d-Galactose induced early aging in human erythrocytes: Role of band 3 protein.

Aging, a time-dependent multifaceted process, affects both cell structure and function and involves oxidative stress as well as glycation. The present investigation focuses on the role of the band 3 protein (B3p), an anion exchanger essential to red cells homeostasis, in a d-galactose ( d-Gal)-induced aging model. Anion exchange capability, measured by the rate constant of SO4²- uptake through B3p, levels of lipid peroxidation, oxidation of membrane sulfhydryl groups, B3p expression, methemoglobin, glycated hemoglobin (Hb), and the reduced glutathione/oxidized glutathione ratio were determined after exposure of human erythrocytes to 25, 35, 50, and 100 mmol/L d-Gal for 24 h. Our results show that: (i) in vitro application of d-Gal is useful to model early aging in human erythrocytes; (ii) assessment of B3p ion transport function is a sensitive tool to monitor aging development; (iii) d-Gal leads to Hb glycation and produces substantial changes on the endogenous antioxidant system; (iv) the impact of aging on B3p function proceeds through steps, first involving Hb glycation and then oxidative events at the membrane level. These findings offer a useful tool to understand the mechanisms of aging in human erythrocytes and propose B3p as a possible target for new therapeutic strategies to counteract age-related disturbances.

Correlation of online assessment parameters with summative exam performance in undergraduate medical education of pharmacology: a prospective cohort study.

BACKGROUND: Learning analytics aims to improve learning outcomes through the systematic measurement and analysis of learning-related data. However, which parameters have the highest predictive power for academic performance remains to be elucidated. The aim of this study was to investigate the correlation of different online assessment parameters with summative exam performance in undergraduate medical education of pharmacology. METHODS: A prospective study was conducted with a cohort of undergraduate medical students enrolled in a pharmacology course at Technical University of Munich, Germany. After a four-week teaching and learning period, students were given access to an online assessment platform consisting of 440 multiple choice (MC) questions. After 12 days, a final written summative exam was performed. Bivariate correlation and multiple regression analyses were performed for different online assessment parameters as predictors and summative exam performance as dependent variable. Self-perceived pharmacology competence was measured by questionnaires pre- and postintervention. RESULTS: A total of 224 out of 393 (57%) students participated in the study and were included in the analysis. There was no significant correlation for the parameters "number of logins" (r = 0.01, p = 0.893), "number of MC-questions answered" (r = 0.02, p = 0.813) and "time spent on the assessment platform" (r = - 0.05, p = 0.459) with exam performance. The variable "time per question" was statistically significant (p = 0.006), but correlated negatively (r = - 0.18) with academic performance of study participants. Only "total score" (r = 0.71, p < 0.001) and the "score of first attempt" (r = 0.72, p < 0.001) were significantly correlated with final grades. In a multiple regression analysis, "score first attempt" accounted for 52% of the variation of "score final exam", and "time per question" and "total score" for additional 5 and 1.4%, respectively. No gender-specific differences were observed. Finally, online assessments resulted in improved self-perceived pharmacology competence of students. CONCLUSION: In this prospective cohort study, we systematically assessed the correlation of different online assessments parameters with exam performance and their gender-neutrality. Our findings may help to improve predictive models of academic performance in undergraduate medical education of pharmacology.

Inhibition of Cullin-RING E3 ubiquitin ligase 7 by simian virus 40 large T antigen.

Simian virus 40 (SV40) large tumor antigen (LT) triggers oncogenic transformation by inhibition of key tumor suppressor proteins, including p53 and members of the retinoblastoma family. In addition, SV40 transformation requires binding of LT to Cullin 7 (CUL7), a core component of Cullin-RING E3 ubiquitin ligase 7 (CRL7). However, the pathomechanistic effects of LT-CUL7 interaction are mostly unknown. Here we report both in vitro and in vivo experimental evidence that SV40 LT suppresses the ubiquitin ligase function of CRL7. We show that SV40 LT, but not a CUL7 binding-deficient mutant (LT(Δ69-83)), impaired 26S proteasome-dependent proteolysis of the CRL7 target protein insulin receptor substrate 1 (IRS1), a component of the insulin and insulin-like growth factor 1 signaling pathway. SV40 LT expression resulted in the accumulation and prolonged half-life of IRS1. In vitro, purified SV40 LT reduced CRL7-dependent IRS1 ubiquitination in a concentration-dependent manner. Expression of SV40 LT, or depletion of CUL7 by RNA interference, resulted in the enhanced activation of IRS1 downstream signaling pathways phosphatidylinositol-3-kinase/AKT and Erk mitogen-activated pathway kinase, as well as up-regulation of the downstream target gene c-fos. Finally, SV40 LT-positive carcinoma of carcinoembryonic antigen 424/SV40 LT transgenic mice displayed elevated IRS1 protein levels and activation of downstream signaling. Taken together, these data suggest that SV40 LT protects IRS1 from CRL7-mediated degradation, thereby sustaining high levels of promitogenic IRS1 downstream signaling pathways.

The CUL7 E3 ubiquitin ligase targets insulin receptor substrate 1 for ubiquitin-dependent degradation.

Recent genetic studies have documented a pivotal growth-regulatory role played by the Cullin 7 (CUL7) E3 ubiquitin ligase complex containing the Fbw8-substrate-targeting subunit, Skp1, and the ROC1 RING finger protein. In this report, we identified insulin receptor substrate 1 (IRS-1), a critical mediator of the insulin/insulin-like growth factor 1 signaling, as a proteolytic target of the CUL7 E3 ligase in a manner that depends on mammalian target of rapamycin and the p70 S6 kinase activities. Interestingly, while embryonic fibroblasts of Cul7-/- mice were found to accumulate IRS-1 and exhibit increased activation of IRS-1's downstream Akt and MEK/ERK pathways, these null cells grew poorly and displayed phenotypes reminiscent of those associated with oncogene-induced senescence. Taken together, our findings demonstrate a key role for the CUL7 E3 in targeting IRS-1 for degradation, a process that may contribute to the regulation of cellular senescence.

Identification of the degradation determinants of insulin receptor substrate 1 for signaling cullin-RING E3 ubiquitin ligase 7-mediated ubiquitination.

BACKGROUND: Negative feedback regulation of insulin signaling involves ubiquitin-dependent degradation of insulin receptor substrate 1 (IRS1). RESULTS: Cullin-RING E3 ubiquitin ligase 7 (CRL7) mediates the ubiquitination of IRS1 in hyperphosphorylated form. CONCLUSION: Multisite IRS1 phosphorylation triggers interactions with CRL7 for ubiquitin modification. SIGNIFICANCE: Insulin signaling is self-restrained when its downstream effector kinases are hyperactivated to trigger the negative feedback inhibition. Hyperactivation of mechanistic target of rapamycin complex 1 (mTORC1) and its effector kinase S6 kinase 1 (S6K1) is known to trigger multisite seryl phosphorylation of insulin receptor substrate 1 (IRS1), leading to its ubiquitination and degradation. This negative feedback inhibition functions to restrain PI3K activity and plays critical roles in the pathogenesis of cancer and type II diabetes. Recent work has implicated a role for cullin-RING E3 ubiquitin ligase 7 (CRL7) in targeting IRS1 for mTORC1/S6K1-dependent degradation. In the present study we have employed both cell-based degradation and reconstituted ubiquitination approaches to define molecular features associated with IRS1 critical for CRL7-mediated ubiquitination and degradation. We have mapped IRS1 degradation signal sequence to its N-terminal 574 amino acid residues, of which the integrity of Ser-307/Ser-312 and Ser-527, each constituting a S6K1 phosphorylation consensus site, was indispensible for supporting CRL7-forced degradation. In vitro, S6K1 was able to support the ubiquitination of bacterially expressed IRS1 N-terminal fragment by CRL7 but at low levels. In contrast, CRL7 supported efficient ubiquitination of IRS1 N-terminal fragment in hyperphosphorylated form, which was isolated from infected insect cells, suggesting requirement of additional phosphorylation by kinases yet to be identified. Finally, removal of IRS1 amino acids 1-260 led to substantial reduction of ubiquitination efficiency, suggesting a role for this region in mediating productive interactions with CRL7. The requirement of multisite phosphorylation and the N terminus of IRS1 for its turnover may ensure that complete IRS1 degradation occurs only when mTORC1 and S6K1 reach exceedingly high levels.

Pharmacases.de - a student-centered e-learning project of clinical pharmacology.

AIM: The aim of the project Pharmacases.de was to develop an innovative concept for creating high-quality e-learning content which integrates and promotes the theoretical and cooperative skills of final-year medical students and is easily adoptable by cooperating institutes and hospitals. METHODS AND RESULTS: A peer-teaching concept was developed in which final-year medical students with the elective pharmacology independently researched and wrote e-learning cases of clinical pharmacology ("pharmacases"). Subject-specific expertise was acquired by consulting a peer network of elective students of other disciplines. The created material was subjected to a multi-step peer review and published on the open-access internet platform http://www.pharmacases.de . At present, the website contains 45 e-learning cases, 27 quizzes, and a student-managed discussion forum. Each month, approximately 1200 students access the e-learning content on the website with above-average evaluation results. SUMMARY AND CONCLUSION: The didactic concept of Pharmacases.de enabled the efficient generation of high-quality e-learning content in a student-centered and interdisciplinary manner and was well received by the students. It will likely facilitate the transfer of theoretical pharmacological knowledge into clinical practice.

Identification of hypertrophy-modulating Cullin-RING ubiquitin ligases in primary cardiomyocytes.

Cullin-RING ubiquitin ligases (CRL) regulate numerous biological processes in the heart and have been implicated in regulating cardiac hypertrophy. This study aimed to identify novel hypertrophy-modulating CRLs in cardiomyocytes (CM). A functional genomic approach using siRNA-mediated depletion and automated microscopy was employed to screen for cell size-modulating CRLs in neonatal rat CM. Screening hits were confirmed by 3H-isoleucine incorporation. Of 43 targets screened, siRNA-mediated depletion of Fbxo6, Fbxo45, and Fbxl14 resulted in decreased cell size, whereas depletion of Fbxo9, Fbxo25, Fbxo30, Fbxo32, Fbxo33, Cullin1, Roc1, Ddb1, Fbxw4, and Fbxw5 led to a markedly increased cell size under basal conditions. In CM stimulated with phenylephrine (PE), depletion of Fbxo6, Fbxo25, Fbxo33, Fbxo45, and Fbxw4 further augmented PE-induced hypertrophy. As a proof-of-concept, the CRLFbox25 was analysed by transverse aortic constriction (TAC) resulting in a 4.5-fold increase in Fbxo25 protein concentrations compared to control animals. In cell culture, siRNA-mediated depletion of Fbxo25 resulted in a ∼ 37% increase in CM cell size and ∼41% increase in 3H-isoleucine incorporation. Depleting Fbxo25 resulted in upregulation of Anp and Bnp. In summary, we identified 13 novel CRLs as positive or negative regulators of CM hypertrophy. Of these, CRLFbox25 was further characterized, as a potential modulator of cardiac hypertrophy.

Role of SLC4 and SLC26 solute carriers during oxidative stress.

Bicarbonate is one of the major anions in mammalian tissues and fluids, is utilized by various exchangers to transport other ions and organic substrates across cell membranes and plays a critical role in cell and systemic pH homoeostasis. Chloride/bicarbonate (Cl- /HCO3- ) exchangers are abundantly expressed in erythrocytes and epithelial cells and, as a consequence, are particularly exposed to oxidants in the systemic circulation and at the interface with the external environment. Here, we review the physiological functions and pathophysiological alterations of Cl- /HCO3- exchangers belonging to the solute carriers SLC4 and SLC26 superfamilies in relation to oxidative stress. Particularly well studied is the impact of oxidative stress on the red blood cell SLC4A1/AE1 (Band 3 protein), of which the function seems to be directly affected by oxidative stress and possibly involves oxidation of the transporter itself or its interacting proteins, with detrimental consequences in oxidative stress-related diseases including inflammation, metabolic dysfunctions and ageing. The effect of oxidative stress on SLC26 members was less extensively explored. Indirect evidence suggests that SLC26 transporters can be target as well as determinants of oxidative stress, especially when their expression is abolished or dysregulated.

Novel POU3F4 variants identified in patients with inner ear malformations exhibit aberrant cellular distribution and lack of SLC6A20 transcriptional upregulation.

Hearing loss (HL) is the most common sensory defect and affects 450 million people worldwide in a disabling form. Pathogenic sequence alterations in the POU3F4 gene, which encodes a transcription factor, are causative of the most common type of X-linked deafness (X-linked deafness type 3, DFN3, DFNX2). POU3F4-related deafness is characterized by a typical inner ear malformation, namely an incomplete partition of the cochlea type 3 (IP3), with or without an enlargement of the vestibular aqueduct (EVA). The pathomechanism underlying POU3F4-related deafness and the corresponding transcriptional targets are largely uncharacterized. Two male patients belonging to a Caucasian cohort with HL and EVA who presented with an IP3 were submitted to genetic analysis. Two novel sequence variants in POU3F4 were identified by Sanger sequencing. In cell-based assays, the corresponding protein variants (p.S74Afs*8 and p.C327*) showed an aberrant expression and subcellular distribution and lack of transcriptional activity. These two protein variants failed to upregulate the transcript levels of the amino acid transporter gene SLC6A20, which was identified as a novel transcriptional target of POU3F4 by RNA sequencing and RT-qPCR. Accordingly, POU3F4 silencing by siRNA resulted in downregulation of SLC6A20 in mouse embryonic fibroblasts. Moreover, we showed for the first time that SLC6A20 is expressed in the mouse cochlea, and co-localized with POU3F4 in the spiral ligament. The findings presented here point to a novel role of amino acid transporters in the inner ear and pave the way for mechanistic studies of POU3F4-related HL.

LPA receptor 1 (LPAR1) is a novel interaction partner of Filamin A that promotes Filamin A phosphorylation, MRTF-A transcriptional activity and oncogene-induced senescence.

Myocardin-related transcription factors A and B (MRTFs) are coactivators of Serum Response Factor (SRF), which controls fundamental biological processes such as cell growth, migration, and differentiation. MRTF and SRF transcriptional activity play an important role in hepatocellular carcinoma (HCC) growth, which represents the second leading cause of cancer-related mortality in humans worldwide. We, therefore, searched for druggable targets in HCC that regulate MRTF/SRF transcriptional activity and can be exploited therapeutically for HCC therapy. We identified the G protein-coupled lysophosphatidic acid receptor 1 (LPAR1) as a novel interaction partner of MRTF-A and Filamin A (FLNA) using fluorescence resonance energy transfer-(FRET) and proximity ligation assay (PLA) in vitro in HCC cells and in vivo in organoids. We found that LPAR1 promotes FLNA phosphorylation at S2152 which enhances the complex formation of FLNA and MRTF-A, actin polymerization, and MRTF transcriptional activity. Pharmacological blockade or depletion of LPAR1 prevents FLNA phosphorylation and complex formation with MRTF-A, resulting in reduced MRTF/SRF target gene expression and oncogene-induced senescence. Thus, inhibition of the LPAR1-FLNA-MRTF-A interaction represents a promising strategy for HCC therapy.

Autoinhibitory regulation of SCF-mediated ubiquitination by human cullin 1's C-terminal tail.

SCF (Skp1 x CUL1 x F-box protein x ROC1) E3 ubiquitin ligase and Cdc34 E2-conjugating enzyme catalyze polyubiquitination in a precisely regulated fashion. Here, we describe biochemical evidence suggesting an autoinhibitory role played by the human CUL1 ECTD (extreme C-terminal domain; spanning the C-terminal 50 amino acids), a region that is predicted to contact the ROC1 RING finger protein by structural studies. We showed that ECTD did not contribute to CUL1's stable association with ROC1. Remarkably, deletion of ECTD, or missense mutations designed to disrupt the predicted ECTD x ROC1 interaction, markedly increased the ability of SCF(betaTrCP2) to promote IkappaB alpha polyubiquitination and polyubiquitin chain assembly by Cdc34 in vitro. Thus, disruption of ECTD yields in vitro effects that parallel SCF activation by Nedd8 conjugation to CUL1. We propose that SCF may be subject to autoinhibitory regulation, in which Nedd8 conjugation acts as a molecular switch to drive the E3 into an active state by diminishing the inhibitory ECTD x ROC1 interaction.

Genetic Determinants of Non-Syndromic Enlarged Vestibular Aqueduct: A Review.

Hearing loss is the most common sensorial deficit in humans and one of the most common birth defects. In developed countries, at least 60% of cases of hearing loss are of genetic origin and may arise from pathogenic sequence alterations in one of more than 300 genes known to be involved in the hearing function. Hearing loss of genetic origin is frequently associated with inner ear malformations; of these, the most commonly detected is the enlarged vestibular aqueduct (EVA). EVA may be associated to other cochleovestibular malformations, such as cochlear incomplete partitions, and can be found in syndromic as well as non-syndromic forms of hearing loss. Genes that have been linked to non-syndromic EVA are SLC26A4, GJB2, FOXI1, KCNJ10, and POU3F4. SLC26A4 and FOXI1 are also involved in determining syndromic forms of hearing loss with EVA, which are Pendred syndrome and distal renal tubular acidosis with deafness, respectively. In Caucasian cohorts, approximately 50% of cases of non-syndromic EVA are linked to SLC26A4 and a large fraction of patients remain undiagnosed, thus providing a strong imperative to further explore the etiology of this condition.

d-Galactose Decreases Anion Exchange Capability through Band 3 Protein in Human Erythrocytes.

d-Galactose (d-Gal), when abnormally accumulated in the plasma, results in oxidative stress production, and may alter the homeostasis of erythrocytes, which are particularly exposed to oxidants driven by the blood stream. In the present investigation, the effect of d-Gal (0.1 and 10 mM, for 3 and 24 h incubation), known to induce oxidative stress, has been assayed on human erythrocytes by determining the rate constant of SO42- uptake through the anion exchanger Band 3 protein (B3p), essential to erythrocytes homeostasis. Moreover, lipid peroxidation, membrane sulfhydryl groups oxidation, glycated hemoglobin (% A1c), methemoglobin levels (% MetHb), and expression levels of B3p have been verified. Our results show that d-Gal reduces anion exchange capability of B3p, involving neither lipid peroxidation, nor oxidation of sulfhydryl membrane groups, nor MetHb formation, nor altered expression levels of B3p. d-Gal-induced %A1c, known to crosslink with B3p, could be responsible for rate of anion exchange alteration. The present findings confirm that erythrocytes are a suitable model to study the impact of high sugar concentrations on cell homeostasis; show the first in vitro effect of d-Gal on B3p, contributing to the understanding of mechanisms underlying an in vitro model of aging; demonstrate that the first impact of d-Gal on B3p is mediated by early Hb glycation, rather than by oxidative stress, which may be involved on a later stage, possibly adding more knowledge about the consequences of d-Gal accumulation.

Genetic ablation of Cullin-RING E3 ubiquitin ligase 7 restrains pressure overload-induced myocardial fibrosis.

Fibrosis is a pathognomonic feature of structural heart disease and counteracted by distinct cardioprotective mechanisms, e.g. activation of the phosphoinositide 3-kinase (PI3K) / AKT pro-survival pathway. The Cullin-RING E3 ubiquitin ligase 7 (CRL7) was identified as negative regulator of PI3K/AKT signalling in skeletal muscle, but its role in the heart remains to be elucidated. Here, we sought to determine whether CRL7 modulates to cardiac fibrosis following pressure overload and dissect its underlying mechanisms. For inactivation of CRL7, the Cullin 7 (Cul7) gene was deleted in cardiac myocytes (CM) by injection of adeno-associated virus subtype 9 (AAV9) vectors encoding codon improved Cre-recombinase (AAV9-CMV-iCre) in Cul7flox/flox mice. In addition, Myosin Heavy Chain 6 (Myh6; alpha-MHC)-MerCreMer transgenic mice with tamoxifen-induced CM-specific expression of iCre were used as alternate model. After transverse aortic constriction (TAC), causing chronic pressure overload and fibrosis, AAV9-CMV-iCre induced Cul7-/- mice displayed a ~50% reduction of interstitial cardiac fibrosis when compared to Cul7+/+ animals (6.7% vs. 3.4%, p<0.01). Similar results were obtained with Cul7flox/flox Myh6-Mer-Cre-MerTg(1/0) mice which displayed a ~30% reduction of cardiac fibrosis after TAC when compared to Cul7+/+ Myh6-Mer-Cre-MerTg(1/0) controls after TAC surgery (12.4% vs. 8.7%, p<0.05). No hemodynamic alterations were observed. AKTSer473 phosphorylation was increased 3-fold (p<0.01) in Cul7-/- vs. control mice, together with a ~78% (p<0.001) reduction of TUNEL-positive apoptotic cells three weeks after TAC. In addition, CM-specific expression of a dominant-negative CUL71152stop mutant resulted in a 16.3-fold decrease (p<0.001) of in situ end-labelling (ISEL) positive apoptotic cells. Collectively, our data demonstrate that CM-specific ablation of Cul7 restrains myocardial fibrosis and apoptosis upon pressure overload, and introduce CRL7 as a potential target for anti-fibrotic therapeutic strategies of the heart.

O-GlcNAcylation Suppresses the Ion Current IClswell by Preventing the Binding of the Protein ICln to α-Integrin.

O-GlcNAcylation is a post-translational modification of proteins that controls a variety of cellular processes, is chronically elevated in diabetes mellitus, and may contribute to the progression of diabetic complications, including diabetic nephropathy. Our previous work showed that increases in the O-GlcNAcylation of cellular proteins impair the homeostatic reaction of the regulatory volume decrease (RVD) after cell swelling by an unknown mechanism. The activation of the swelling-induced chloride current IClswell is a key step in RVD, and ICln, a ubiquitous protein involved in the activation of IClswell, is O-GlcNAcylated. Here, we show that experimentally increased O-GlcNAcylation of cellular proteins inhibited the endogenous as well as the ICln-induced IClswell current and prevented RVD in a human renal cell line, while decreases in O-GlcNAcylation augmented the current magnitude. In parallel, increases or decreases in O-GlcNAcylation, respectively, weakened or stabilized the binding of ICln to the intracellular domain of α-integrin, a process that is essential for the activation of IClswell. Mutation of the putative YinOYang site at Ser67 rendered the ICln-induced IClswell current unresponsive to O-GlcNAc variations, and the ICln interaction with α-integrin insensitive to O-GlcNAcylation. In addition, exposure of cells to a hypotonic solution reduced the O-GlcNAcylation of cellular proteins. Together, these findings show that O-GlcNAcylation affects RVD by influencing IClswell and further indicate that hypotonicity may activate IClswell by reducing the O-GlcNAcylation of ICln at Ser67, therefore permitting its binding to α-integrin. We propose that disturbances in the regulation of cellular volume may contribute to disease in settings of chronically elevated O-GlcNAcylation, including diabetic nephropathy.

The ubiquitin-proteasome system: focus on the heart.

Proteasomes are the main non-lysosomal multicatalytic protease complexes that are involved in the degradation of most intracellular proteins. The substrate proteins are marked by ubiquitin, which serves as a tag for their regulated proteasomal destruction. One major function of the ubiquitin-proteasome system (UPS) is to prevent accumulation of non-functional, potentially toxic proteins. Moreover, it has become clear that the UPS is involved in most aspects of eukaryotic biology, such as intracellular signaling, transcriptional control, or regulation of cell death. Recent studies demonstrated that the UPS regulates receptor internalization, hypertrophic response, apoptosis, and tolerance to ischemia and reperfusion in cardiomyocytes. Since structural remodeling of the myocardium, ischemia-reperfusion injury, and myocardial cell loss are important components in the genesis of progressive heart failure, these findings suggest a pathophysiological role of the UPS. This review briefly summarizes present knowledge about structure and function of the proteasome in the heart and discusses the relevance of the UPS for cardiac diseases.

Impairment of the ubiquitin-proteasome system by truncated cardiac myosin binding protein C mutants.

OBJECTIVE: Most cardiac myosin binding protein C (cMyBP-C) gene mutations causing familial hypertrophic cardiomyopathy (FHC) result in C-terminal truncated proteins. However, truncated cMyBP-Cs were undetectable in myocardial tissue of FHC patients. In the present study, we investigated whether truncated cMyBP-Cs are subject to accelerated degradation by the lysosome or ubiquitin-proteasome system (UPS). METHODS AND RESULTS: By using an adenovirus-based approach, we analyzed expression and localization of myc-tagged truncated proteins (M6t 3%, M7t 80% truncation, both mutations have been identified in FHC patients) compared to wild type (WT) in neonatal rat cardiomyocytes. Despite similar mRNA levels, protein expression of M6t and M7t was markedly lower than WT (70+/-4% and 11+/-5% of WT, respectively, p<0.05). M6t exhibited weak incorporation in the sarcomere, whereas M7t was mis-incorporated at the Z-disk and formed ubiquitin-positive aggregates. The lysosome inhibitor bafilomycin only slightly raised the protein level of M7t, whereas the UPS inhibitors lactacystin or MG132 markedly raised M6t and M7t to WT level. Using an adenovirus encoding a fluorescent reporter of UPS activity, we demonstrate that mutant cMyBP-Cs impair the proteolytic capacity of the UPS. CONCLUSION: Truncated cMyBP-Cs are preferentially degraded by the UPS, which, in turn, may competitively inhibit breakdown of other UPS substrates. Since the UPS plays an important role in a variety of fundamental cellular processes, we propose impairment of this system by mutant cMyBP-Cs as a contributing factor to the pathogenesis of FHC.

Essential Role for Premature Senescence of Myofibroblasts in Myocardial Fibrosis.

BACKGROUND: Fibrosis is a hallmark of many myocardial pathologies and contributes to distorted organ architecture and function. Recent studies have identified premature senescence as a regulatory mechanism of tissue fibrosis, but its relevance in the heart remains to be established. OBJECTIVES: This study investigated the role of premature senescence in myocardial fibrosis. METHODS: Murine models of cardiac diseases and human heart biopsies were analyzed for characteristics of premature senescence and fibrosis. Loss-of-function and gain-of-function models of premature senescence were used to determine its pathophysiological role in myocardial fibrosis. RESULTS: Senescence markers p21(CIP1/WAF1), senescence-associated ß-galactosidase (SA-ß-gal), and p16(INK4a) were increased 2-, 8-, and 20-fold (n = 5 to 7; p < 0.01), respectively, in perivascular fibrotic areas after transverse aortic constriction compared with sham-treated control subjects. Similar results were observed with cardiomyocyte-specific ß1-adrenoceptor transgenic mice and human heart biopsies. Senescent cells were positive for platelet-derived growth factor receptor-α, vimentin, and α-smooth muscle actin, specifying myofibroblasts as the predominant cell population undergoing premature senescence in the heart. Inactivation of the premature senescence program by genetic ablation of p53 and p16(INK4a) (Trp53(-/-)Cdkn2a(-/-) mice) resulted in aggravated fibrosis after transverse aortic constriction, when compared with wild-type control subjects (49 ± 4.9% vs. 33 ± 2.7%; p < 0.01), and was associated with impaired cardiac function. Conversely, cardiac-specific expression of CCN1 (CYR61), a potent inducer of premature senescence, by adeno-associated virus serotype 9 gene transfer, resulted in ∼50% reduction of perivascular fibrosis after transverse aortic constriction, when compared with mock- or dominant-negative CCN1-infected control subjects, and improved cardiac function. CONCLUSIONS: Our data establish premature senescence of myofibroblasts as an essential antifibrotic mechanism and potential therapeutic target in myocardial fibrosis.

An Introduction to the Inverted/Flipped Classroom Model in Education and Advanced Training in Medicine and in the Healthcare Professions.

In describing the inverted classroom model (ICM), the following paper is meant to provide an introduction to the subject matter and to serve as a practical guide for those wishing to employ its methods in basic and advanced medical training and education. The ICM is a blended-learning method in which a self-directed learning phase (individual phase) precedes the classroom-instruction phase. During the online phase, factual knowledge is imparted that serves as a basis for the classroom phase. The classroom phase should subsequently be used to assimilate and implement the previously gained knowledge. In contrast, traditional course concepts impart factual knowledge in lectures, for example, or in other face-to-face teaching formats and are followed by the students' self-instruction in order to assimilate this knowledge. The goal of the ICM is the shift from passive learning to accelerated learning in order to foster learning at cognitively demanding levels such as analysis, synthesis and evaluation. The concurrent increase in production and use of screencasts and educational videos, the Open Educational Resources "movement" and the widespread use of Massive Open Online Courses (MOOCS) have contributed to the increased dissemination of the inverted-classroom method. The intention of the present paper is to provide an introduction to the subject matter and simultaneously to offer a short overview of important projects and research results in the field of medical education and other health professions. Furthermore, an outline is given of the advantages and disadvantages of the model as well as its potential benefit to the future of medical education and training.

Use of learning media by undergraduate medical students in pharmacology: a prospective cohort study.

The ubiquity of the internet and computer-based technologies has an increasing impact on higher education and the way students access information for learning. Moreover, there is a paucity of information about the quantitative and qualitative use of learning media by the current student generation. In this study we systematically analyzed the use of digital and non-digital learning resources by undergraduate medical students. Daily online surveys and semi-structured interviews were conducted with a cohort of 338 third year medical students enrolled in a general pharmacology course. Our data demonstrate a predominant use of digital over non-digital learning resources (69 ± 7% vs. 31 ± 7%; p < 0.01) by students. Most used media for learning were lecture slides (26.8 ± 3.0%), apps (22.0 ± 3.7%) and personal notes (15.5 ± 2.7%), followed by textbooks (> 300 pages) (10.6 ± 3.3%), internet search (7.9 ± 1.6%) and e-learning cases (7.6 ± 3.0%). When comparing learning media use of teaching vs. pre-exam self-study periods, textbooks were used significantly less during self-study (-55%; p < 0.01), while exam questions (+334%; p < 0.01) and e-learning cases (+176%; p < 0.01) were utilized more. Taken together, our study revealed a high prevalence and acceptance of digital learning resources by undergraduate medical students, in particular mobile applications.