (OMA) was recognized during the first year of life and confined to horizontal pursuit in all patients. OMA attenuated over the years in most cases, regressed completely in two siblings, and persisted unimproved in one individual. Accompanying clinical features included early onset ataxia in most patients and cognitive impairment with learning disability (n = 6) or intellectual disability (n = 4). Re-evaluation of MRI data sets revealed a hitherto unrecognized molar tooth sign diagnostic for Joubert syndrome in 11 patients, neuroimaging features of Poretti-Boltshauser syndrome in one case and cerebral malformation suspicious of a tubulinopathy in another subject. In the remainder, MRI showed vermian hypo-/dysplasia in 4 and no abnormalities in another 4 patients. There was a strong trend to more severe cognitive impairment in patients with Joubert syndrome compared to those with inconclusive MRI, but otherwise no significant difference in clinical phenotypes between these two groups.
Conclusions: Systematical renewed analysis of neuroimaging data resulted in a diagnostic reappraisal in the majority of patients with early-onset OMA in the cohort reported here. This finding poses a further challenge to the notion of COMA constituting a separate entity and underlines the need for an expert assessment of neuroimaging in children with COMA, especially if they show cognitive impairment.
Targeted redox inhibition of protein phosphatase 1 by Nox4 regulates eIF2α‐mediated stress signaling
(2016)
Phosphorylation of translation initiation factor 2α (eIF2α) attenuates global protein synthesis but enhances translation of activating transcription factor 4 (ATF4) and is a crucial evolutionarily conserved adaptive pathway during cellular stresses. The serine–threonine protein phosphatase 1 (PP1) deactivates this pathway whereas prolonging eIF2α phosphorylation enhances cell survival. Here, we show that the reactive oxygen species‐generating NADPH oxidase‐4 (Nox4) is induced downstream of ATF4, binds to a PP1‐targeting subunit GADD34 at the endoplasmic reticulum, and inhibits PP1 activity to increase eIF2α phosphorylation and ATF4 levels. Other PP1 targets distant from the endoplasmic reticulum are unaffected, indicating a spatially confined inhibition of the phosphatase. PP1 inhibition involves metal center oxidation rather than the thiol oxidation that underlies redox inhibition of protein tyrosine phosphatases. We show that this Nox4‐regulated pathway robustly enhances cell survival and has a physiologic role in heart ischemia–reperfusion and acute kidney injury. This work uncovers a novel redox signaling pathway, involving Nox4–GADD34 interaction and a targeted oxidative inactivation of the PP1 metal center, that sustains eIF2α phosphorylation to protect tissues under stress.
Background: Preclinical studies demonstrate synergism between cancer immunotherapy and local radiation, enhancing anti-tumor effects and promoting immune responses. BI1361849 (CV9202) is an active cancer immunotherapeutic comprising protamine-formulated, sequence-optimized mRNA encoding six non-small cell lung cancer (NSCLC)-associated antigens (NY-ESO-1, MAGE-C1, MAGE-C2, survivin, 5T4, and MUC-1), intended to induce targeted immune responses.
Methods: We describe a phase Ib clinical trial evaluating treatment with BI1361849 combined with local radiation in 26 stage IV NSCLC patients with partial response (PR)/stable disease (SD) after standard first-line therapy. Patients were stratified into three strata (1: non-squamous NSCLC, no epidermal growth factor receptor (EGFR) mutation, PR/SD after ≥4 cycles of platinum- and pemetrexed-based treatment [n = 16]; 2: squamous NSCLC, PR/SD after ≥4 cycles of platinum-based and non-platinum compound treatment [n = 8]; 3: non-squamous NSCLC, EGFR mutation, PR/SD after ≥3 and ≤ 6 months EGFR-tyrosine kinase inhibitor (TKI) treatment [n = 2]). Patients received intradermal BI1361849, local radiation (4 × 5 Gy), then BI1361849 until disease progression. Strata 1 and 3 also had maintenance pemetrexed or continued EGFR-TKI therapy, respectively. The primary endpoint was evaluation of safety; secondary objectives included assessment of clinical efficacy (every 6 weeks during treatment) and of immune response (on Days 1 [baseline], 19 and 61).
Results: Study treatment was well tolerated; injection site reactions and flu-like symptoms were the most common BI1361849-related adverse events. Three patients had grade 3 BI1361849-related adverse events (fatigue, pyrexia); there was one grade 3 radiation-related event (dysphagia). In comparison to baseline, immunomonitoring revealed increased BI1361849 antigen-specific immune responses in the majority of patients (84%), whereby antigen-specific antibody levels were increased in 80% and functional T cells in 40% of patients, and involvement of multiple antigen specificities was evident in 52% of patients. One patient had a partial response in combination with pemetrexed maintenance, and 46.2% achieved stable disease as best overall response. Best overall response was SD in 57.7% for target lesions.
Conclusion: The results support further investigation of mRNA-based immunotherapy in NSCLC including combinations with immune checkpoint inhibitors.
Trial registration: ClinicalTrials.gov identifier: NCT01915524.
Tolerizing CTL by sustained hepatic PD-L1 expression provides a new therapy spproach in mouse sepsis
(2019)
Cytotoxic T lymphocyte (CTL) activation contributes to liver damage during sepsis, but the mechanisms involved are largely unknown. Understanding the underlying principle will permit interference with CTL activation and thus, provide a new therapeutic option.
Methods: To elucidate the mechanism leading to CTL activation we used the Hepa1-6 cell line in vitro and the mouse model of in vivo polymicrobial sepsis, following cecal-ligation and -puncture (CLP) in wildtype, myeloid specific NOX-2, global NOX2 and NOX4 knockout mice, and their survival as a final readout. In this in vivo setting, we also determined hepatic mRNA and protein expression as well as clinical parameters of liver damage - aspartate- and alanine amino-transaminases. Hepatocyte specific overexpression of PD-L1 was achieved in vivo by adenoviral infection and transposon-based gene transfer using hydrodynamic injection.
Results: We observed downregulation of PD-L1 on hepatocytes in the murine sepsis model. Adenoviral and transposon-based gene transfer to restore PD-L1 expression, significantly improved survival and reduced the release of liver damage, as PD-L1 is a co-receptor that negatively regulates T cell function. Similar protection was observed during pharmacological intervention using recombinant PD-L1-Fc. N-acetylcysteine blocked the downregulation of PD-L1 suggesting the involvement of reactive oxygen species. This was confirmed in vivo, as we observed significant upregulation of PD-L1 expression in NOX4 knockout mice, following sham operation, whereas its expression in global as well as myeloid lineage NOX2 knockout mice was comparable to that in the wild type animals. PD-L1 expression remained high following CLP only in total NOX2 knockouts, resulting in significantly reduced release of liver damage markers.
Conclusion: These results suggest that, contrary to common assumption, maintaining PD-L1 expression on hepatocytes improves liver damage and survival of mice during sepsis. We conclude that administering recombinant PD-L1 or inhibiting NOX2 activity might offer a new therapeutic option in sepsis.
Objectives: There is sparse information on the safety of early primary discharge from the emergency department (ED) after rule-out of myocardial infarction in suspected acute coronary syndrome (ACS). This prospective registry aimed to confirm randomised study results in patients at low-to-intermediate risk, with a broader spectrum of symptoms, across different institutional standards and with a range of local troponin assays including high-sensitivity cTn (hs-cTn), cardiac troponin (cTn) and point-of-care troponin (POC Tn).
Design: Prospective, multicentre European registry.
Setting: 18 emergency departments in nine European countries (Germany, Austria, Switzerland, France, Spain, UK, Turkey, Lithuania and Hungary)
Participants: The final study cohort consisted of 2294 patients (57.2% males, median age 57 years) with suspected ACS.
Interventions: Using the new dual markers strategy, 1477 patients were eligible for direct discharge, which was realised in 974 (42.5%) of patients.
Main outcome measures: The primary endpoint was all-cause mortality at 30 days.
Results: Compared with conventional workup after dual marker measurement, the median length of ED stay was 60 min shorter (228 min, 95% CI: 219 to 239 min vs 288 min, 95% CI: 279 to 300 min) in the primary dual marker strategy (DMS) discharge group. All-cause mortality was 0.1% (95% CI: 0% to 0.6%) in the primary DMS discharge group versus 1.1% (95% CI: 0.6% to 1.8%) in the conventional workup group after dual marker measurement. Conventional workup instead of discharge despite negative DMS biomarkers was observed in 503 patients (21.9%) and associated with higher prevalence of ACS (17.1% vs 0.9%, p<0.001), cardiac diagnoses (55.2% vs 23.5%, p<0.001) and risk factors (p<0.01), but with a similar all-cause mortality of 0.2% (95% CI: 0% to 1.1%) versus primary DMS discharge (p=0.64).
Conclusions: Copeptin on top of cardiac troponin supports safe discharge in patients with chest pain or other symptoms suggestive of ACS under routine conditions with the use of a broad spectrum of local standard POC, conventional and high-sensitivity troponin assays.
Trial registration number NCT02490969.
The Kinase Chemogenomic Set (KCGS): An open science resource for kinase vulnerability identification
(2019)
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) is the most highly annotated set of selective kinase inhibitors available to researchers for use in cell-based screens.
Extracellular signal-regulated kinase 3 (ERK3), known also as mitogen-activated protein kinase 6 (MAPK6), is an atypical member of MAPK kinase family, which has been poorly studied. Little is known regarding its function in biological processes, yet this atypical kinase has been suggested to play important roles in the migration and invasiveness of certain cancers. The lack of tools, such as a selective inhibitor, hampers the study of ERK3 biology. Here, we report the crystal structure of the kinase domain of this atypical MAPK kinase, providing molecular insights into its distinct ATP binding pocket compared to the classical MAPK ERK2, explaining differences in their inhibitor binding properties. Medium-scale small molecule screening identified a number of inhibitors, several of which unexpectedly exhibited remarkably high inhibitory potencies. The crystal structure of CLK1 in complex with CAF052, one of the most potent inhibitors identified for ERK3, revealed typical type-I binding mode of the inhibitor, which by structural comparison could likely be maintained in ERK3. Together with the presented structural insights, these diverse chemical scaffolds displaying both reversible and irreversible modes of action, will serve as a starting point for the development of selective inhibitors for ERK3, which will be beneficial for elucidating the important functions of this understudied kinase.
Aim: Assessment of the effect of nonsurgical periodontal therapy on haematological parameters in patients with grades B (BP) and C periodontitis (CP).
Methods: Eight BP and 46 CP patients received full-mouth periodontal debridement within 48 h, if positive for Aggregatibacter actinomycetemcomitans with adjunctive systemic antibiotics (4 BP, 17 CP). Clinical data were collected prior and 12 weeks after periodontal therapy. Blood was sampled prior to and 1 day as well as 6 and 12 weeks after the first SD visit. Erythrocyte count, haemoglobin value, haematocrit (HCT), mean erythrocyte volume (MCV), mean corpuscular haemoglobin (MCH), MCH concentration (MCHC), platelets (PLT) and heat shock protein 27 (Hsp27) were assessed.
Results: Both groups showed significant clinical improvement (p < 0.05). Using univariate analysis, MCV was noticeably lower in CP than BP at all examinations, HCT only at baseline. For CP, MCHC was noticeably higher 12 weeks after SD than at baseline and 1 day (p ≤ 0.005) and Hsp27 increased noticeably at 1 day (p < 0.05). Repeated measures analysis of variance revealed African origin to be associated with lower MCV and female sex with lower MCHC.
Conclusion: Based on multivariate analysis, periodontal diagnosis (BP/CP) was not associated with haematological parameters measured in this study or serum Hsp27. In CP, nonsurgical periodontal therapy improved MCHC 12 weeks after SD. Also in CP Hsp27 was increased 1 day after SD.
Selectivity remains a challenge for ATP-mimetic kinase inhibitors, an issue that may be overcome by targeting unique residues or binding pockets. However, to date only few strategies have been developed. Here we identify that bulky residues located N-terminal to the DFG motif (DFG-1) represent an opportunity for designing highly selective inhibitors with unexpected binding modes. We demonstrate that several diverse inhibitors exerted selective, noncanonical binding modes that exclusively target large hydrophobic DFG-1 residues present in many kinases including PIM, CK1, DAPK, and CLK. By use of the CLK family as a model, structural and biochemical data revealed that the DFG-1 valine controlled a noncanonical binding mode in CLK1, providing a rationale for selectivity over the closely related CLK3 which harbors a smaller DFG-1 alanine. Our data suggest that targeting the restricted back pocket in the small fraction of kinases that harbor bulky DFG-1 residues offers a versatile selectivity filter for inhibitor design.