Protective effects of Gαi3 deficiency in a murine heart-failure model of ß1-adrenoceptor overexpression.

We have shown that in murine cardiomyopathy caused by overexpression of the ß1-adrenoceptor, Gαi2-deficiency is detrimental. Given the growing evidence for isoform-specific Gαi-functions, we now examined the consequences of Gαi3 deficiency in the same heart-failure model. Mice overexpressing cardiac ß1-adrenoceptors with (ß1-tg) or without Gαi3-expression (ß1-tg/Gαi3-/-) were compared to C57BL/6 wildtypes and global Gαi3-knockouts (Gαi3-/-). The life span of ß1-tg mice was significantly shortened but improved when Gαi3 was lacking (95% CI: 592-655 vs. 644-747 days). At 300 days of age, left-ventricular function and survival rate were similar in all groups. At 550 days of age, ß1-tg but not ß1-tg/Gαi3-/- mice displayed impaired ejection fraction (35 ± 18% vs. 52 ± 16%) compared to wildtype (59 ± 4%) and Gαi3-/- mice (60 ± 5%). Diastolic dysfunction of ß1-tg mice was prevented by Gαi3 deficiency, too. The increase of ANP mRNA levels and ventricular fibrosis observed in ß1-tg hearts was significantly attenuated in ß1-tg/Gαi3-/- mice. Transcript levels of phospholamban, ryanodine receptor 2, and cardiac troponin I were similar in all groups. However, Western blots and phospho-proteomic analyses showed that in ß1-tg, but not ß1-tg/Gαi3-/- ventricles, phospholamban protein was reduced while its phosphorylation increased. Here, we show that in mice overexpressing the cardiac ß1-adrenoceptor, Gαi3 deficiency slows or even prevents cardiomyopathy and increases shortened life span. Previously, we found Gαi2 deficiency to aggravate cardiac dysfunction and mortality in the same heart-failure model. Our findings indicate isoform-specific interventions into Gi-dependent signaling to be promising cardio-protective strategies.

The role of research competence as an influencing factor for the careers of young academics. Findings and implications from studies on doctorates in medicine and life sciences in Germany.

Background: When viewed internationally, Germany boasts a high rate of doctoral candidates. Fields such as medicine and life sciences have a notably high proportion of doctoral students, a trend rooted in historical factors. Despite this, comprehensive empirical studies concerning the doctoral phase and early-career researchers, especially in relation to the rise of structured doctoral programmes, have only recently gained traction. Methods: We present findings from a project investigating young scientists in medicine and life sciences. Postdoctoral graduates from these disciplines were examined both quantitatively and qualitatively within the E-Prom projects, emphasizing the primary domain of research. Results: Our analysis indicates some benefits of structured doctoral programmes over traditional individual doctorates. However, the disparities between these doctoral approaches are less pronounced than anticipated. We also identified discrepancies between the programme descriptions and their actual execution. Integration into the scientific community and research-related self-efficacy are potential indicators of publication output and inclination towards a scientific career. Physicians exhibited lower research-related self-efficacy and a lesser tendency towards a scientific career than biologists. Notably, we found gender disparities disadvantaging female graduates, with these disparities being more marked in medicine. Conclusions: There is evidence to suggest that official representations of structured doctoral programmes do not always align with their practical applications, limiting their potential effectiveness. Therefore, resources should be allocated to ensure the consistent execution of these programmes. Given the empirical evidence supporting the benefits of community integration for junior researchers, efforts should be made to facilitate their networking. Additionally, our findings emphasize the necessity of providing enhanced support for young female scientists.

Therapy-Related Transcriptional Subtypes in Matched Primary and Recurrent Head and Neck Cancer.

PURPOSE: The genetic relatedness between primary and recurrent head and neck squamous cell carcinomas (HNSCC) reflects the extent of heterogeneity and therapy-driven selection of tumor subpopulations. Yet, current treatment of recurrent HNSCC ignores the molecular characteristics of therapy-resistant tumor populations. EXPERIMENTAL DESIGN: From 150 tumors, 74 primary HNSCCs were RNA sequenced and 38 matched primary/recurrent tumor pairs were both whole-exome and RNA sequenced. Transcriptome analysis determined the predominant classical (CL), basal (BA), and inflamed-mesenchymal (IMS) transcriptional subtypes according to an established classification. Genomic alterations and clonal compositions of tumors were evaluated from whole-exome data. RESULTS: Although CL and IMS subtypes were more common in primary HNSCC with low recurrence rates, the BA subtype was more prevalent and stable in recurrent tumors. The BA subtype was associated with a transcriptional signature of partial epithelial-to-mesenchymal transition (p-EMT) and early recurrence. In 44% of matched cases, the dominant subtype changed from primary to recurrent tumors, preferably from IMS to BA or CL. Expression analysis of prognostic gene sets identified upregulation of hypoxia, p-emt, and radiotherapy resistance signatures and downregulation of tumor inflammation in recurrences compared with index tumors. A relevant subset of primary/recurrent tumor pairs presented no evidence for a common clonal origin. CONCLUSIONS: Our study showed a high degree of genetic and transcriptional heterogeneity between primary/recurrent tumors, suggesting therapy-related selection of a transcriptional subtype with characteristics unfavorable for therapy. We conclude that therapy decisions should be based on genetic and transcriptional characteristics of recurrences rather than primary tumors to enable optimally tailored treatment strategies.

Regulations and practices of structured doctoral education in the life sciences in Germany.

Structured doctoral education is increasingly preferred compared to the individual model. Several science policy organisations give recommendations on how to structure doctoral education. However, there is little research on to what extent these recommendations find their way into practice. In our study, we first compared European and German recommendations on doctoral education with, second, the institutional regulations of structured doctoral programmes (N = 98) in the life sciences at twelve different German universities. Additionally, we third asked doctoral graduates (N = 1796) of these structured doctoral programmes and graduates of individual doctoral studies about their experience in doctoral education. Fourth, we contrasted the regulations of structured doctoral programmes with the reported experiences of their graduates. We found significant deviations of the reported practices of graduates from the regulations of their organisations, regarding the student admission, supervision and curricular activities of doctoral candidates. The efficacy of structured versus traditional doctoral education should be examined based on reported practice rather than on the respective written regulations.

Autism-associated mutations in the CaVß2 calcium-channel subunit increase Ba2+-currents and lead to differential modulation by the RGK-protein Gem.

Voltage-gated calcium-channels (VGCCs) are heteromers consisting of several subunits. Mutations in the genes coding for VGCC subunits have been reported to be associated with autism spectrum disorder (ASD). In a previous study, we identified electrophysiologically relevant missense mutations of CaVß2 subunits of VGCCs. From this, we derived the hypothesis that several CaVß2-mutations associated with ASD show common features sensitizing LTCCs and/or enhancing currents. Using a CaVß2d backbone, we performed extensive whole-cell and single-channel patch-clamp analyses of Ba2+ currents carried by Cav1.2 pore subunits co-transfected with the previously described CaVß2 mutations (G167S, S197F) as well as a recently identified point mutation (V2D). Furthermore, the interaction of the mutated CaVß2d subunits with the RGK protein Gem was analyzed by co-immunoprecipitation assays and electrophysiological studies. Patch-clamp analyses revealed that all mutations increase Ba2+ currents, e.g. by decreasing inactivation or increasing fraction of active sweeps. All CaVß2 mutations interact with Gem, but differ in the extent and characteristics of modulation by this RGK protein (e.g. decrease of fraction of active sweeps: CaVß2d_G167S > CaVß2d_V2D > CaVß2d_S197F). In conclusion, patch-clamp recordings of ASD-associated CaVß2d mutations revealed differential modulation of Ba2+ currents carried by CaV1.2 suggesting kind of an "electrophysiological fingerprint" each. The increase in current finally observed with all CaVß2d mutations analyzed might contribute to the complex pathophysiology of ASD and by this indicate a possible underlying molecular mechanism.

Fifteen years of the cologne medical model study course: has the expectation of increasing student interest in general practice specialization been fulfilled?

Background: The 2002 Medical Licensure Act gave German universities certain freedoms for reforming their medical degree courses. The Medical Faculty of the University of Cologne took advantage of this opportunity and introduced a model study course in the winter semester 2003/04 through §41 of the Medical Licensure Act. One of the main reasons for this was that back then there was an increasing shortage of doctors in clinical curative medicine and GP primary care. This study investigates whether the introduction of the Cologne Model Study Course (MSG) can show stronger interest in curative medical work (especially General Practice) compared to students of the standard degree course (RSG). Methodology: The proof of added value was examined through graduate surveys conducted at the University of Cologne and through the proportion of students who completed the PY elective rotation "General Practice". The students of the standard degree course (start of studies prior to winter semester 2003/2004) were compared with students of the model study course (start of studies from winter semester 2003/04 onwards). Measurements were carried out using descriptive frequency tables and correlation analyzes according to Spearman. Results: The students' interest in curative medicine was already high (91%) even before the model study course was introduced and increased only slightly (to 91.9%). There is also only a slight increase in specialization in General Practice (RSG=5.9% vs. MSG=9.2%). However, selection of rotations in General Practice was significantly increased (RSG=1.9% vs. MSG=3.4%, r=0.046 **, p<0.005). Conclusion: The Cologne Model Study Course in Human Medicine has increased awareness of the subject of General Practice among students through a large number of curricular changes. The fact that only marginal effects can be demonstrated shows once more the strong dependence of choosing General Medicine as a career path on other factors (such as gender or the presence of positive role models) and emphasizes the necessity of promoting General Practice student education not only through increased curricular mapping but by additional innovative concepts to maximize the status of General Practice from the perspective of students.

Direct, gabapentin-insensitive interaction of a soluble form of the calcium channel subunit α2δ-1 with thrombospondin-4.

The α2δ-1 subunit of voltage-gated calcium channels binds to gabapentin and pregabalin, mediating the analgesic action of these drugs against neuropathic pain. Extracellular matrix proteins from the thrombospondin (TSP) family have been identified as ligands of α2δ-1 in the CNS. This interaction was found to be crucial for excitatory synaptogenesis and neuronal sensitisation which in turn can be inhibited by gabapentin, suggesting a potential role in the pathogenesis of neuropathic pain. Here, we provide information on the biochemical properties of the direct TSP/α2δ-1 interaction using an ELISA-style ligand binding assay. Our data reveal that full-length pentameric TSP-4, but neither TSP-5/COMP of the pentamer-forming subgroup B nor TSP-2 of the trimer-forming subgroup A directly interact with a soluble variant of α2δ-1 (α2δ-1S). Interestingly, this interaction is not inhibited by gabapentin on a molecular level and is not detectable on the surface of HEK293-EBNA cells over-expressing α2δ-1 protein. These results provide biochemical evidence that supports a specific role of TSP-4 among the TSPs in mediating the binding to neuronal α2δ-1 and suggest that gabapentin does not directly target TSP/α2δ-1 interaction to alleviate neuropathic pain.

"Hopefully, I will never forget that again" - sensitizing medical students for drug safety by working on cases and simulating doctor-patient communication.

Objective: This project is part of the "PJ-STArT-Block", a one-week course preparing 10th semester medical students for their final practical year. The focus is on sensitizing students to aspects of medication safety by becoming aware of their skills and their deficits in terms of application and communication of pharmacological knowledge. The modules were evaluated regarding feasibility, acceptance and possible effects. Furthermore, the areas in which students see their pharmacological deficits or learning successes were gathered. Methods: In simulated physician-patient conversations, the students are to identify drug-related problems such as medication errors, adverse drug events or interactions. Together with their fellow students and under medical or pharmaceutical moderation, they then have to find solutions for the identified problems and communicate these solutions to the patients. Based on paper cases, students practice, reflect, and discuss the research of reliable information about drugs and medication therapy. The written evaluation included the evaluation by school grades and the possibility of comments in free text. A content analysis of interviews with students at the beginning of the project aimed to identify areas of pharmacology in which they see their own deficits. Results: Evaluation results including the free text comments indicate students' acceptance of our pharmacology modules. According to this, the students realize the importance of aspects relevant for medication safety. The areas mentioned in 35 interviews in which students localize deficits, correspond to the topics that were intended when conceiving the modules and which are important for medication safety (e.g. interactions, adverse drug effects, dosages). Conclusion: Implementation of context-based, application-oriented teaching formats as recently claimed for pharmacological education to improve the quality of prescriptions, is possible, as the Cologne example shows. The student evaluation turns out positively and indicates a critical self-reflection. The students identified various pharmacological deficits in themselves, which have since been confirmed and quantified in another study.

Single-Channel Resolution of the Interaction between C-Terminal CaV1.3 Isoforms and Calmodulin.

Voltage-dependent calcium (CaV) 1.3 channels are involved in the control of cellular excitability and pacemaking in neuronal, cardiac, and sensory cells. Various proteins interact with the alternatively spliced channel C-terminus regulating gating of CaV1.3 channels. Binding of a regulatory calcium-binding protein calmodulin (CaM) to the proximal C-terminus leads to the boosting of channel activity and promotes calcium-dependent inactivation (CDI). The C-terminal modulator domain (CTM) of CaV1.3 channels can interfere with the CaM binding, thereby inhibiting channel activity and CDI. Here, we compared single-channel gating behavior of two natural CaV1.3 splice isoforms: the long CaV1.342 with the full-length CTM and the short CaV1.342A with the C-terminus truncated before the CTM. We found that CaM regulation of CaV1.3 channels is dynamic on a minute timescale. We observed that at equilibrium, single CaV1.342 channels occasionally switched from low to high open probability, which perhaps reflects occasional binding of CaM despite the presence of CTM. Similarly, when the amount of the available CaM in the cell was reduced, the short CaV1.342A isoform showed patterns of the low channel activity. CDI also underwent periodic changes with corresponding kinetics in both isoforms. Our results suggest that the competition between CTM and CaM is influenced by calcium, allowing further fine-tuning of CaV1.3 channel activity for particular cellular needs.

Development of perceived pharmacological deficits of medical students and alumni supports claim for continuous and more application-oriented education.

Medical students' prescribing competencies are insufficient. So far, surveys focused on final-year students. Knowledge and confidence seem important, but their development during medical studies are unclear. This study investigated whether students perceived deficits in pharmacological knowledge change during medical studies. Alumni were included to look for changes occurring after graduation. Medical students at different stages of their studies were invited to fill in paper-and-pencil (6th-, 8th-, 9th- and 10th-term students) or online questionnaires (final-year students and alumni) regarding their self-assessed deficits in pharmacology. Questionnaires have been developed based on previous interviews with 10th-term students. We differentiated between declarative and application-oriented knowledge. In total, data from 816 participants could be analysed. Self-assessment regarding declarative knowledge changed during medical studies, being more sceptical in terms without pharmacology courses. Of note, self-assessment of application-oriented knowledge remained constantly low throughout, although our pharmacology courses use problem-based learning. Tenth-term students were most sceptical, perhaps influenced by an obligatory, formative, simulation-based, 1-week course, preparing students for their final practical year. Compared to students, alumni were significantly less sceptical regarding application-oriented knowledge. Students' self-assessment of deficits in pharmacological knowledge changes throughout their studies, presumably in association with pharmacology courses. Overall, students are rather sceptical, especially with regard to application-oriented knowledge. Our data further substantiate the European Association for Clinical Pharmacology and Therapeutics (EACPT) recommendations to improve pharmacology education throughout the entire medical curriculum, e.g. by providing more training in simulated and clinical environments.

Alternative Splicing at N Terminus and Domain I Modulates CaV1.2 Inactivation and Surface Expression.

The CaV1.2 L-type calcium channel is a key conduit for Ca2+ influx to initiate excitation-contraction coupling for contraction of the heart and vasoconstriction of the arteries and for altering membrane excitability in neurons. Its α1C pore-forming subunit is known to undergo extensive alternative splicing to produce many CaV1.2 isoforms that differ in their electrophysiological and pharmacological properties. Here, we examined the structure-function relationship of human CaV1.2 with respect to the inclusion or exclusion of mutually exclusive exons of the N-terminus exons 1/1a and IS6 segment exons 8/8a. These exons showed tissue selectivity in their expression patterns: heart variant 1a/8a, one smooth-muscle variant 1/8, and a brain isoform 1/8a. Overall, the 1/8a, when coexpressed with CaVß2a, displayed a significant and distinct shift in voltage-dependent activation and inactivation and inactivation kinetics as compared to the other three splice variants. Further analysis showed a clear additive effect of the hyperpolarization shift in V1/2inact of CaV1.2 channels containing exon 1 in combination with 8a. However, this additive effect was less distinct for V1/2act. However, the measured effects were ß-subunit-dependent when comparing CaVß2a with CaVß3 coexpression. Notably, calcium-dependent inactivation mediated by local Ca2+-sensing via the N-lobe of calmodulin was significantly enhanced in exon-1-containing CaV1.2 as compared to exon-1a-containing CaV1.2 channels. At the cellular level, the current densities of the 1/8a or 1/8 variants were significantly larger than the 1a/8a and 1a/8 variants when coexpressed either with CaVß2a or CaVß3 subunit. This finding correlated well with a higher channel surface expression for the exon 1-CaV1.2 isoform that we quantified by protein surface-expression levels or by gating currents. Our data also provided a deeper molecular understanding of the altered biophysical properties of alternatively spliced human CaV1.2 channels by directly comparing unitary single-channel events with macroscopic whole-cell currents.

The prescription talk - an approach to teach patient-physician conversation about drug prescription to medical students.

Background: Medication communication from physicians to patients often is poor, by this among others enhancing the risk of non-adherence. In this context, a neglect regarding the prescription talk has been complained. Aim of the project: In a newly developed elective medical students work on physician-patient conversations dealing with drug prescription. Essential aspects related to an effective and safe drug treatment are combined with steps of shared decision-making. Together with a tutor, students develop a (model) conversation guide that might be tailored according to individual needs and views. Description/Methods: In a one-week course 3rd-5th year medical students treat a paper case according to problem-based learning. This is accompanied by a one-hour lecture and literature provided on an online learning platform (ILIAS). During a workshop, aspects of drug treatment and patient participation are integrated into a guide for a prescription talk. At the end of the week the students are invited to apply the (if need be individualized) guide in a simulated physician-patient communication with an actor. The conversation is evaluated using a checklist based upon the (model) conversation guide. Results: Informal and formalized feedback indicate high acceptance and satisfaction of participants with this elective. The checklist turned out to be of acceptable to good reliability with mostly selective items. Portfolio entries and written evaluation suggest that participants' positions and attitudes are influenced.

A multi-centre student survey on weighing disciplines in medical curricula - a pilot study.

Aim: Initiated by students, this pilot study examines how obtaining medical students' perspectives via a structured online survey may prove useful for curriculum deliberation. Methods: In 2012, 747 students of 32 medical faculties in Germany assessed disciplines specified in the Medical Licensure Act (AÄpprO) thereby concerning the allocation of teaching time, perceived usefulness regarding preparation for state examination and medical practice, their interest and motivation for studying as well as consideration for future work. Results: Internal medicine, surgery, paediatrics, gynaecology/obstetrics and general medicine rank amongst the upper third regarding allocation of teaching time and perceived usefulness for future medical practice. Concerning both preparation for state examination and medical practice internal medicine ranks second, while surgery only 22nd and 28th of 32, respectively. Some clinical-theoretical disciplines (e.g. pharmacology) are in the top ten regarding perceived preparation for state examination, too. Students who consider choosing internal medicine for future work rate associated disciplines significantly higher regarding usefulness for clinical practice (e.g. pharmacology) or motivation for studying (e.g. microbiology) than other students do. Conclusion: A simple survey reveals interesting data on students' perceptions and ideas of medical studies. Though the data are plausible, interpretations should be done with caution. Nonetheless, data like these should give rise to further questions and discussions, e.g. as part of curriculum deliberation.

Exclusion of alternative exon 33 of CaV1.2 calcium channels in heart is proarrhythmogenic.

Alternative splicing changes the CaV1.2 calcium channel electrophysiological property, but the in vivo significance of such altered channel function is lacking. Structure-function studies of heterologously expressed CaV1.2 channels could not recapitulate channel function in the native milieu of the cardiomyocyte. To address this gap in knowledge, we investigated the role of alternative exon 33 of the CaV1.2 calcium channel in heart function. Exclusion of exon 33 in CaV1.2 channels has been reported to shift the activation potential -10.4 mV to the hyperpolarized direction, and increased expression of CaV1.2Δ33 channels was observed in rat myocardial infarcted hearts. However, how a change in CaV1.2 channel electrophysiological property, due to alternative splicing, might affect cardiac function in vivo is unknown. To address these questions, we generated mCacna1c exon 33-/--null mice. These mice contained CaV1.2Δ33 channels with a gain-of-function that included conduction of larger currents that reflects a shift in voltage dependence and a modest increase in single-channel open probability. This altered channel property underscored the development of ventricular arrhythmia, which is reflected in significantly more deaths of exon 33-/- mice from ß-adrenergic stimulation. In vivo telemetric recordings also confirmed increased frequencies in premature ventricular contractions, tachycardia, and lengthened QT interval. Taken together, the significant decrease or absence of exon 33-containing CaV1.2 channels is potentially proarrhythmic in the heart. Of clinical relevance, human ischemic and dilated cardiomyopathy hearts showed increased inclusion of exon 33. However, the possible role that inclusion of exon 33 in CaV1.2 channels may play in the pathogenesis of human heart failure remains unclear.

An expert protocol for immunofluorescent detection of calcium channels in tsA-201 cells.

INTRODUCTION: Pore-forming subunits of voltage gated calcium channels (VGCC) are large membrane proteins (260kDa) containing 24 transmembrane domains. Despite transfection with viral promoter driven vectors, biochemical analysis of VGCC is often hampered by rather low expression levels in heterologous systems rendering VGCC challenging targets. Especially in immunofluorescent detection, calcium channels are demanding proteins. METHODS: We provide an expert step-by-step protocol with adapted conditions for handling procedures (tsA-201 cell culture, transient transfection, incubation time and temperature at 28°C or 37°C and immunostaining) to address the L-type calcium-channel pore Cav1.2 in an immunofluorescent approach. RESULTS: We performed immunocytochemical analysis of Cav1.2 expression at single-cell level in combination with detection of different markers for cellular organelles. We show confluency levels and shapes of tsA-201 cells at different time points during an experiment. Our experiments reveal sufficient levels of Cav1.2 protein and a correct Cav1.2 expression pattern in polygonal shaped cells already 12h after transfection. DISCUSSION: A sequence of elaborated protocol modifications allows subcellular localization analysis of Cav1.2 in an immunocytochemical approach. We provide a protocol that may be used to achieve insights into physiological and pathophysiological processes involving voltage gated calcium channels. Our protocol may be used for expression analysis of other challenging proteins and efficient overexpression may be exploited in related biochemical techniques requiring immunolabels.

Outcome-Relevant Effects of Shared Decision Making.

BACKGROUND: Shared decision making (SDM) is considered a gold standard for the cooperation of doctor and patient. SDM improves patients' overall satisfaction and their confidence in decisions that have been taken. The extent to which it might also positively affect patient-relevant, disease-related endpoints is a matter of debate. METHODS: We systematically searched the PubMed database and the Cochrane Library for publications on controlled intervention studies of SDM. The quality of the intervention and the risk of bias in each publication were assessed on the basis of pre-defined inclusion and exclusion criteria. The effects of SDM on patient-relevant, disease-related endpoints were compared, and effect sizes were calculated. RESULTS: We identified 22 trials that differed widely regarding the patient populations studied, the types of intervention performed, and the mode of implementation of SDM. In ten articles, 57% of the endpoints that were considered relevant were significantly improved by the SDM intervention compared to the control group. The median effect size (Cohen's d) was 0.53 (0.14-1.49). In 12 trials, outcomes did not differ between the two groups. In all 22 studies identified, 39% of the relevant outcomes were significantly improved compared with the control groups. CONCLUSION: The trials performed to date to addressing the effect of SDM on patient-relevant, disease-related endpoints are insufficient in both quantity and quality. Although just under half of the trials reviewed here indicated a positive effect, no final conclusion can be drawn. A consensus-based standardization of both SDM-promoting measures and appropriate clinical studies are needed.

Lack of Gαi2 leads to dilative cardiomyopathy and increased mortality in ß1-adrenoceptor overexpressing mice.

AIMS: Inhibitory G (Gi) proteins have been proposed to be cardioprotective. We investigated effects of Gαi2 knockout on cardiac function and survival in a murine heart failure model of cardiac ß1-adrenoceptor overexpression. METHODS AND RESULTS: ß1-transgenic mice lacking Gαi2 (ß1-tg/Gαi2 (-/-)) were compared with wild-type mice and littermates either overexpressing cardiac ß1-adrenoceptors (ß1-tg) or lacking Gαi2 (Gαi2 (-/-)). At 300 days, mortality of mice only lacking Gαi2 was already higher compared with wild-type or ß1-tg, but similar to ß1-tg/Gαi2 (-/-), mice. Beyond 300 days, mortality of ß1-tg/Gαi2 (-/-) mice was enhanced compared with all other genotypes (mean survival time: 363 ± 21 days). At 300 days of age, echocardiography revealed similar cardiac function of wild-type, ß1-tg, and Gαi2 (-/-) mice, but significant impairment for ß1-tg/Gαi2 (-/-) mice (e.g. ejection fraction 14 ± 2 vs. 40 ± 4% in wild-type mice). Significantly increased ventricle-to-body weight ratio (0.71 ± 0.06 vs. 0.48 ± 0.02% in wild-type mice), left ventricular size (length 0.82 ± 0.04 vs. 0.66 ± 0.03 cm in wild types), and atrial natriuretic peptide and brain natriuretic peptide expression (mRNA: 2819 and 495% of wild-type mice, respectively) indicated hypertrophy. Gαi3 was significantly up-regulated in Gαi2 knockout mice (protein compared with wild type: 340 ± 90% in Gαi2 (-/-) and 394 ± 80% in ß1-tg/Gαi2 (-/-), respectively). CONCLUSIONS: Gαi2 deficiency combined with cardiac ß1-adrenoceptor overexpression strongly impaired survival and cardiac function. At 300 days of age, ß1-adrenoceptor overexpression alone had not induced cardiac hypertrophy or dysfunction while there was overt cardiomyopathy in mice additionally lacking Gαi2. We propose an enhanced effect of increased ß1-adrenergic drive by the lack of protection via Gαi2. Gαi3 up-regulation was not sufficient to compensate for Gαi2 deficiency, suggesting an isoform-specific or a concentration-dependent mechanism.

Voltage-gated Calcium Channels and Autism Spectrum Disorders.

Autism spectrum disorder is a complex-genetic disease and its etiology is unknown for the majority of cases. So far, more than one hundred different susceptibility genes were detected. Voltage-gated calcium channels are among the candidates linked to autism spectrum disorder by results of genetic studies. Mutations of nearly all pore-forming and some auxiliary subunits of voltage gated calcium channels have been revealed from investigations of autism spectrum disorder patients and populations. Though there are only few electrophysiological characterizations of voltage-gated calcium channel mutations found in autistic patients these studies suggest their functional relevance. In summary, both genetic and functional data suggest a potential role of voltage-gated calcium channels in autism spectrum disorder. Future studies require refinement of the clinical and systems biological concepts of autism spectrum disorder and an appropriate holistic approach at the molecular level, e.g. regarding all facets of calcium channel functions.

Development of a high-throughput screening-compatible assay to identify inhibitors of the CK2α/CK2ß interaction.

Increased activity of protein kinase CK2 is associated with various types of cancer, neurodegenerative diseases, and chronic inflammation. In the search for CK2 inhibitors, attention has expanded toward compounds disturbing the interaction between CK2α and CK2ß in addition to established active site-directed approaches. The current article describes the development of a fluorescence anisotropy-based assay that mimics the principle of CK2 subunit interaction by using CK2α(1-335) and the fluorescent probe CF-Ahx-Pc as a CK2ß analog. In addition, we identified new inhibitors able to displace the fluorescent probe from the subunit interface on CK2α(1-335). Both CF-Ahx-Pc and the inhibitors I-Pc and Cl-Pc were derived from the cyclic peptide Pc, a mimetic of the C-terminal CK2α-binding motif of CK2ß. The design of the two inhibitors was based on docking studies using the known crystal structure of the Pc/CK2α(1-335) complex. The dissociation constants obtained in the fluorescence anisotropy assay for binding of all compounds to human CK2α(1-335) were validated by isothermal titration calorimetry. I-Pc was identified as the tightest binding ligand with a KD value of 240nM and was shown to inhibit the CK2 holoenzyme-dependent phosphorylation of PDX-1, a substrate requiring the presence of CK2ß, with an IC50 value of 92µM.