Selective inhibition of protein secretion by abrogating receptor-coat interactions during ER export.

Protein secretion is an essential process that drives cell growth, movement, and communication. Protein traffic within the secretory pathway occurs via transport intermediates that bud from one compartment and fuse with a downstream compartment to deliver their contents. Here, we explore the possibility that protein secretion can be selectively inhibited by perturbing protein-protein interactions that drive capture into transport vesicles. Human proprotein convertase subtilisin/kexin type 9 (PCSK9) is a determinant of cholesterol metabolism whose secretion is mediated by a specific cargo adaptor protein, SEC24A. We map a series of protein-protein interactions between PCSK9, its endoplasmic reticulum (ER) export receptor SURF4, and SEC24A that mediate secretion of PCSK9. We show that the interaction between SURF4 and SEC24A can be inhibited by 4-phenylbutyrate (4-PBA), a small molecule that occludes a cargo-binding domain of SEC24. This inhibition reduces secretion of PCSK9 and additional SURF4 clients that we identify by mass spectrometry, leaving other secreted cargoes unaffected. We propose that selective small-molecule inhibition of cargo recognition by SEC24 is a potential therapeutic intervention for atherosclerosis and other diseases that are modulated by secreted proteins.

Incidence and outcomes of surgical pulmonary embolectomy in the UK.

BACKGROUND: Surgical pulmonary embolectomy is rarely used for the treatment of massive acute pulmonary embolism. The aim of this study was to assess the incidence and outcomes of this operation by undertaking a retrospective analysis of a large national registry in the UK. METHODS: All acute pulmonary embolectomies performed between 1996 and 2018 were captured in the National Institute of Cardiovascular Outcomes Research central database. Trends in the number of operations performed during this interval and reported in-hospital outcomes were analysed retrospectively. Multivariable logistic regression was used to identify independent risk factors for in-hospital death. RESULTS: All 256 patients treated surgically for acute pulmonary embolism during the study interval were included in the analysis. Median age at presentation was 54 years, 55.9% of the patients were men, 48.0% had class IV heart failure symptoms, and 37.5% had preoperative cardiogenic shock. The median duration of bypass was 73 min, and median cross-clamp time was 19 min. Cardioplegic arrest was used in 53.1% of patients. The median duration of hospital stay was 11 days. The in-hospital mortality rate was 25%, postoperative stroke occurred in 5.4%, postoperative dialysis was required in 16%, and the reoperation rate for bleeding was 7.5%. Risk-adjusted multivariable analysis revealed cardiogenic shock (OR 2.54, 95% c.i. 1.05 to 6.21; P = 0.038), preoperative ventilation (OR 5.85, 2.22 to 16.35; P < 0.001), and duration of cardiopulmonary bypass exceeding 89 min (OR 7.82, 3.25 to 20.42; P < 0.001) as significant independent risk factors for in-hospital death. CONCLUSION: Surgical pulmonary embolectomy is rarely performed in the UK, and is associated with significant mortality and morbidity. Preoperative ventilation, cardiogenic shock, and increased duration of bypass were significant predictors of in-hospital death.

A blood clot in the lung can prevent the lungs from working properly and put pressure on the heart to work harder. Small clots can be treated with medications taken at home and are not a danger to life. Larger blood clots can put a lot of pressure on the heart and need immediate hospital treatment. Large blood clots can be treated with 'clot busting' medications, the delivery of a small tube into the blood vessels of the lung to suck up the clot or deliver medications directly on to its surface, and finally a form of open-heart surgery. With this surgery, a surgeon opens the chest, make a cut into the large vessels containing the clot, and physically removes the large piece of obstructing clot. The aim of this study was to describe and analyse the outcomes of this operation done in the UK over a long period. A database was used to find out how often and where this operation took place and its results. The available data were studied to try to understand how helpful this operation is to patients with lung blood clots. Between 1996 and 2018, 256 people had this operation. One in four patients did not survive the operation, 5.4% developed a clot or bleed in the brain, 16% needed to go on to a dialysis machine, and 7.5% had to be rushed back into theatre because of bleeding. Needing a ventilator machine for help with breathing, being in a sudden state of heart failure, and a long time on the heart bypass machine were all linked with patients who did not survive. This operation is rarely performed in the UK, and is often linked to a high chance of death or serious complication. In this study, the points described above were linked to a bad outcome.

Adeno-Associated Virus-Mediated Dorsal Root Ganglion Toxicity in the New Zealand White Rabbit.

Recombinant adeno-associated virus (AAV)-mediated degeneration of sensory neurons in the dorsal root ganglia (DRG) and trigeminal ganglia (TG) has been observed in non-human primates (NHPs) following intravenous (IV) and intrathecal (IT) delivery. Administration of recombinant AAV encoding a human protein transgene via a single intra-cisterna magna (ICM) injection in New Zealand white rabbits resulted in histopathology changes very similar to NHPs: mononuclear cell infiltration, degeneration/necrosis of sensory neurons, and nerve fiber degeneration of sensory tracts in the spinal cord and of multiple nerves. AAV-associated clinical signs and incidence/severity of histologic findings indicated that rabbits were equally or more sensitive than NHPs to sensory neuron damage. Another study using human and rabbit transgene constructs of the same protein demonstrated comparable changes suggesting that the effects are not an immune response to the non-self protein transgene. Rabbit has not been characterized as a species for general toxicity testing of AAV gene therapies, but these studies suggest that it may be an alternative model to investigate mechanisms of AAV-mediated neurotoxicity and test novel AAV designs mitigating these adverse effects.

CRISPR gRNA phenotypic screening in zebrafish reveals pro-regenerative genes in spinal cord injury.

Zebrafish exhibit robust regeneration following spinal cord injury, promoted by macrophages that control post-injury inflammation. However, the mechanistic basis of how macrophages regulate regeneration is poorly understood. To address this gap in understanding, we conducted a rapid in vivo phenotypic screen for macrophage-related genes that promote regeneration after spinal injury. We used acute injection of synthetic RNA Oligo CRISPR guide RNAs (sCrRNAs) that were pre-screened for high activity in vivo. Pre-screening of over 350 sCrRNAs allowed us to rapidly identify highly active sCrRNAs (up to half, abbreviated as haCRs) and to effectively target 30 potentially macrophage-related genes. Disruption of 10 of these genes impaired axonal regeneration following spinal cord injury. We selected 5 genes for further analysis and generated stable mutants using haCRs. Four of these mutants (tgfb1a, tgfb3, tnfa, sparc) retained the acute haCR phenotype, validating the approach. Mechanistically, tgfb1a haCR-injected and stable mutant zebrafish fail to resolve post-injury inflammation, indicated by prolonged presence of neutrophils and increased levels of il1b expression. Inhibition of Il-1ß rescues the impaired axon regeneration in the tgfb1a mutant. Hence, our rapid and scalable screening approach has identified functional regulators of spinal cord regeneration, but can be applied to any biological function of interest.

Transcriptional plasticity promotes primary and acquired resistance to BET inhibition.

Following the discovery of BRD4 as a non-oncogene addiction target in acute myeloid leukaemia (AML), bromodomain and extra terminal protein (BET) inhibitors are being explored as a promising therapeutic avenue in numerous cancers. While clinical trials have reported single-agent activity in advanced haematological malignancies, mechanisms determining the response to BET inhibition remain poorly understood. To identify factors involved in primary and acquired BET resistance in leukaemia, here we perform a chromatin-focused RNAi screen in a sensitive MLL-AF9;Nras(G12D)-driven AML mouse model, and investigate dynamic transcriptional profiles in sensitive and resistant mouse and human leukaemias. Our screen shows that suppression of the PRC2 complex, contrary to effects in other contexts, promotes BET inhibitor resistance in AML. PRC2 suppression does not directly affect the regulation of Brd4-dependent transcripts, but facilitates the remodelling of regulatory pathways that restore the transcription of key targets such as Myc. Similarly, while BET inhibition triggers acute MYC repression in human leukaemias regardless of their sensitivity, resistant leukaemias are uniformly characterized by their ability to rapidly restore MYC transcription. This process involves the activation and recruitment of WNT signalling components, which compensate for the loss of BRD4 and drive resistance in various cancer models. Dynamic chromatin immunoprecipitation sequencing and self-transcribing active regulatory region sequencing of enhancer profiles reveal that BET-resistant states are characterized by remodelled regulatory landscapes, involving the activation of a focal MYC enhancer that recruits WNT machinery in response to BET inhibition. Together, our results identify and validate WNT signalling as a driver and candidate biomarker of primary and acquired BET resistance in leukaemia, and implicate the rewiring of transcriptional programs as an important mechanism promoting resistance to BET inhibitors and, potentially, other chromatin-targeted therapies.

Correction to: The Peacock study: feasibility of the dynamic characterisation of the paediatric hypothalamic-pituitary-adrenal function during and after cardiac surgery.

An amendment to this paper has been published and can be accessed via the original article.

The Peacock study: feasibility of the dynamic characterisation of the paediatric hypothalamic-pituitary-adrenal function during and after cardiac surgery.

BACKGROUND: Cortisol is the main stress hormone mobilised during surgery to establish homeostasis. Our current understanding of the hypothalamic-pituitary-adrenal axis physiology in children undergoing cardiopulmonary bypass is very limited due to: (1) very few cortisol time point measurements over long periods (2) difficulties of sampling in low weight babies and (3) the concomitant use of glucocorticoids at anaesthesia induction. This lack of understanding is reflected in a lack of consensus on the utility of glucocorticoids perioperatively in cardiac surgery with the use of cardiopulmonary bypass. METHODS: The Peacock Study is a prospective, two-centre, observational cohort study of 78 children (undergoing cardiopulmonary bypass procedures and non-surgical procedures - split by age/cyanosis) that aims to characterise in detail the hypothalamic-pituitary-adrenal axis physiology of children using the stress model of paediatric cardiac surgery. Also, we aim to correlate cortisol profiles with clinical outcome data. We herein describe the main study design and report the full cortisol profile of one child undergoing heart surgery, thus proving the feasibility of the method. RESULTS: We used an automated, 24-h tissue microdialysis system to measure cortisol and cortisone, every 20 min. We herein report one cortisol profile of a child undergoing heart surgery. Besides, we measured serum cortisol and adrenocorticotrophic hormone at seven-time points for correlation. Tissue concentrations of cortisol increased markedly several hours after the end of surgery. We also noted an increase in the tissue cortisol/cortisone ratio during this response. CONCLUSION: We report for the first time, the use of an automated microdialysis sampling system to evaluate the paediatric adrenal response in children. Changes in cortisol and cortisone could be measured, and the concentration of cortisol in the tissues increased after the end of cardiac surgery. The method has wide application to measure other hormones dynamically and frequently without the limitation of the circulating blood volume. The data from the main study will clarify how these cortisol profiles vary with age, pathology, type of procedure and correlation to clinical outcomes. TRIAL REGISTRATION: ISCRTN registry, number: 982586.

Evoked potentials as a translatable biomarker to track functional remyelination.

Enhancing remyelination is a key therapeutic strategy for demyelinating diseases such as multiple sclerosis. To achieve this goal, a central challenge is being able to quantitatively and longitudinally track functional remyelination, especially with translatable biomarkers that can be performed in both preclinical models and in the clinic. We developed the methodology to stably measure multi-modal sensory evoked potentials from the skull surface over the course of months in individual mice and applied it to a genetic mouse model of oligodendrocyte ablation and demyelination. We found that auditory and somatosensory evoked potential latencies reliably increased over time during the early phase of the model and recovered spontaneously and almost completely during a later phase. Histological examination supported the interpretation that the evoked potential latency changes dynamically reflect changes in CNS myelination. Specifically, we found reduction of myelination in corresponding brain regions at the time that sensory evoked potentials were maximally impacted. Importantly, we also found that myelination levels recovered when evoked potential latencies recovered. Other changes known to associate with demyelination were also observed at the time of delayed evoked potentials, including the emergence of white matter vacuoles and increased markers for activated microglia and macrophages; these changes also fully reversed by the time that evoked potentials recovered. Our results support the hypothesis that skull-surface recorded evoked potential latencies can dynamically track CNS myelination changes. The methods developed here allow for longitudinally tracking functional myelination changes in vivo in preclinical rodent models with a quantitative biomarker that can also be applied clinically and will facilitate translational development of CNS remyelinating therapies.

The Pan London Emergency Cardiac Surgery service blueprint.

The authors share their experience of managing the cardiac surgery services across London during the challenging Covid-19 pandemic. The Pan London Emergency Cardiac Surgery Service model could serve as a blueprint to design policies applicable to other surgical specialities and parts of the UK and worldwide.

Triple Angiokinase Inhibitor Nintedanib Directly Inhibits Tumor Cell Growth and Induces Tumor Shrinkage via Blocking Oncogenic Receptor Tyrosine Kinases.

The triple-angiokinase inhibitor nintedanib is an orally available, potent, and selective inhibitor of tumor angiogenesis by blocking the tyrosine kinase activities of vascular endothelial growth factor receptor (VEGFR) 1-3, platelet-derived growth factor receptor (PDGFR)-α and -ß, and fibroblast growth factor receptor (FGFR) 1-3. Nintedanib has received regulatory approval as second-line treatment of adenocarcinoma non-small cell lung cancer (NSCLC), in combination with docetaxel. In addition, nintedanib has been approved for the treatment of idiopathic lung fibrosis. Here we report the results from a broad kinase screen that identified additional kinases as targets for nintedanib in the low nanomolar range. Several of these kinases are known to be mutated or overexpressed and are involved in tumor development (discoidin domain receptor family, member 1 and 2, tropomyosin receptor kinase A (TRKA) and C, rearranged during transfection proto-oncogene [RET proto oncogene]), as well as in fibrotic diseases (e.g., DDRs). In tumor cell lines displaying molecular alterations in potential nintedanib targets, the inhibitor demonstrates direct antiproliferative effects: in the NSCLC cell line NCI-H1703 carrying a PDGFRα amplification (ampl.); the gastric cancer cell line KatoIII and the breast cancer cell line MFM223, both driven by a FGFR2 amplification; AN3CA (endometrial carcinoma) bearing a mutated FGFR2; the acute myeloid leukemia cell lines MOLM-13 and MV-4-11-B with FLT3 mutations; and the NSCLC adenocarcinoma LC-2/ad harboring a CCDC6-RET fusion. Potent kinase inhibition does not, however, strictly translate into antiproliferative activity, as demonstrated in the TRKA-dependent cell lines CUTO-3 and KM-12. Importantly, nintedanib treatment of NCI-H1703 tumor xenografts triggered effective tumor shrinkage, indicating a direct effect on the tumor cells in addition to the antiangiogenic effect on the tumor stroma. These findings will be instructive in guiding future genome-based clinical trials of nintedanib.

Stanniocalcin1 is a key mediator of amyloidogenic light chain induced cardiotoxicity.

Immunoglobulin light chain (LC) amyloidosis (AL) results from overproduction of circulating amyloidogenic LC proteins and subsequent amyloid fibril deposition in organs. Mortality in AL amyloidosis patients is highly associated with a rapidly progressive AL cardiomyopathy, marked by profound impairment of diastolic and systolic cardiac function and significant early mortality. While myocardial fibril deposition contributes to the severe diastolic dysfunction seen in AL cardiomyopathy patients, the degree of fibril deposition has not been found to correlate with prognosis. Previously, we and others showed a direct cardiotoxic effect of amyloidogenic LC proteins (AL-LC), which may contribute to the pathophysiology and mortality observed in AL cardiomyopathy patients. However, the mechanisms underlying AL-LC related cardiotoxicity remain unknown. Mammalian stanniocalcin1 (STC1) is associated with a number of cellular processes including oxidative stress and cell death. Herein, we find that STC1 expression is elevated in cardiac tissue from AL cardiomyopathy patients, and is induced in isolated cardiomyocytes in response to AL-LC, but not non-amyloidogenic LC. STC1 overexpression in vitro recapitulates the pathophysiology of AL-LC mediated cardiotoxicity, with increased ROS production, contractile dysfunction and cell death. Overexpression of STC1 in vivo results in significant cardiac dysfunction and cell death. Genetic silencing of STC1 prevents AL-LC induced cardiotoxicity in cardiomyocytes and protects against AL-LC induced cell death and early mortality in zebrafish. The cardiotoxic effects of STC1 appears to be mediated via mitochondrial dysfunction as indicated by loss of mitochondrial membrane potential, ROS production and increased mitochondrial calcium levels. Collectively, this work identifies STC1 as a critical determinant of AL-LC cardiotoxicity.

Microvesicle-associated AAV vector as a novel gene delivery system.

Adeno-associated virus (AAV) vectors have shown remarkable efficiency for gene delivery to cultured cells and in animal models of human disease. However, limitations to AAV vectored gene transfer exist after intravenous transfer, including off-target gene delivery (e.g., liver) and low transduction of target tissue. Here, we show that during production, a fraction of AAV vectors are associated with microvesicles/exosomes, termed vexosomes (vector-exosomes). AAV capsids associated with the surface and in the interior of microvesicles were visualized using electron microscopy. In cultured cells, vexosomes outperformed conventionally purified AAV vectors in transduction efficiency. We found that purified vexosomes were more resistant to a neutralizing anti-AAV antibody compared to conventionally purified AAV. Finally, we show that vexosomes bound to magnetic beads can be attracted to a magnetized area in cultured cells. Vexosomes represent a unique entity which offers a promising strategy to improve gene delivery.

A multi-parametric high throughput assay for detecting beta-cell proliferation in dispersed primary islets.

Identification of novel compounds to selectively induce pancreatic beta-cell proliferation has the potential to restore functional beta-cell mass and insulin secretory demand in type 2 diabetes. The rarity of islet cell clusters (comprising of only 1% of the total pancreas mass) makes such a discovery a challenge. To address this obstacle a high throughput, 384 well, plate-based multi-parametric imaging assay was developed to capture ex vivo primary islet proliferation, allowing positive identification of compounds that can selectively enhance islet beta-cell proliferation. The use of microscopy-based, high-content imaging technology enables acquisition of additional multi-parametric information such as proliferating populations in the islet beta and non beta-cells, insulin intensity, and cell counts, improving understanding of on and off target effects in primary tissue. The protocol requires access to a high-throughput microscopy platform for automated image acquisition of treated islet cells in assay plates. High content image analysis software is required to extract multiparametric cellular features and aid identification of therapeutically relevant small molecules and perturbants. Several putative beta-cell proliferative compounds have validated in this high throughput assay format, including the pleiotropic hormone prolactin [1] and the small molecule DYRK1A inhibitor harmine [2]. It is recommended to include one, or both, as positive controls to provide a reference for image analysis, give confidence in assay performance and capture potential assay variability during experimental runs. The protocol outlined specifically focuses on the multiparametric assessment of betacell proliferation in mouse and rat ex vivo islets and provides the methodology required for the collection of high quality cellular material. The high throughput, plate based assay can additionally be adapted to evaluate and quantify other disease relevant endpoints by high content microscopy and be applied to other downstream measurements. One of the caveats of a high-throughput, 384 microplate beta-cell proliferative assay is its limitations to facilitate human beta-cell proliferation detection, especially for weak activators. Adult human beta-cell proliferation is an extremely rare biological event and assessment experimentally can be donor dependent. In addition lower human islet beta-cell subpopulations require large numbers of cells for accurate rare event measurement.

Highly scalable arrayed CRISPR mediated gene silencing in primary lung small airway epithelial cells.

Small airway epithelial cells (SAECs) play a central role in the pathogenesis of lung diseases and are now becoming a crucial cellular model for target identification and validation in drug discovery. However, primary cell lines such as SAECs are often difficult to transfect using traditional lipofection methods; therefore, gene editing using CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 is often carried out through ribonucleoprotein (RNP) electroporation. Here we have established a robust, scalable, and automated arrayed CRISPR nuclease (CRISPRn) screening workflow for SAECs which can be combined with a myriad of disease-specific endpoint assays.

Impact of Preoperative Atrial Fibrillation on In-Hospital Outcomes of Coronary Artery Bypass Grafting.

Perioperative atrial fibrillation (AF) is associated with increased mortality, morbidity, and excess healthcare costs. The objective of our study was to assess if preoperative AF in patients undergoing coronary artery bypass grafting is a predictor of operative mortality, postoperative stroke, and need for postoperative dialysis by interrogating a large registry database. We included all isolated procedures performed between February 1996 and March 2019. We used a generalized linear mixed model to assess the effect of preoperative AF on mortality stroke and the need for postoperative dialysis after adjusting for the relevant confounders derived from EuroSCORE 2. Confounders considered included age, gender, neurological dysfunction, renal dysfunction, recent myocardial infarction, pulmonary disease, unstable angina, NYHA class, pulmonary hypertension, diabetes on insulin and peripheral vascular disease, and urgency of the operation. We treated the hospital and operating consultant as random effect variables. We also performed LV function subgroup analyses to assess the effect of preoperative AF on the outcomes of interest. The incidence of pre-existent AF in the cohort of patients we analyzed (N = 356,040 patients) was 3.5% (N = 12,664). In the unadjusted baseline characteristics, preoperative AF patients had more associated comorbidities. After adjustment, preoperative AF remained a significant predictor of increased mortality (odds ratio [OR]: 1.63, confidence interval [CI] 1.48-1.79, p < 0.001), stroke (OR: 1.33, CI 1.16-1.54, p = 0.001), and need for renal dialysis (OR:1.61, CI 1.46-1.78, p < 0.001). Preoperative AF was a significant predictor of adverse outcomes in patients with moderate and good LV function but not in patients with poor LV function (EF <30%). Our study suggests that preoperative AF is associated with an increased risk for perioperative mortality and stroke in patients undergoing coronary artery bypass grafting.

Fragment-based screening: A new paradigm for ligand and target discovery.

Significant advances in fragment-based screening, including the emergence of Fully Functionalised Fragments (FFFs) and innovations in Covalent Fragment screening are providing a new paradigm for ligand and target discovery. FFFs offer some key distinct advantages over other screening modalities such as small molecules and genetic screens, including 1) An ability to access diverse chemical space employing a relatively small compound set 2) Ease of screen optimisation given there is no requirement for genetic manipulation and 3) Built-in proteomics tools to facilitate rapid target deconvolution directly in cells. Covalent fragments enable exploration of novel druggable nodes through irreversible fragment-cysteine interactions, complementing their fully functionalized counterparts. Both FFFs and Covalent fragments present the phenotypic screening community with an additional and complementary approach for disease centric target identification.

Weekday and outcomes of elective cardiac surgery in the UK: a large retrospective database analysis.

OBJECTIVES: Several studies have shown worse outcomes in patients operated on later in the week. We tested this hypothesis in a large UK national audit database in elective patients undergoing adult cardiac surgery. METHODS: We used a generalized additive model to evaluate the effect of the day of the week on the following postoperative outcomes: 30-day mortality, stroke, need for dialysis and return to theatre for bleeding. We have adjusted for the relevant European System for Cardiac Operative Risk Evaluation (EuroSCORE) II covariates, plus responsible consultant, hospital and year of operation and performed subgroup analysis for isolated coronary artery bypass grafting (CABG) procedures. RESULTS: Out of 371 500 patients, 60 555 (16.3%) underwent AVR, 36 553 (9.8%) AVR plus CABG, 238 812 (64.3%) isolated CABG, 26 517 (7.1%) isolated mitral valve repair or replacement and 9063 (2.4%) mitral valve plus CABG. A total of 13 997 (3%) had surgery over the weekend. After covariate adjustment, we found no effect of day of surgery on mortality (P = 0.081), stroke (P = 0.137) and need for postop dialysis (P = 0.732). However, across all operations, there was evidence of a lower rate of return to theatre for bleeding/tamponade at the weekend (P = 0.039). In subgroup analysis of isolated CABG, the day of the week did not affect any outcomes. CONCLUSIONS: We found no effect of the day of the week on risk-adjusted short-term mortality, stroke, and the requirement for postoperative dialysis after elective cardiac surgery. Overall, the patients operated on during the weekdays were less likely to return to theatre for bleeding. In isolated CABG, the day of the week did not affect any outcomes.

Selecting the right therapeutic target for kidney disease.

Kidney disease is a complex disease with several different etiologies and underlying associated pathophysiology. This is reflected by the lack of effective treatment therapies in chronic kidney disease (CKD) that stop disease progression. However, novel strategies, recent scientific breakthroughs, and technological advances have revealed new possibilities for finding novel disease drivers in CKD. This review describes some of the latest advances in the field and brings them together in a more holistic framework as applied to identification and validation of disease drivers in CKD. It uses high-resolution 'patient-centric' omics data sets, advanced in silico tools (systems biology, connectivity mapping, and machine learning) and 'state-of-the-art' experimental systems (complex 3D systems in vitro, CRISPR gene editing, and various model biological systems in vivo). Application of such a framework is expected to increase the likelihood of successful identification of novel drug candidates based on strong human target validation and a better scientific understanding of underlying mechanisms.

Protein aggregates and novel presenilin gene variants in idiopathic dilated cardiomyopathy.

BACKGROUND: Heart failure is a debilitating condition resulting in severe disability and death. In a subset of cases, clustered as idiopathic dilated cardiomyopathy (iDCM), the origin of heart failure is unknown. In the brain of patients with dementia, proteinaceous aggregates and abnormal oligomeric assemblies of beta-amyloid impair cell function and lead to cell death. METHODS AND RESULTS: We have similarly characterized fibrillar and oligomeric assemblies in the hearts of iDCM patients, pointing to abnormal protein aggregation as a determinant of iDCM. We also showed that oligomers alter myocyte Ca(2+) homeostasis. Additionally, we have identified 2 new sequence variants in the presenilin-1 (PSEN1) gene promoter leading to reduced gene and protein expression. We also show that presenilin-1 coimmunoprecipitates with SERCA2a. CONCLUSIONS: On the basis of these findings, we propose that 2 mechanisms may link protein aggregation and cardiac function: oligomer-induced changes on Ca(2+) handling and a direct effect of PSEN1 sequence variants on excitation-contraction coupling protein function.

Different Fumaric Acid Esters Elicit Distinct Pharmacologic Responses.

OBJECTIVE: To test the hypothesis that dimethyl fumarate (DMF, Tecfidera) elicits different biological changes from DMF combined with monoethyl fumarate (MEF) (Fumaderm, a psoriasis therapy), we investigated DMF and MEF in rodents and cynomolgus monkeys. Possible translatability of findings was explored with lymphocyte counts from a retrospective cohort of patients with MS. METHODS: In rodents, we evaluated pharmacokinetic and pharmacodynamic effects induced by DMF and MEF monotherapies or in combination (DMF/MEF). Clinical implications were investigated in a retrospective, observational analysis of patients with MS treated with DMF/MEF (n = 36). RESULTS: In rodents and cynomolgus monkeys, monomethyl fumarate (MMF, the primary metabolite of DMF) exhibited higher brain penetration, whereas MEF was preferentially partitioned into the kidney. In mice, transcriptional profiling for DMF and MEF alone identified both common and distinct pharmacodynamic responses, with almost no overlap between DMF- and MEF-induced differentially expressed gene profiles in immune tissues. The nuclear factor (erythroid-derived 2)-like 2 (Nrf2)-mediated oxidative stress response pathway was exclusively regulated by DMF, whereas apoptosis pathways were activated by MEF. DMF/MEF treatment demonstrated that DMF and MEF functionally interact to modify DMF- and MEF-specific responses in unpredictable ways. In patients with MS, DMF/MEF treatment led to early and pronounced suppression of lymphocytes, predominantly CD8+ T cells. In a multivariate regression analysis, the absolute lymphocyte count (ALC) was associated with age at therapy start, baseline ALC, and DMF/MEF dosage but not with previous immunosuppressive medication and sex. Furthermore, the ALC increased in a small cohort of patients with MS (n = 6/7) after switching from DMF/MEF to DMF monotherapy. CONCLUSIONS: Fumaric acid esters exhibit different biodistribution and may elicit different biological responses; furthermore, pharmacodynamic effects of combinations differ unpredictably from monotherapy. The strong potential to induce lymphopenia in patients with MS may be a result of activation of apoptosis pathways by MEF compared with DMF.