Regulation of Locomotor activity in fed, fasted, and food-restricted mice lacking tissue-type plasminogen activator.

BACKGROUND: Circadian rhythms of physiology and behavior are driven by a circadian clock located in the suprachiasmatic nucleus of the hypothalamus. This clock is synchronized to environmental day/night cycles by photic input, which is dependent on the presence of mature brain-derived neurotrophic factor (BDNF) in the SCN. Mature BDNF is produced by the enzyme plasmin, which is converted from plasminogen by the enzyme tissue-type plasminogen activator (tPA). In this study, we evaluate circadian function in mice lacking functional tPA. RESULTS: tPA-/- mice have normal circadian periods, but show decreased nocturnal wheel-running activity. This difference is eliminated or reversed on the second day of a 48-h fast. Similarly, when placed on daily cycles of restricted food availability the genotypic difference in total wheel-running activity disappears, and tPA-/- mice show equivalent amounts of food anticipatory activity to wild type mice. CONCLUSIONS: These data suggest that tPA regulates nocturnal wheel-running activity, and that tPA differentially affects SCN-driven nocturnal activity rhythms and activity driven by fasting or temporal food restriction.

Identification of Viral and Host Proteins That Interact with Murine Gammaherpesvirus 68 Latency-Associated Nuclear Antigen during Lytic Replication: a Role for Hsc70 in Viral Replication.

UNLABELLED: Latency-associated nuclear antigen (LANA) is a conserved, multifunctional protein encoded by members of the rhadinovirus subfamily of gammaherpesviruses, including Kaposi sarcoma-associated herpesvirus (KSHV) and murine gammaherpesvirus 68 (MHV68). We previously demonstrated that MHV68 LANA (mLANA) is required for efficient lytic replication. However, mechanisms by which mLANA facilitates viral replication, including interactions with cellular and viral proteins, are not known. Thus, we performed a mass spectrometry-based interaction screen that defined an mLANA protein-protein interaction network for lytic viral replication consisting of 15 viral proteins and 191 cellular proteins, including 19 interactions previously reported in KSHV LANA interaction studies. We also employed a stable-isotope labeling technique to illuminate high-priority mLANA-interacting host proteins. Among the top prioritized mLANA-binding proteins was a cellular chaperone, heat shock cognate protein 70 (Hsc70). We independently validated the mLANA-Hsc70 interaction through coimmunoprecipitation and in vitro glutathione S-transferase (GST) pulldown assays. Immunofluorescence and cellular fractionation analyses comparing wild-type (WT) to mLANA-null MHV68 infections demonstrated mLANA-dependent recruitment of Hsc70 to nuclei of productively infected cells. Pharmacologic inhibition and small hairpin RNA (shRNA)-mediated knockdown of Hsc70 impaired MHV68 lytic replication, which functionally correlated with impaired viral protein expression, reduced viral DNA replication, and failure to form viral replication complexes. Replication of mLANA-null MHV68 was less affected than that of WT virus by Hsc70 inhibition, which strongly suggests that Hsc70 function in MHV68 lytic replication is at least partially mediated by its interaction with mLANA. Together these experiments identify proteins engaged by mLANA during the MHV68 lytic replication cycle and define a previously unknown role for Hsc70 in facilitating MHV68 lytic replication. IMPORTANCE: Latency-associated nuclear antigen (LANA) is a conserved gamma-2-herpesvirus protein important for latency maintenance and pathogenesis. For MHV68, this includes regulating lytic replication and reactivation. While previous studies of KSHV LANA defined interactions with host cell proteins that impact latency, interactions that facilitate productive viral replication are not known. Thus, we performed a differential proteomics analysis to identify and prioritize cellular and viral proteins that interact with the MHV68 LANA homolog during lytic infection. Among the proteins identified was heat shock cognate protein 70 (Hsc70), which we determined is recruited to host cell nuclei in an mLANA-dependent process. Moreover, Hsc70 facilitates MHV68 protein expression and DNA replication, thus contributing to efficient MHV68 lytic replication. These experiments expand the known LANA-binding proteins to include MHV68 lytic replication and demonstrate a previously unappreciated role for Hsc70 in regulating viral replication.

The role of the exosporium in the environmental distribution of anthrax.

AIMS: To determine the contribution of the exosporium, the outer layer of the Bacillus anthracis spore, to soil attachment. Persistence of spores in soil and their ability to infect animals has been linked to a range of factors which include the presence of organic material and calcium (OMC), pH > 6.0, temperatures above 15.5°C and cycles of local flooding which are thought to transport buried spores to the surface. METHODS AND RESULTS: The ability of wild type (exosporium +ve) and sonicated (exosporium -ve) spores to bind to soils which differed in their composition was determined using a flow-through soil column-based method. A statistically significant difference (P < 0.05) in the binding of wild type spores was observed with spores adhering more firmly to the soil with the highest OMC content. We also found that the removal of the exosporium increased the ability of the spore to adhere to both soil types. CONCLUSION: Structures within the exosporium affected the ability of B. anthracis spores to bind to different soil types. Not surprisingly, wild type spores adhered to soil which has been shown to favour the persistence of the pathogen. SIGNIFICANCE AND IMPACT OF THE STUDY: The ability to persist in and colonise the soil surface is a key requirement of a pathogen which infects grazing animals. By characterising the process involved, we will be better placed to develop strategies to disrupt the infection cycle.

Community seroprevalence of SARS-CoV-2 in children and adolescents in England, 2019-2021.

OBJECTIVE: To understand community seroprevalence of SARS-CoV-2 in children and adolescents. This is vital to understanding the susceptibility of this cohort to COVID-19 and to inform public health policy for disease control such as immunisation. DESIGN: We conducted a community-based cross-sectional seroprevalence study in participants aged 0-18 years old recruiting from seven regions in England between October 2019 and June 2021 and collecting extensive demographic and symptom data. Serum samples were tested for antibodies against SARS-CoV-2 spike and nucleocapsid proteins using Roche assays processed at UK Health Security Agency laboratories. Prevalence estimates were calculated for six time periods and were standardised by age group, ethnicity and National Health Service region. RESULTS: Post-first wave (June-August 2020), the (anti-spike IgG) adjusted seroprevalence was 5.2%, varying from 0.9% (participants 10-14 years old) to 9.5% (participants 5-9 years old). By April-June 2021, this had increased to 19.9%, varying from 13.9% (participants 0-4 years old) to 32.7% (participants 15-18 years old). Minority ethnic groups had higher risk of SARS-CoV-2 seropositivity than white participants (OR 1.4, 95% CI 1.0 to 2.0), after adjusting for sex, age, region, time period, deprivation and urban/rural geography. In children <10 years, there were no symptoms or symptom clusters that reliably predicted seropositivity. Overall, 48% of seropositive participants with complete questionnaire data recalled no symptoms between February 2020 and their study visit. CONCLUSIONS: Approximately one-third of participants aged 15-18 years old had evidence of antibodies against SARS-CoV-2 prior to the introduction of widespread vaccination. These data demonstrate that ethnic background is independently associated with risk of SARS-CoV-2 infection in children. TRIAL REGISTRATION NUMBER: NCT04061382.

Lethal and edema toxins of anthrax induce distinct hemodynamic dysfunction.

Fatalities due to anthrax are associated with severe hypotension suggesting that the toxins generated from Bacillus anthracis, lethal toxin (LeTx) and edema toxin (EdTx), have cardiovascular effects. Here, we demonstrate the effects of these toxins and characterize their effects by echocardiography. LeTx leads to a significant reduction in ejection fraction, decreased velocity of propagation (diastolic dysfunction), decreased velocity of circumferential fiber shortening (decreased contractility), and increased LV systolic area (pathophysiology). EdTx leads to a significant reduction in left ventricular volumes and cardiac output (reduced stroke volume) but does not cause significant change in ejection fraction or contractility. These results indicate that LeTx reduces left ventricular systolic function and EdTx reduces preload but does not have direct myocardial effects. Together, these findings suggest that LeTx and EdTx exert distinct hemodynamic dysfunction associated with anthrax infection.

Single coronary artery with an ectopic origin and interarterial course: a case report and review of the literature.

A previously unreported variant of a single coronary artery arising between the aorta and pulmonary artery is presented. This variant had many high-risk features, so prophylactic coronary artery bypass grafting was recommended.

Baseline echocardiographic values for adult male rats.

BACKGROUND: Because of safety, repeatability, and portability, clinical echocardiography is well established as a standard for cardiac anatomy, cardiac function, and hemodynamics. Similarly, application of echocardiography in commonly used rat experimental models would be worthwhile. The use of noninvasive ultrasound imaging in the rat is a potential replacement for more invasive terminal techniques. Although echocardiography has become commonly used in the rat, normal parameters for cardiac anatomy and function, and comparison with established human values, have not been reported. METHODS: A total of 44 Sprague-Dawley male rats had baseline echocardiography replicating a protocol for clinical echocardiography. RESULTS: Complete 2-dimensional echocardiography for cardiac anatomy and function was obtained in 44 rats. Hemodynamic parameters could be recorded in 85% of rats. The ejection fraction and fractional shortening values of the left ventricle were similar to those reported for healthy human beings. Pulsed Doppler velocities of atrial systole for mitral valve inflow, pulmonary vein reversal, and Doppler tissue of the lateral mitral valve annulus also had similar means as healthy human beings. The calculated left ventricular mass was at the same order of magnitude as a proportion of body weight of rat to man. All other observations in the clinical protocol were different from those reported in healthy human beings. CONCLUSION: The use of echocardiography for assessment of cardiac anatomy, function, and hemodynamics can be consistently applied to the rat and replicates much of the information used routinely in human echocardiography.

Comparison of lipid and non-lipid markers of atherosclerosis in arterial versus venous blood.

BACKGROUND: Early studies have suggested that there may be differences in the concentration of lipoprotein particles and their associated apolipoproteins in arterial and venous blood and that this gradient might explain a proclivity to develop atherosclerotic lesions. The aim of this study was to use current methods of analysis to determine levels of these components, including particle densities and several common inflammatory markers in arterial and venous blood. METHODS: Samples of arterial and venous blood were obtained nearly simultaneously in 26 patients undergoing right and left heart catheterization. Analyses were performed using enzymatic, immunoturbidimetric and ultracentrifugation assays. RESULTS: Data obtained for total cholesterol, triglyceride, HDL-cholesterol, LDL-cholesterol, HDL and LDL particle density, high sensitivity C-reactive protein, serum amyloid-A and apoprotein B-100 concentrations in arterial and venous blood did not demonstrate any significant difference in the means. CONCLUSION: Arterial and venous blood can be used interchangeably to study the effect of blood concentrations of common soluble surrogate markers of atherosclerosis.

Intravenous immunoglobulin-related acute coronary syndrome and coronary angiography in idiopathic thrombocytopenic purpura--a case report and literature review.

In acute coronary syndromes, GPIIb/IIIa platelet inhibitors have demonstrated a reduction in recurrent myocardial ischemia. Conversely, one might expect that enhancing platelet activity in patients in acute coronary syndromes would have the opposite effect. We report a patient with idiopathic thrombocytopenic purpura (ITP) that had recurrent myocardial ischemia associated with administration of intravenous immunogloblin (IVIG). Literature is reviewed.

Echocardiographic effects of eplerenone and aldosterone in hypertensive rats.

The effects of aldosterone receptor blockade on echocardiography in spontaneously hypertensive rats (SHR) are not fully characterized. In this study, multiple echocardiographic parameters were compared for 42 weeks between SHR versus Wistar-Kyoto rats (WKY) serving as normotensive controls. In addition, echocardiographic parameters were compared for 28 weeks between the SHR versus SHR treated with eplerenone 100 mg/kg/day or spironolactone 50 mg/kg/day. Compared to normotensive WKY rats, SHRs had significantly increased systolic blood pressure, increased cardiac mass, increased isovolumic relaxation time (IVRT), decreased E/A ratio, increased mitral closure opening time interval (MCO) and increased Tei index. Both eplerenone and spironolactone significantly decreased systolic blood pressure compared to the SHR controls. The spironolactone treatment group demonstrated significant increases in heart rate and cardiac output and a decrease in cardiac index compared to SHR controls. Any aldosterone blockade in SHR protected against the increased cardiac mass. Similar to clinical echocardiographic observations, hypertension in rats results in left ventricular hypertrophy (LVH) and diastolic dysfunction and aldosterone receptor blockade reduces LVH in SHR.

Quantitative Proteomics Identifies Activation of Hallmark Pathways of Cancer in Patient Melanoma.

Molecular pathways regulating melanoma initiation and progression are potential targets of therapeutic development for this aggressive cancer. Identification and molecular analysis of these pathways in patients has been primarily restricted to targeted studies on individual proteins. Here, we report the most comprehensive analysis of formalin-fixed paraffin-embedded human melanoma tissues using quantitative proteomics. From 61 patient samples, we identified 171 proteins varying in abundance among benign nevi, primary melanoma, and metastatic melanoma. Seventy-three percent of these proteins were validated by immunohistochemistry staining of malignant melanoma tissues from the Human Protein Atlas database. Our results reveal that molecular pathways involved with tumor cell proliferation, motility, and apoptosis are mis-regulated in melanoma. These data provide the most comprehensive proteome resource on patient melanoma and reveal insight into the molecular mechanisms driving melanoma progression.

Anthrax lethal toxin induces acute diastolic dysfunction in rats through disruption of the phospholamban signaling network.

BACKGROUND: Anthrax lethal toxin (LT), secreted by Bacillus anthracis, causes severe cardiac dysfunction by unknown mechanisms. LT specifically cleaves the docking domains of MAPKK (MEKs); thus, we hypothesized that LT directly impairs cardiac function through dysregulation of MAPK signaling mechanisms. METHODS AND RESULTS: In a time-course study of LT toxicity, echocardiography revealed acute diastolic heart failure accompanied by pulmonary regurgitation and left atrial dilation in adult Sprague-Dawley rats at time points corresponding to dysregulated JNK, phospholamban (PLB) and protein phosphatase 2A (PP2A) myocardial signaling. Using isolated rat ventricular myocytes, we identified the MEK7-JNK1-PP2A-PLB signaling axis to be important for regulation of intracellular calcium (Ca(2+)(i)) handling, PP2A activation and targeting of PP2A-B56α to Ca(2+)(i) handling proteins, such as PLB. Through a combination of gain-of-function and loss-of-function studies, we demonstrated that over-expression of MEK7 protects against LT-induced PP2A activation and Ca(2+)(i) dysregulation through activation of JNK1. Moreover, targeted phosphorylation of PLB-Thr(17) by Akt improved sarcoplasmic reticulum Ca(2+)(i) release and reuptake during LT toxicity. Co-immunoprecipitation experiments further revealed the pivotal role of MEK7-JNK-Akt complex formation for phosphorylation of PLB-Thr(17) during acute LT toxicity. CONCLUSIONS: Our findings support a cardiogenic mechanism of LT-induced diastolic dysfunction, by which LT disrupts JNK1 signaling and results in Ca(2+)(i) dysregulation through diminished phosphorylation of PLB by Akt and increased dephosphorylation of PLB by PP2A. Integration of the MEK7-JNK1 signaling module with Akt represents an important stress-activated signalosome that may confer protection to sustain cardiac contractility and maintain normal levels of Ca(2+)(i) through PLB-T(17) phosphorylation.

A Proteomic Study of Human Merkel Cell Carcinoma.

Merkel Cell Carcinoma (MCC) is an aggressive neuroendocrine cancer of the skin. The incidence has been quadrupled with a 5-year mortality rate of 46%, presently there is no cure for metastatic disease. Despite the contribution of Merkel cell polyomavirus, the molecular events of MCC carcinogenesis are poorly defined. To better understand MCC carcinogensis, we have performed the first quantitative proteomic comparison of formalin-fixed, paraffin-embedded (FFPE) MCC tissues using another neuroendocrine tumor (carcinoid tumor of the lung) as controls. Bioinformatic analysis of the proteomic data has revealed that MCCs carry distinct protein expression patterns. Further analysis of significantly over-expressed proteins suggested the involvement of MAPK, PI3K/Akt/mTOR, wnt, and apoptosis signaling pathways. Our previous study and that from others have shown mTOR activation in MCCs. Therefore, we have focused on two downstream molecules of the mTOR pathway, lactate dehydrogenase B (LDHB) and heterogeneous ribonucleoprotein F (hnRNPF). We confirm over-expression of LDHB and hnRNPF in two primary human MCC cell lines, 16 fresh tumors, and in the majority of 80 tissue microarray samples. Moreover, mTOR inhibition suppresses LDHB and hnRNPF expression in MCC cells. The results of the current study provide insight into MCC carcinogenesis and provide rationale for mTOR inhibition in pre-clinical studies.

Norepinephrine induces systolic failure and inhibits antiapoptotic genes in a polymicrobial septic rat model.

AIMS: We examined the effect of norepinephrine (NE) infusion on left ventricular function and apoptotic genes during progression of polymicrobial sepsis. METHODS: Male Sprague-Dawley rats (350-400 g) were made septic by intraperitoneal (i.p.) administration of 200mg/kg cecal inoculum. Sham animals received 5% dextrose water, i.p. Echocardiography was performed at baseline, 3 days and 7 days post-sepsis/sham. NE (0.6 µgkg(-1)h(-1)) was infused for 2h, before the end of day 3 of echocardiography. At the end of day 7, rats were euthanized and heart tissues harvested for isolation of total RNA. PCR was performed using RT(2) profiler™ PCR array PARN-012 (Rat apoptosis array; SuperArray, MD) using RT(2) Real-Time™ SYBR Green PCR master mix PA-012. KEY FINDINGS: NE-infusion resulted in a significant decrease in the left ventricular ejection fraction (EF) (62.56±2.07 from the baseline 71.11±3.23, p<0.05) and fractional shortening (FS) (39.90±2.64 from the sham group 54.41±2.19, p<0.05) at 7 days post-sepsis, respectively. Super Array data revealed that during sepsis, tumor necrosis factor (TNF-α) (2.85±0.07 fold, p<0.0001), anti-apoptotic molecules, Prok2 (16.07±0.48 fold, p<0.0001) and interleukin-10 (IL-10) (23.5±0.57 fold, p<0.0001) were up regulated at day 1. At 7-days post-sepsis, CD40l g (2.49±0.54 fold, p<0.08) and Birc1b (17.8±0.58 fold, p<0.0001) were up regulated compared to the sham, 1 and 3-days post-sepsis groups. SIGNIFICANCE: The data suggest that upregulation of a series of pro-apoptotic molecules could be responsible for systolic and diastolic dysfunction during 3 and 7 days post sepsis.

Enhanced external counterpulsation is a regenerative therapy.

Enhanced external counterpulsation (EECP) is used for the treatment of severe angina and heart failure in patients who are not candidates for revascularization. The clinical benefits of EECP extend well beyond the time period of any hemodynamic effects, but the cause of this prolonged effect is not understood. The prolonged clinical benefits suggest EECP could be a regenerative therapy. This study was performed to determine whether EECP increased circulating hematopoietic progenitor cells (HPCs) or endothelial progenitor cells (EPCs) and thus be a possible regenerative therapy. The proposed mechanism of the increase in regenerative circulating stem cells is the enhanced shear forces induced on the endothelial boundary by the flow reversal produced by the sequential inflation of the pneumatic cuffs during EECP therapy.

Integrins and proximal signaling mechanisms in cardiovascular disease.

Integrins are heterodimeric cell-surface molecules, which act as the principle mediators of molecular dialog between a cell and its extracellular matrix environment. In addition to their structural functions, integrins mediate signaling from the extracellular space into the cell through integrin-associated signaling and adaptor molecules such as FAK (focal adhesion kinase), ILK (integrin-linked kinase), PINCH (particularly interesting new cysteine-histidine rich protein) and Nck2 (non-catalytic (region of) tyrosine kinase adaptor protein-2). Via these molecules, integrin signaling tightly and cooperatively interacts with receptor tyrosine kinases (RTKs) signaling to regulate survival, proliferation and cell shape as well as polarity, adhesion, migration and differentiation. In the heart and blood vessels, the function and regulation of these molecules can be partially disturbed and thus contribute to cardiovascular diseases such as cardiac hypertrophy and atherosclerosis. In this review, we discuss the primary mechanisms of action and signaling of integrins in the cardiac and vascular system in normal and pathological states, as well as therapeutic strategies for targeting these systems (1).

Anthrax toxin: pathologic effects on the cardiovascular system.

Anthrax is a disease caused by infection with spores from the bacteria Bacillus anthracis. After entering the body, the spores germinate into bacteria and secrete a toxin that causes local edema and, in systemic infections, cardiovascular collapse and death. The toxin is a tripartite polypeptide, consisting of protective antigen (PA), lethal factor (LF) and edema factor (EF), which have key roles in the bacterial pathogenesis and disease progression. PA facilitates transfer of LF and EF to the cytosol. Lethal toxin is a zinc metalloproteinase, which has the capacity to inactivate mitogen-activated protein (MAP) kinase kinase (MEK) and stimulates the release of sepsis-related cytokines tumor necrosis factor-alpha and interleukin-1beta. Edema factor is a calmodulin (CaM)-dependent adenylate cyclase, which increases levels of cyclic AMP, causing impaired neutrophil function and disruption of water balance that ultimately results in massive tissue edema. Together, the toxins effectively inhibit host innate and adaptive immune responses, allowing the bacteria to grow unrestrained and overwhelming any resistance. Clinically, inhalational anthrax presents in a biphasic pattern with initial nonspecific "flu-like" symptoms nausea and vomiting 1 to 4 days after exposure, followed by severe illness with dyspnea, high fever and circulatory shock. The latter symptoms represent a terminal stage and treatment is often ineffective when started at that time. Key indicators of early anthrax cardiovascular-related pathogenesis include mediastinal widening in association with pleural effusion and edema. In this review, we describe the current understanding of anthrax toxins on cellular function in the context of cardiovascular function and discuss potential therapeutic strategies.

Guidewire pacing safely and effectively treats bradyarrhythmias induced by rheolytic thrombectomy and precludes the need for transvenous pacing: the Scott & White experience.

Rheolytic thrombectomy (RT) is useful in certain percutaneous coronary interventions but may be associated with transient bradyarrhythmias. Clinicians have devised numerous strategies to deal with these arrhythmias apart from transvenous right ventricular pacing, some of which are described in other parts of this supplement. We report the Scott & White experience utilizing guidewire pacing to quickly and safely pace the heart in the event of bradyarrhythmia. We found this method to be safe and reliable (96.2% successful) during RT and now use this technique almost exclusively in the cardiac catheterization lab to deal with transient bradyarrhythmias during RT or due to any other cause.We also report an increased incidence of bradyarrhythmia occurring during RT when it is performed in the right coronary artery, with a trend toward an increased incidence during the clinical presentation of ST-elevation myocardial infarction.

Integrins: novel therapeutic targets for cardiovascular diseases.

Integrins are the principle mediators of molecular dialog between a cell and its extracellular matrix environment. The unique combinations of integrin subunits determine which extracellular matrix molecules are recognized by a cell. Recent studies have demonstrated that remodeling in heart and vasculature is linked to alterations in extracellular matrix and integrin expression. The roles of integrins in controlling cellular behavior have made these molecules highly attractive drug targets. New insights into mechanisms whereby the extracellular matrix takes part in the control of smooth muscle cell proliferation and cardiac growth suggest a number of putative targets for future therapies that can be applied to increase plaque stability, prevent the clinical consequences of atherosclerosis and improve outcomes after interventional procedures such as cardiac transplantation. Therapeutic candidates include antibodies, cyclic peptides, peptidomimetics and small molecules. The integrin inhibitors Integrilin and ReoPro have been approved as blood thinners in cardiovascular disease, and newer agents are undergoing testing. Although integrin function is important in the cardiovascular system, there are wide gaps in knowledge. In this review, we discuss the primary mechanisms of action and signaling of integrins in the cardiac and vascular system in normal and pathological states, as well as therapeutic strategies for targeting these molecules in the cardiovascular system.