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Coronary microvascular dysfunction (CMD) is associated with cardiac dysfunction and predictive of cardiac mortality in obesity, especially in females. Clinical data further support that CMD associates with development of heart failure with preserved ejection fraction and that mineralocorticoid receptor (MR) antagonism may be more efficacious in obese female, versus male, HFpEF patients. Accordingly, we examined the impact of smooth muscle cell (SMC)-specific MR deletion on obesity-associated coronary and cardiac diastolic dysfunction in female mice. Obesity was induced in female mice via western diet (WD) feeding alongside littermates fed standard diet. Global MR blockade with spironolactone prevented coronary and cardiac dysfunction in obese females and specific deletion of SMC-MR was sufficient to prevent obesity-associated coronary and cardiac diastolic dysfunction. Cardiac gene expression profiling suggested reduced cardiac inflammation in WD-fed mice with SMC-MR deletion independent of blood pressure, aortic stiffening, and cardiac hypertrophy. Further mechanistic studies utilizing single-cell RNA sequencing of non-cardiomyocyte cell populations revealed novel impacts of SMC-MR deletion on the cardiac cellulome in obese mice. Specifically, WD feeding induced inflammatory gene signatures in non-myocyte populations including B/T cells, macrophages, and endothelium as well as increased coronary VCAM-1 protein expression, independent of cardiac fibrosis, that was prevented by SMC-MR deletion. Further, SMC-MR deletion induced a basal reduction in cardiac mast cells and prevented WD-induced cardiac pro-inflammatory chemokine expression and leukocyte recruitment. These data reveal a central role for SMC-MR signaling in obesity-associated coronary and cardiac dysfunction, thus supporting the emerging paradigm of a vascular origin of cardiac dysfunction in obesity.

Coronary microvascular dysfunction (CMD) is associated with cardiac dysfunction and predictive of cardiac mortality in obesity, especially in females. Emerging evidence suggests development of heart failure with preserved ejection fraction in females with CMD and that mineralocorticoid receptor (MR) antagonism may be more efficacious in obese female, versus male, HFpEF patients. Accordingly, we examined the hypothesis that smooth muscle cell (SMC)-specific MR deletion prevents obesity-associated coronary and cardiac diastolic dysfunction in females. Obesity was induced in female mice via western diet (WD) feeding alongside littermates fed standard diet. Initial studies revealed that global MR blockade with spironolactone prevented impaired coronary vasodilation and diastolic dysfunction in obese females. Importantly, specific deletion of SMC-MR similarly prevented obesity-associated coronary and cardiac dysfunction. Cardiac gene expression profiling suggested reduced cardiac inflammation in WD-fed mice with SMC-MR deletion independent of blood pressure, aortic stiffening, and cardiac hypertrophy. Further mechanistic studies utilizing single-cell RNA sequencing of non-cardiomyocyte cell populations revealed novel impacts of SMC-MR deletion on the cardiac cellulome in obese mice. Specifically, WD feeding induced inflammatory gene signatures in multiple non-myocyte populations (B/T cells, macrophages, and endothelium), independent of cardiac fibrosis, that was prevented by SMC-MR deletion. Further, SMC-MR deletion induced a basal reduction in cardiac mast cells and prevented WD-induced cardiac pro-inflammatory chemokine expression and leukocyte recruitment. These data reveal a central role for SMC-MR signaling in obesity-associated coronary and cardiac dysfunction thus supporting the emerging paradigm of a vascular origin of cardiac dysfunction in obesity.

Human exposure to carbon black (CB) is inevitable due to its widespread applications in the medical, industrial and consumer sectors. With an ageing population, it is imperative that the effects of (nano)particle exposure in individuals with compromised immunity or infection are considered. Since barrier immunity provides the first line of defence against CB and the human skin and lung pathogen, Staphylococcus aureus, this work focuses on studying the impact of CB exposure upon compromised immunity during infection on human skin and lung epithelial cells in vitro. The principal aim of the work was to develop an epithelial cell model to characterise (co-)exposure to CB and S. aureus. The work used two human epithelial cell lines, HaCaT (skin) and A549 (lung), ELISA technology to assess the (pro-)inflammatory response, aseptic microbiology techniques to grow S. aureus and a Zetasizer, EDX spectroscopy, and both scanning and transmission electron microscopy (SEM and TEM) to characterise the CB under the conditions used in the study. Physicochemical characterisation of CB confirmed its shape, dramatic polydispersity and potential to aggregate. CB significantly inhibited S. aureus growth, but in a biological media dependent manner. CB did not induce cytokines or antimicrobial peptides from skin and lung epithelial cells, when given alone. In contrast, S. aureus induced a robust interleukin (IL)-8 response in both skin and lung epithelial cells. IL-6 and human beta defensin (hβD)-2 could only be detected when cells were stimulated with S. aureus. However, co-exposure to CB (100µg/ml) and S. aureus resulted in significant inhibition of IL-8 (compared to S. aureus only induced levels). Furthermore, the same co-exposure induced significantly more hβD-2 (compared to S. aureus alone). The ability to detect pathogen responses to particle, in addition to epithelial responses to particle and pathogen is an advance on assessing cell responses under ‘healthy’ conditions and supports the need for developing exposure models under stressed or immunosuppressed conditions. This model will be useful for studying mechanisms of exposure in at-risk groups, including factory workers, the elderly and immunocompromised. Advanced models, that better represent human pathophysiology are essential for understanding cellular mechanisms of toxicity in the 21st Century.

Impaired coronary microvascular function (e.g., reduced dilation and coronary flow reserve) predicts cardiac mortality in obesity, yet underlying mechanisms and potential therapeutic strategies remain poorly understood. Mineralocorticoid receptor (MR) antagonism improves coronary microvascular function in obese humans and animals. Whether MR blockade improves in vivo regulation of coronary flow, a process involving voltage-dependent K+ (Kv) channel activation, or reduces coronary structural remodeling in obesity is unclear. Thus, the goals of this investigation were to determine the effects of obesity on coronary responsiveness to reductions in arterial PO2 and potential involvement of Kv channels and whether the benefit of MR blockade involves improved coronary Kv function or altered passive structural properties of the coronary microcirculation. Hypoxemia increased coronary blood flow similarly in lean and obese swine; however, baseline coronary vascular resistance was significantly higher in obese swine. Inhibition of Kv channels reduced coronary blood flow and augmented coronary resistance under baseline conditions in lean but not obese swine and had no impact on hypoxemic coronary vasodilation. Chronic MR inhibition in obese swine normalized baseline coronary resistance, did not influence hypoxemic coronary vasodilation, and did not restore coronary Kv function (assessed in vivo, ex vivo, and via patch clamping). Lastly, MR blockade prevented obesity-associated coronary arteriolar stiffening independent of cardiac capillary density and changes in cardiac function. These data indicate that chronic MR inhibition prevents increased coronary resistance in obesity independent of Kv channel function and is associated with mitigation of obesity-mediated coronary arteriolar stiffening.

Impaired coronary microvascular function (e.g., reduced dilation and coronary flow reserve) predicts cardiac mortality in obesity, yet underlying mechanisms and potential therapeutic strategies remain poorly understood. Mineralocorticoid receptor (MR) antagonism improves coronary microvascular function in obese humans and animals. Whether MR blockade improves in vivo regulation of coronary flow, a process involving voltage-dependent K(+) (K(v)) channel activation, or reduces coronary structural remodeling in obesity is unclear. Thus, the goals of this investigation were to determine the effects of obesity on coronary responsiveness to reductions in arterial PO(2) and potential involvement of K(v) channels and whether the benefit of MR blockade involves improved coronary K(v) function or altered passive structural properties of the coronary microcirculation. Hypoxemia increased coronary blood flow similarly in lean and obese swine, however baseline coronary vascular resistance was significantly higher in obese swine. Inhibition of K(v) channels reduced coronary blood flow and augmented coronary resistance under baseline conditions in lean but not obese swine and had no impact on hypoxemic coronary vasodilation. Chronic MR inhibition in obese swine normalized baseline coronary resistance, did not influence hypoxemic coronary vasodilation, and did not restore coronary K(v) function (assessed in vivo, ex vivo, and via patch clamping). Lastly, MR blockade prevented obesity-associated coronary arteriolar stiffening independent of cardiac capillary density and changes in cardiac function. These data indicate that chronic MR inhibition prevents increased coronary resistance in obesity independent of K(v) channel function and is associated with mitigation of obesity-mediated coronary arteriolar stiffening.

Elevated plasma aldosterone (Aldo) levels are associated with greater risk of cardiac ischemic events and cardiovascular mortality. Adenosine-mediated coronary vasodilation is a critical cardioprotective mechanism during ischemia; however, whether this response is impaired by increased Aldo is unclear. We hypothesized that chronic Aldo impairs coronary adenosine-mediated vasodilation via downregulation of vascular K(+) channels. Male C57BL/6J mice were treated with vehicle (Con) or subpressor Aldo for 4 wk. Coronary artery function, assessed by wire myography, revealed Aldo-induced reductions in vasodilation to adenosine and the endothelium-dependent vasodilator acetylcholine but not to the nitric oxide donor sodium nitroprusside. Coronary vasoconstriction to endothelin-1 and the thromboxane A(2) mimetic U-46619 was unchanged by Aldo. Additional mechanistic studies revealed impaired adenosine A(2A), not A(2B), receptor-dependent vasodilation by Aldo with a tendency for Aldo-induced reduction of coronary A(2A) gene expression. Adenylate cyclase inhibition attenuated coronary adenosine dilation but did not eliminate group differences, and adenosine-stimulated vascular cAMP production was similar between Con and Aldo mice. Similarly, blockade of inward rectifier K(+) channels reduced but did not eliminate group differences in adenosine dilation whereas group differences were eliminated by blockade of Ca(2+)-activated K(+) (K(Ca)) channels that blunted and abrogated adenosine and A(2A)-dependent dilation, respectively. Gene expression of several coronary K(Ca) channels was reduced by Aldo. Together, these data demonstrate Aldo-induced impairment of adenosine-mediated coronary vasodilation involving blunted A(2A)-K(Ca)-dependent vasodilation, independent of blood pressure, providing important insights into the link between plasma Aldo and cardiac mortality and rationale for aldosterone antagonist use to preserve coronary microvascular function. NEW & NOTEWORTHY Increased plasma aldosterone levels are associated with worsened cardiac outcomes in diverse patient groups by unclear mechanisms. We identified that, in male mice, elevated aldosterone impairs coronary adenosine-mediated vasodilation, an important cardioprotective mechanism. This aldosterone-induced impairment involves reduced adenosine A(2A), not A(2B), receptor-dependent vasodilation associated with downregulation of coronary K(Ca) channels and does not involve altered adenylate cyclase/cAMP signaling. Importantly, this effect of aldosterone occurred independent of changes in coronary vasoconstrictor responsiveness and blood pressure. Listen to this article’s corresponding podcast at https://ajpheart.podbean.com/e/attenuation-of-coronary-adenosine-dilation-by-aldosterone/.

Prevalence studies of Borrelia burgdorferi and Anaplasma phagocytophilum have been rare for ticks from southwestern Pennsylvania. We collected 325 Ixodes scapularis ticks between 2011 and 2012 from four counties in southwestern Pennsylvania. We tested for the presence of Borrelia burgdorferi and Anaplasma phagocytophilum using PCR. Of the ticks collected from Pennsylvania, B. burgdorferi (causative agent of Lyme disease) was present in 114/325 (35%) and Anaplasma phagocytophilum (causative agent of Human Granulocytic Anaplasmosis) was present in 48/325 (15%) as determined by PCR analysis.

Costimulatory ligands, B7.1 and B7.2, have been incorporated into viral and DNA vectors as potential nonchemical adjuvants to enhance CTL and humoral immune responses against viral pathogens. In addition, soluble B7 proteins, minus their transmembrane and cytoplasmic domains, have been shown to block the down regulation of T-cell activation through blockade of B7/CTLA-4 interactions in mouse tumor models. Recently, we developed swinepox virus (SPV) vectors for delivery of feline leukemia antigens for vaccine use in cats [Winslow, B.J., Cochran, M.D., Holzenburg, A., Sun, J., Junker, D.E., Collisson, E.W., 2003. Replication and expression of a swinepox virus vector delivering feline leukemia virus Gag and Env to cell lines of swine and feline origin. Virus Res. 98, 1–15]. To explore the use of feline B7.1 and B7.2 ligands as nonchemical adjuvants, SPV vectors containing full-length feline B7.1 and B7.2 ligands were constructed and analyzed. Full-length feline B7.1 and B7.2 produced from SPV vectors were natively processed and costimulated Jurkat cells to produce IL-2, in vitro. In addition, we explored the feasibility of utilizing SPV as a novel expression vector to produce soluble forms of feline B7.1 (sB7.1) and B7.2 (sB7.2) in tissue culture. The transmembrane and cytoplasmic regions of the B7.1 and B7.2 genes were replaced with a poly-histidine tag and purified via a two-step chromatography procedure. Receptor binding and costimulation activity was measured. Although feline sB7.1-his and sB7.2-his proteins bound to the human homolog receptors, CTLA-4 and CD28, both soluble ligands possessed greater affinity for CTLA-4, compared to CD28. However, both retained the ability to partially block CD28-mediated costimulation in vitro. Results from these studies establish the use of SPV as a mammalian expression vector and suggest that full-length-vectored and purified soluble feline B7 ligands may be valuable, nonchemical immune-modulators.

In order for vacuum-assisted venous return (VAVR) to be used safely and efficiently during cardiopulmonary bypass (CPB), a full understanding of venous return is necessary. The focus of this work was to use the concepts of energy conservation and viscous energy dissipation in the development of a theoretical model of venous return utilizing vacuum assist. The effectiveness and accuracy of this model has been verified through in vitro laboratory investigations and statistical analysis. Although VAVR can provide higher flows through smaller venous cannula, vacuum assist may lead to increased levels of wall shear stress as shown in this work. The clinical implications of VAVR have yet to be investigated, but may lead to an exacerbation of the detrimental effects of CPB during cardiac surgery.

This paper highlights some of the intricacies and complexities associated with patent drafting, particularly in unpredictable sciences such as biotechnology. The paper draws on the new guidelines issued by the Patent Office regarding compliance with the 'utility' and 'written description' requirements of the Patent Statutes to illustrate some of the thorny issues applicants must thoughtfully consider during drafting as well as the pitfalls applicants will encounter when failing to do so. The paper then reviews the recent decision in Festo Corp. v Shoketsu Kinzoku Kogyo Kabushiki Co, Ltd to underscore how a lack of forethought can result in the issuance of claims that provide a compromised scope of protection.

The current state of user interfaces for large information spaces imposes an unmanageable cognitive burden upon the user. Determining how to get the right information into the right form with the right tool at the right time has become a monumental task. Interface agents address the problem of increasing task load by serving as either an assistant or associate, extracting and analyzing relevant information, providing information abstractions of that information, and providing timely, beneficial assistance to suers. Interface agents communicate with the user through the existing user interface and also adapt to user needs and behaviors. User modeling, on the other hand, is concerned with how to represent users' knowledge and interaction within a system to adapt the system to the needs of users. The inclusion of a user model within the overall system architecture allows the system to adapt its response to the preferences, biases, expertise level, goals and needs.

Knowledge elicitation/acquisition continues to be a bottleneck to constructing, decision-theoretic systems. Methodologies and techniques for incremental elicitation/acquisition of knowledge especially under uncertainty in support of users' current goals is desirable. This paper presents PESKI, a probabilistic expert system development environment. PESKI provides users with a highly interactive and integrated suite of intelligent knowledge engineering tools for decision-theoretic systems. From knowledge acquisition, data mining, and verification and validation to a distributed inference engine for querying knowledge, PESKI is based on the concept of active user interfaces actuators to the human-machine interface. PESKI uses a number of techniques to reduce the inherent complexity of developing a cohesive, real-world knowledge-based system. This is accomplished by providing multiple communication modes for human-computer interaction and the use of a knowledge representation endowed with the ability to detect problems with the knowledge acquired and alert the user to these possible problems. We discuss PESKI's use of these intelligent assistants to help users with the acquisition of knowledge especially in the presence of uncertainty.

This paper describes two forms of information visualization for mixed-initiative systems associated with team collaboration and begins to discuss how plans might be formulated to achieve the visualizations. Common understanding visualization is concerned with visualizing the information a team employs, whereas visual collaboration is concerned with visualizing the ongoing, incremental information collection, the credibility and origins of that information, and the dynamic interpersonal relationships of the team itself. The first is the more "classic" form of visualization where data and information is collected, analyzed, abstracted, and tailored for display to the user. We are concerned not only with visualization for the single user, but also with visualizing the relationship the information holds in regard to the entire team. At the level of the individual user, a mixed-initiative system must consider how to tailor the appropriate information given the user's skill, expertise, and preferences. At the corporate level, a system must manage the display of information across multiple human users and system components that share a common goal. The second form of visualization deals with the collaboration between a user and his/her mixed-initiative system, between users, and between systems. That is, the user needs to understand and visualize the collaboration, how the user fits into it, and the associated human/information interaction and processes involved in human/information interaction and processes involved in such collaboration. We claim both types of visualization are important for effective collaboration.

An underlying problem of current interface agent research is the failure to adequately address effective and efficient knowledge representations and associated methodologies suitable for modeling the users' interactions with the system. These user models lack the representational complexity to manage the uncertainty and dynamics involved in predicting user intent and modeling user behavior. A utility theory-based approach is presented for effective user intent prediction by incorporating the ability to explicitly model users' goals, the uncertainty in the users' intent in pursuing these goals, and the dynamics of users' behavior. We present an interface agent architecture, CIaA, that incorporates our approach and discuss the integration of CIaA with three disparate domains — a probabilistic expert system shell, a natural language input database query system, and a virtual space plane —that are being used as test beds for our interface agent research.

A phage-neutralizing rabbit antiserum collected after immunization with tail-fiberless bacteriophage T4 particles was adsorbed with complete T4 phage. The resulting adsorbed serum inhibited tail fiber attachment in vitro. To identify the antigens against which this inhibitory activity was directed, blocking experiments were carried out with the adsorbed serum. Isolated complete baseplates and mutant-infected-cell extracts lacking known baseplate gene products but containing gene 9 product showed similar high levels of blocking activity. By contrast, both tail-fiberless particles lacking gene 9 product and infected-cell extracts made with gene 9 mutants showed 30-fold to 100-fold lower blocking activity. These results strongly support the conclusion that gene 9 product is the baseplate protein to which tail fibers attach.

Evidence is presented that p40 affects the solubility of p20 inside the T4-infected cell. In the absence of p20 and p40 function at high temperature, p20 is located specifically and exclusively in the cell envelope. It is possible that p40 may interact with p20 before head assembly can be initiated. Cleavage of the major head protein in gene 40 mutant-infected cells is also inhibited. In addition to producing single-layered polyheads and phage at 30°, am restrictive cells infected with gene 40 am mutants produce some empty capsids. It is likely that p40 function is only strictly required for phage growth at high temperature. Electron microscopy of thin sections of crude cell envelopes showed few recognizable structural components, although most phage structural proteins were present upon sodium dodecyl sulfate-gel electrophoresis of envelope preparations. Extraction with sodium chloride selectively removes some of the viral proteins from the crude cell envelope preparation, while extraction with 4 M guanidine · HCl greatly reduces the amount of T4 protein in the cell envelopes. The envelope association of most of these proteins appears to be nonspecific.