Syntrophin mutation associated with long QT syndrome through activation of the nNOS-SCN5A macromolecular complex.

Mutations in 11 genes that encode ion channels or their associated proteins cause inherited long QT syndrome (LQTS) and account for approximately 75-80% of cases (LQT1-11). Direct sequencing of SNTA1, the gene encoding alpha1-syntrophin, was performed in a cohort of LQTS patients that were negative for mutations in the 11 known LQTS-susceptibility genes. A missense mutation (A390V-SNTA1) was found in a patient with recurrent syncope and markedly prolonged QT interval (QTc, 530 ms). SNTA1 links neuronal nitric oxide synthase (nNOS) to the nNOS inhibitor plasma membrane Ca-ATPase subtype 4b (PMCA4b); SNTA1 also is known to associate with the cardiac sodium channel SCN5A. By using a GST-fusion protein of the C terminus of SCN5A, we showed that WT-SNTA1 interacted with SCN5A, nNOS, and PMCA4b. In contrast, A390V-SNTA1 selectively disrupted association of PMCA4b with this complex and increased direct nitrosylation of SCN5A. A390V-SNTA1 expressed with SCN5A, nNOS, and PMCA4b in heterologous cells increased peak and late sodium current compared with WT-SNTA1, and the increase was partially inhibited by NOS blockers. Expression of A390V-SNTA1 in cardiac myocytes also increased late sodium current. We conclude that the A390V mutation disrupted binding with PMCA4b, released inhibition of nNOS, caused S-nitrosylation of SCN5A, and was associated with increased late sodium current, which is the characteristic biophysical dysfunction for sodium-channel-mediated LQTS (LQT3). These results establish an SNTA1-based nNOS complex attached to SCN5A as a key regulator of sodium current and suggest that SNTA1 be considered a rare LQTS-susceptibility gene.

Blastomycosis granuloma involving the cranial vena cava associated with chylothorax and cranial vena caval syndrome in a dog

A four-year-old, sexually intact, male dachshund was diagnosed with pulmonary blastomycosis. Itraconazole was administered for 60 days, and the dog was considered to be disease-free at three- and 12-month reevaluations. Two years following discontinuation of itraconazole, the dog developed a granuloma of the cranial vena cava resulting in chylothorax and cranial vena caval obstruction. To the authors' knowledge, this is the first case of a blastomycotic granuloma involving the vena cava reported in the dog. Blastomycosis should be considered as a differential diagnosis for both chylothorax and cranial vena caval syndrome in the dog.

Targeting membrane-bound viral RNA synthesis reveals potent inhibition of diverse coronaviruses including the middle East respiratory syndrome virus.

Coronaviruses raise serious concerns as emerging zoonotic viruses without specific antiviral drugs available. Here we screened a collection of 16671 diverse compounds for anti-human coronavirus 229E activity and identified an inhibitor, designated K22, that specifically targets membrane-bound coronaviral RNA synthesis. K22 exerts most potent antiviral activity after virus entry during an early step of the viral life cycle. Specifically, the formation of double membrane vesicles (DMVs), a hallmark of coronavirus replication, was greatly impaired upon K22 treatment accompanied by near-complete inhibition of viral RNA synthesis. K22-resistant viruses contained substitutions in non-structural protein 6 (nsp6), a membrane-spanning integral component of the viral replication complex implicated in DMV formation, corroborating that K22 targets membrane bound viral RNA synthesis. Besides K22 resistance, the nsp6 mutants induced a reduced number of DMVs, displayed decreased specific infectivity, while RNA synthesis was not affected. Importantly, K22 inhibits a broad range of coronaviruses, including Middle East respiratory syndrome coronavirus (MERS-CoV), and efficient inhibition was achieved in primary human epithelia cultures representing the entry port of human coronavirus infection. Collectively, this study proposes an evolutionary conserved step in the life cycle of positive-stranded RNA viruses, the recruitment of cellular membranes for viral replication, as vulnerable and, most importantly, druggable target for antiviral intervention. We expect this mode of action to serve as a paradigm for the development of potent antiviral drugs to combat many animal and human virus infections.

A deleterious gene-by-environment interaction imposed by calcium channel blockers in Marfan syndrome.

Calcium channel blockers (CCBs) are prescribed to patients with Marfan syndrome for prophylaxis against aortic aneurysm progression, despite limited evidence for their efficacy and safety in the disorder. Unexpectedly, Marfan mice treated with CCBs show accelerated aneurysm expansion, rupture, and premature lethality. This effect is both extracellular signal-regulated kinase (ERK1/2) dependent and angiotensin-II type 1 receptor (AT1R) dependent. We have identified protein kinase C beta (PKCβ) as a critical mediator of this pathway and demonstrate that the PKCβ inhibitor enzastaurin, and the clinically available anti-hypertensive agent hydralazine, both normalize aortic growth in Marfan mice, in association with reduced PKCβ and ERK1/2 activation. Furthermore, patients with Marfan syndrome and other forms of inherited thoracic aortic aneurysm taking CCBs display increased risk of aortic dissection and need for aortic surgery, compared to patients on other antihypertensive agents.

Comparison of prasugrel and clopidogrel-treated patients with acute coronary syndrome undergoing percutaneous coronary intervention: A propensity score-matched analysis of the Acute Myocardial Infarction in Switzerland (AMIS)-Plus Registry

OBJECTIVE The purpose of this study was to investigate outcomes of patients treated with prasugrel or clopidogrel after percutaneous coronary intervention (PCI) in a nationwide acute coronary syndrome (ACS) registry. BACKGROUND Prasugrel was found to be superior to clopidogrel in a randomized trial of ACS patients undergoing PCI. However, little is known about its efficacy in everyday practice. METHODS All ACS patients enrolled in the Acute Myocardial Infarction in Switzerland (AMIS)-Plus registry undergoing PCI and being treated with a thienopyridine P2Y12 inhibitor between January 2010-December 2013 were included in this analysis. Patients were stratified according to treatment with prasugrel or clopidogrel and outcomes were compared using propensity score matching. The primary endpoint was a composite of death, recurrent infarction and stroke at hospital discharge. RESULTS Out of 7621 patients, 2891 received prasugrel (38%) and 4730 received clopidogrel (62%). Independent predictors of in-hospital mortality were age, Killip class >2, STEMI, Charlson comorbidity index >1, and resuscitation prior to admission. After propensity score matching (2301 patients per group), the primary endpoint was significantly lower in prasugrel-treated patients (3.0% vs 4.3%; p=0.022) while bleeding events were more frequent (4.1% vs 3.0%; p=0.048). In-hospital mortality was significantly reduced (1.8% vs 3.1%; p=0.004), but no significant differences were observed in rates of recurrent infarction (0.8% vs 0.7%; p=1.00) or stroke (0.5% vs 0.6%; p=0.85). In a predefined subset of matched patients with one-year follow-up (n=1226), mortality between discharge and one year was not significantly reduced in prasugrel-treated patients (1.3% vs 1.9%, p=0.38). CONCLUSIONS In everyday practice in Switzerland, prasugrel is predominantly used in younger patients with STEMI undergoing primary PCI. A propensity score-matched analysis suggests a mortality benefit from prasugrel compared with clopidogrel in these patients.

Reasons for discontinuation of recommended therapies according to the patients after acute coronary syndromes.

BACKGROUND The prescription of recommended medical therapies is a key factor to improve prognosis after acute coronary syndromes (ACS). However, reasons for cardiovascular therapies discontinuation after hospital discharge are poorly reported in previous studies. METHODS We enrolled 3055 consecutive patients hospitalized with a main diagnosis of ACS in four Swiss university hospitals with a prospective one-year follow-up. We assessed the self-reported use of recommended therapies and the reasons for medication discontinuation according to the patient interview performed at one-year follow-up. RESULTS 3014 (99.3%) patients were discharged with aspirin, 2983 (98.4%) with statin, 2464 (81.2%) with beta-blocker, 2738 (90.3%) with ACE inhibitors/ARB and 2597 (100%) with P2Y12 inhibitors if treated with coronary stent. At the one-year follow-up, the discontinuation percentages were 2.9% for aspirin, 6.6% for statin, 11.6% for beta-blocker, 15.1% for ACE inhibitor/ARB and 17.8% for P2Y12 inhibitors. Most patients reported having discontinued their medication based on their physicians' decision: 64 (2.1%) for aspirin, 82 (2.7%) for statin, 212 (8.6%) for beta-blocker, 251 (9.1% for ACE inhibitor/ARB) and 293 (11.4%) for P2Y12 inhibitors, while side effect, perception that medication was unnecessary and medication costs were uncommon reported reasons (<2%) according to the patients. CONCLUSIONS Discontinuation of recommended therapies after ACS differs according the class of medication with the lowest percentages for aspirin. According to patients, most stopped their cardiovascular medication based on their physician's decision, while spontaneous discontinuation was infrequent.

Rebound-associated vertebral fractures after discontinuation of denosumab-from clinic and biomechanics.

Rebound-associated vertebral fractures may follow treatment discontinuation of highly potent reversible bone antiresorptives, resulting from the synergy of rapid bone resorption and accelerated microdamage accumulation in trabecular bone. INTRODUCTION The purposes of this study are to characterize rebound-associated vertebral fractures following the discontinuation of a highly potent reversible antiresorptive therapy based on clinical observation and propose a pathophysiological rationale. METHODS This study is a case report of multiple vertebral fractures early after discontinuation of denosumab therapy in a patient with hormone receptor-positive non-metastatic breast cancer treated with an aromatase inhibitor. RESULTS Discontinuation of highly potent reversible bone antiresorptives such as denosumab may expose patients to an increased fracture risk due to the joined effects of absent microdamage repair during therapy followed by synchronous excess activation of multiple bone remodelling units at the time of loss-of-effect. We suggest the term rebound-associated vertebral fractures (RVF) for this phenomenon characterized by the presence of multiple new clinical vertebral fractures, associated with either no or low trauma, in a context consistent with the presence of high bone turnover and rapid loss of lumbar spine bone mineral density (BMD) occurring within 3 to 12 months after discontinuation (loss-of-effect) of a reversible antiresorptive therapy in the absence of secondary causes of bone loss or fractures. Unlike atypical femoral fractures that emerge from failure of microdamage repair in cortical bone with long-term antiresorptive treatment, RVF originate from the synergy of rapid bone resorption and accelerated microdamage accumulation in trabecular bone triggered by the discontinuation of highly potent reversible antiresorptives. CONCLUSIONS Studies are urgently needed to i) prove the underlying pathophysiological processes suggested above, ii) establish the predictive criteria exposing patients to an increased risk of RVF, and iii) determine appropriate treatment regimens to be applied in such patients.

Clinical Course of acute-on-chronic liver failure syndrome and effects on prognosis

Acute-on-chronic liver failure (ACLF) is characterized by acute decompensation (AD) of cirrhosis, organ failure(s), and high 28-day mortality. We investigated whether assessments of patients at specific time points predicted their need for liver transplantation (LT) or the potential futility of their care. We assessed clinical courses of 388 patients who had ACLF at enrollment, from February through September 2011, or during early (28-day) follow-up of the prospective multicenter European Chronic Liver Failure (CLIF) ACLF in Cirrhosis study. We assessed ACLF grades at different time points to define disease resolution, improvement, worsening, or steady or fluctuating course. ACLF resolved or improved in 49.2%, had a steady or fluctuating course in 30.4%, and worsened in 20.4%. The 28-day transplant-free mortality was low-to-moderate (6%-18%) in patients with nonsevere early course (final no ACLF or ACLF-1) and high-to-very high (42%-92%) in those with severe early course (final ACLF-2 or -3) independently of initial grades. Independent predictors of course severity were CLIF Consortium ACLF score (CLIF-C ACLFs) and presence of liver failure (total bilirubin ≥12 mg/dL) at ACLF diagnosis. Eighty-one percent had their final ACLF grade at 1 week, resulting in accurate prediction of short- (28-day) and mid-term (90-day) mortality by ACLF grade at 3-7 days. Among patients that underwent early LT, 75% survived for at least 1 year. Among patients with ≥4 organ failures, or CLIF-C ACLFs >64 at days 3-7 days, and did not undergo LT, mortality was 100% by 28 days. CONCLUSIONS Assessment of ACLF patients at 3-7 days of the syndrome provides a tool to define the emergency of LT and a rational basis for intensive care discontinuation owing to futility.

MUTATIONS IN STAT3 ASSOCIATED WITH HUMAN HYPER IgE SYNDROME ENHANCE NFκB AND MAPK-MEDIATED GENE EXPRESSION

Hyper IgE syndrome (HIES) is a multisystem disorder resulting in bone and immune system abnormalities. It is associated with mutations in STAT3, which disrupt protein domains responsible for transcriptional function. Patients with HIES display osteoporosis and enhanced inflammatory cytokine production similar to hematopoietic Stat3-deficient mice. Since osteoclast and inflammatory cytokine genes are NFκB targets, these observations indicate a possible deregulation of NFκB signaling in both mice and humans with STAT3-deficiency. Here, we sought to examine the role of STAT3 in the regulation of NFκB-mediated gene expression through analysis of three HIES STAT3 point mutations in both hematopoietic and non- hematopoietic cells. We found that IL-6-induced tyrosine phosphorylation of STAT3 was partially or completely abrogated by HIES mutations in the transactivation domain (V713L) or SH2 domain (V637M), respectively, in both hematopoietic and non- hematopoietic cells. By contrast, IL-6-induced tyrosine phosphorylation of an HIES mutant in the STAT3 DNA-binding domain (R382W) was intact. The R382W and V713L mutants significantly reduced IL-6-dependent STAT3 transcriptional activity in reporter gene assays. Moreover, the R382W and V637M mutants significantly diminished IL-6-responsive expression of the endogenous STAT3 target gene, Socs3, as assessed by quantitative real-time PCR (qPCR) in the RAW macrophage cell line. These observations indicate the HIES mutants dominantly suppress the transcriptional activity of wild type STAT3, albeit to varying degrees. All three HIES mutants enhanced LPS-induced expression of the NFκB target genes IL6 (IL-6), Cxcl10 (IP- 10), and Tnf (TNFα) in RAW cells, as indicated by qPCR. Furthermore, overexpression of wild type STAT3 in Stat3-deficient murine embryonic fibroblasts significantlyreduced LPS-stimulated expression of IL6, Cxcl10, and IL12p35. In addition, in aprimary murine osteoclast differentiation assay, a STAT3-specific SH2 domain inhibitor led to significantly increased levels of osteoclast-specific gene expression. These results suggest that STAT3 serves as a negative regulator of NFκB-mediated gene expression, and furthermore imply that STAT3 mutations associated with HIES contribute to the osteopenia and inflammation observed in HIES patients.

A novel DNA damage inducible inhibitor of p53

p53 is required for the maintenance of the genomic stability of cells. Mutations in the p53 tumor-suppressor gene occur in more than 50% of human cancers of diverse types. In addition, 70% of families with Li-Fraumeni syndrome have a germline mutation in p53, predisposing these individuals to multiple forms of cancer. In response to DNA damage, p53 becomes stabilized and activated. However the exact mechanism by which DNA damage signals the stabilization and activation of p53 still remains elusive. The biochemical activity of p53 that is required for tumor suppression, and presumably the cellular response to DNA damage, involves the ability of the protein to bind to specific DNA sequences and to function as a transcription factor. For the downstream targets, p53 transactivates many genes involved in growth arrest, apoptosis and DNA repair such as p21, Bax and GADD45, respectively. An open question in the field is how cells can determine the downstream effects of p53. ^ We hypothesize that, through its associated proteins, p53 can differentially transactivate its target genes, which determine its downstream effect. Additionally, p53 interacting proteins may be involved in signaling for the stabilization and activation of p53. Therefore, a key aspect to understanding p53 function is the identification and analysis of proteins that interact with it. We have employed the Sos recruitment system (SRS), a cytoplasmic yeast two-hybrid screen to identify p53 interacting proteins. The SRS is based on the ability of Sos to activate Ras when it becomes localized to the plasma membrane. The system takes advantage of an S. cerevisiae strain, cdc25-2 temperature sensitive mutant, harboring a mutation in Sos. In this strain, fusion proteins containing a truncated Sos will only localize to the membrane by protein-protein interaction, which allows growth at non-permissive temperature. This system allows the use of intact transcriptional activators such as p53. ^ To date, using a modified SRS library screen to identify p53 interacting proteins, I have identified p53 (known to interact with itself) and a novel p53-interacting protein (PIP). PIP is a specific p53 interacting protein in the SRS. The interaction of p53 and PIP was further confirmed by performing in vitro and in vivo binding assays. In the in vivo binding study, the interaction can only be detected in the presence of ionizing radiation suggesting that this interaction might be involved in DNA-damage induced p53-signalling pathway. After screening cDNA and genomic libraries, a full-length PIP-cDNA clone ( ∼ 3kb) was obtained which encodes a protein of 429 amino acids with calculated molecular weight of 46 kDa. The results of genebank search indicated that the PIP is an unidentified gene and contains a conserved ring-finger domain, which is present in a diverse family of regulatory proteins involved in different aspects of cellular function. Northern blot analysis revealed that the size of its messenge is approximately 3 kb preferentially expressed in brain, heart, liver and kidney. The PIP protein is mainly located in the cytoplasm as determined by the cellular localization of a green fluorescence fusion protein. Preliminary functional analysis revealed that PIP downregulated the transactivation activity of p53 on both p21 and mdm2 promoters. Thus, PIP may be a novel negative regulator of p53 subsequent to DNA damage. ^

Increased IGF-II protein affects p57kip2 expression in vivo and in vitro: Implications for Beckwith–Wiedemann syndrome

In both human and mouse, the Igf2 gene, localized on chromosomes 11 and 7, respectively, is expressed from the paternally inherited chromosome in the majority of tissues. Insulin-like growth factor-II (IGF-II) plays an important role in embryonic growth, and aberrant IGF2 expression has been documented in several human pathologies, such as Beckwith–Wiedemann syndrome (BWS), and a wide variety of tumors. Human and mouse genetic data strongly implicate another gene, CDKN1C (p57kip2), located in the same imprinted gene cluster on human chromosome II, in BWS. p57KIP2 is a cyclin-dependent kinase inhibitor and is required for normal mouse embryonic development. Mutations in CDKN1C (p57kip2) have been identified in a small proportion of patients with BWS, and removal of the gene from mice by targeted mutagenesis produces a phenotype with elements in common with this overgrowth syndrome. Patients with BWS with biallelic expression of IGF2 or with a CDKN1C (p57kip2) mutation, as well as overlapping phenotypes observed in two types of mutant mice, the p57kip2 knockout and IGF-II-overexpressing mice, strongly suggest that the genes may act in a common pathway of growth control in situations where Igf2 expression is abnormal. Herein, we show that p57kip2 expression is reduced on IGF-II treatment of primary embryo fibroblasts in a dose-dependent manner. In addition, p57kip2 expression is down-regulated in mice with high serum levels of IGF-II. These data suggest that the effects of increased IGF-II in BWS may, in part, be mediated through a decrease in p57kip2 gene expression.

Uncompensated polyuria in a mouse model of Bartter's syndrome

We have used homologous recombination to disrupt the mouse gene coding for the NaK2Cl cotransporter (NKCC2) expressed in kidney epithelial cells of the thick ascending limb and macula densa. This gene is one of several that when mutated causes Bartter's syndrome in humans, a syndrome characterized by severe polyuria and electrolyte imbalance. Homozygous NKCC2−/− pups were born in expected numbers and appeared normal. However, by day 1 they showed signs of extracellular volume depletion (hematocrit 51%; wild type 37%). They subsequently failed to thrive. By day 7, they were small and markedly dehydrated and exhibited renal insufficiency, high plasma potassium, metabolic acidosis, hydronephrosis of varying severity, and high plasma renin concentrations. None survived to weaning. Treatment of −/− pups with indomethacin from day 1 prevented growth retardation and 10% treated for 3 weeks survived, although as adults they exhibited severe polyuria (10 ml/day), extreme hydronephrosis, low plasma potassium, high blood pH, hypercalciuria, and proteinuria. Wild-type mice treated with furosemide, an inhibitor of NaK2Cl cotransporters, have a phenotype similar to the indomethacin-rescued −/− adults except that hydronephrosis was mild. The polyuria, hypercalciuria, and proteinuria of the −/− adults and furosemide-treated wild-type mice were unresponsive to inhibitors of the renin angiotensin system, vasopressin, and further indomethacin. Thus absence of NKCC2 in the mouse causes polyuria that is not compensated elsewhere in the nephron. The NKCC2 mutant animals should be valuable for uncovering new pathophysiologic and therapeutic aspects of genetic disturbances in water and electrolyte recovery by the kidney.

Imprinting of the gene encoding a human cyclin-dependent kinase inhibitor, p57KIP2, on chromosome 11p15.

Parental origin-specific alterations of chromosome 11p15 in human cancer suggest the involvement of one or more maternally expressed imprinted genes involved in embryonal tumor suppression and the cancer-predisposing Beckwith-Wiedemann syndrome (BWS). The gene encoding cyclin-dependent kinase inhibitor p57KIP2, whose overexpression causes G1 phase arrest, was recently cloned and mapped to this band. We find that the p57KIP2 gene is imprinted, with preferential expression of the maternal allele. However, the imprint is not absolute, as the paternal allele is also expressed at low levels in most tissues, and at levels comparable to the maternal allele in fetal brain and some embryonal tumors. The biochemical function, chromosomal location, and imprinting of the p57KIP2 gene match the properties predicted for a tumor suppressor gene at 11p15.5. However, as the p57KIP2 gene is 500 kb centromeric to the gene encoding insulin-like growth factor 2, it is likely to be part of a large domain containing other imprinted genes. Thus, loss of heterozygosity or loss of imprinting might simultaneously affect several genes at this locus that together contribute to tumor and/or growth- suppressing functions that are disrupted in BWS and embryonal tumors.

Multiple genetic loci within 11p15 defined by Beckwith-Wiedemann syndrome rearrangement breakpoints and subchromosomal transferable fragments.

Beckwith-Wiedemann syndrome (BWS) involves fetal overgrowth and predisposition to a wide variety of embryonal tumors of childhood. We have previously found that BWS is genetically linked to 11p15 and that this same band shows loss of heterozygosity in the types of tumors to which children with BWS are susceptible. However, 11p15 contains > 20 megabases, and therefore, the BWS and tumor suppressor genes could be distinct. To determine the precise physical relationship between these loci, we isolated yeast artificial chromosomes, and cosmid libraries from them, within the region of loss of heterozygosity in embryonal tumors. Five germ-line balanced chromosomal rearrangement breakpoint sites from BWS patients, as well as a balanced chromosomal translocation breakpoint from a rhabdoid tumor, were isolated within a 295- to 320-kb cluster defined by a complete cosmid contig crossing these breakpoints. This breakpoint cluster terminated approximately 100 kb centromeric to the imprinted gene IGF2 and 100 kb telomeric to p57KIP2, an inhibitor of cyclin-dependent kinases, and was located within subchromosomal transferable fragments that suppressed the growth of embryonal tumor cells in genetic complementation experiments. We have identified 11 transcribed sequences in this BWS/tumor suppressor coincident region, one of which corresponded to p57KIP2. However, three additional BWS breakpoints were > 4 megabases centromeric to the other five breakpoints and were excluded from the tumor suppressor region defined by subchromosomal transferable fragments. Thus, multiple genetic loci define BWS and tumor suppression on 11p15.

Animal model for the therapy of acquired immunodeficiency syndrome with reverse transcriptase inhibitors.

The reverse transcriptase (RT) of the human immunodeficiency virus type 1 (HIV-1) is the major target for antiretroviral therapy of the acquired immunodeficiency syndrome (AIDS). While some inhibitors exhibit activity against most retroviral RTs, others are specific for the HIV-1 enzyme. To develop an animal model for the therapy of the HIV-1 infection with RT inhibitors, the RT of the simian immunodeficiency virus (SIV) was replaced by the RT of HIV-1. Macaques infected with this SIV/HIV-1 hybrid virus developed AIDS-like symptoms and pathology. The HIV-1-specific RT inhibitor LY300046.HCl, but not zidovudine [3'-azido-3'-deoxythymidine (AZT)] delayed the appearance of plasma antigenemia in macaques infected with a high dose of the chimeric virus. Infection of macaques with the chimeric virus seems to be a valuable model to study the in vivo efficacy of new RT inhibitors, the emergence and reversal of drug resistance, the therapy of infections with drug-resistant viruses, and the efficacy of combination therapy.