Search

The roseolovirus U20 proteins are virus-encoded integral membrane glycoproteins possessing class I major histocompatibility complex (MHC)-like folds. Surprisingly, although U20 proteins from HHV-6A and -6B share 92% identity, recent studies ascribe different functions to HHV6A U20 and HHV6B U20.

Despite recent advances in our understanding of pathogenic access to the central nervous system (CNS), the mechanisms by which intracellular pathogens disseminate within the dense cellular network of neural tissue remain poorly understood. To address this issue, longitudinal analysis of Toxoplasma gondii dissemination in the brain was conducted using 2-photon imaging through a cranial window in living mice that transgenically express enhanced green fluorescent protein (eGFP)-claudin-5. Extracellular T. gondii parasites were observed migrating slowly (1.37 ± 1.28 μm/min) and with low displacement within the brain. In contrast, a population of highly motile infected cells transported vacuoles of T. gondii significantly faster (6.30 ± 3.09 μm/min) and with a higher displacement than free parasites. Detailed analysis of microglial dynamics using CX3CR1-GFP mice revealed that T. gondii-infected microglia remained stationary, and infection did not increase the extension/retraction of microglial processes. The role of infiltrating immune cells in shuttling T. gondii was examined by labeling of peripheral hematopoietic cells with anti-CD45 antibody. Infected CD45+ cells were found crawling along the CNS vessel walls and trafficked T. gondii within the brain parenchyma at significantly higher speeds (3.35 ± 1.70 μm/min) than extracellular tachyzoites. Collectively, these findings highlight a dual role for immune cells in neuroprotection and in facilitating parasite dissemination within the brain. IMPORTANCE T. gondii is a foodborne parasite that infects the brain and can cause fatal encephalitis in immunocompromised individuals. However, there is a limited understanding of how the parasites disseminate through the brain and evade immune clearance. We utilized intravital imaging to visualize extracellular T. gondii tachyzoites and infected cells migrating within the infected mouse brain during acute infection. The infection of motile immune cells infiltrating the brain from the periphery significantly increased the dissemination of T. gondii in the brain compared to that of free parasites migrating using their own motility: the speed and displacement of these infected cells would enable them to cover nearly 1 cm of distance per day! Among the infiltrating cells, T. gondii predominantly infected monocytes and CD8+ T cells, indicating that the parasite can hijack immune cells that are critical for controlling the infection in order to enhance their dissemination within the brain.

Roseoloviruses (HHV-6A, -6B, and -7) infect >90% of the human population during early childhood, and are thought to remain latent or persistent throughout the life of the host. As such,these viruses are among the most pervasive and stealthy of all viruses;they must necessarily excel at escaping immune detection throughout the life of the host, and yet very little is known about how these viruses so successfully escape host defenses. Herein, we characterize the HHV6A and HHV6B U20 gene products, which are encoded within a block of genes unique to the roseoloviruses, and therefore of particular interest. Despite 92% amino acid identity, U20 proteins from HHV6A and 6B have been shown to possess different host evasion functions. Here we characterize expression, trafficking, and post-translational modifications of U20 during HHV6A infection. While U20 localized to lysosomes in HHV-6A-infected cells, HHV-6B U20 trafficked to the cell surface and was rapidly internalized. HHV-6B U20 trafficked slowly through the secretory system, receiving several post translational modifications to its N-linked glycans indicative of surface expressed glycoproteins. Interestingly, U20 is also phosphorylated on at least one Ser, Thr, or Tyr residue. These results provide a framework to understand the role(s) of U20 in evading host defenses.ImportanceHHV6A and HHV6B U20 proteins are virus-encoded integral membrane glycoproteins possessing class I MHC-like folds. As such, it is tempting to speculate that they are involved in host evasion. Indeed, although they share 92% identity, HHV6A U20 has been shown to target NK activating ligands (1) and HHV6B U20 has been shown to inhibit TNF receptor signaling and apoptosis (2). Here, we have performed cell biological and biochemical characterization of the trafficking, glycosylation, and post-translational modifications occurring on HHV6A and -6B U20, and we demonstrate U20 expression in the context of HHV6 infection.

Bio-engineered myocardium has great potential to substitute damaged myocardium and for studies of myocardial physiology and disease, but structural and functional immaturity still implies limitations. Current protocols of engineered heart tissue (EHT) generation fall short of simulating the conditions of postnatal myocardial growth, which are characterized by tissue expansion and increased mechanical load. To investigate whether these two parameters can improve EHT maturation, we developed a new approach for the generation of cardiac tissues based on biomimetic stimulation under application of continuously increasing stretch. Methods: EHTs were generated by assembling cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CM) at high cell density in a low collagen hydrogel. Maturation and growth of the EHTs were induced in a custom-made biomimetic tissue culture system that provided continuous electrical stimulation and medium agitation along with progressive stretch at four different increments. Tissues were characterized after a three week conditioning period. Results: The highest rate of stretch (S3 = 0.32 mm/day) increased force development by 5.1-fold compared to tissue with a fixed length, reaching contractility of 11.28 mN/mm². Importantly, intensely stretched EHTs developed physiological length-dependencies of active and passive forces (systolic/diastolic ratio = 9.47 ± 0.84), and a positive force-frequency relationship (1.25-fold contractility at 180 min-1). Functional markers of stretch-dependent maturation included enhanced and more rapid Ca2+ transients, higher amplitude and upstroke velocity of action potentials, and pronounced adrenergic responses. Stretch conditioned hiPSC-CMs displayed structural improvements in cellular volume, linear alignment, and sarcomere length (2.19 ± 0.1 µm), and an overall upregulation of genes that are specifically expressed in adult cardiomyocytes. Conclusions: With the intention to simulate postnatal heart development, we have established techniques of tissue assembly and biomimetic culture that avoid tissue shrinkage and yield muscle fibers with contractility and compliance approaching the properties of adult myocardium. This study demonstrates that cultivation under progressive stretch is a feasible way to induce growth and maturation of stem cell-derived myocardium. The novel tissue-engineering approach fulfills important requirements of disease modelling and therapeutic tissue replacement.

Human roseolovirus U20 and U21 are type I membrane glycoproteins that have been implicated in immune evasion by interfering with recognition of classical and non-classical MHC proteins. U20 and U21 are predicted to be type I glycoproteins with extracytosolic immunoglobulin-like domains, but detailed structural information is lacking. AlphaFold and RoseTTAfold are next generation machine-learning-based prediction engines that recently have revolutionized the field of computational three-dimensional protein structure prediction. Here, we review the structural biology of viral immunoevasins and the current status of computational structure prediction algorithms. We use these computational tools to generate structural models for U20 and U21 proteins, which are predicted to adopt MHC-Ia-like folds with closed MHC platforms and immunoglobulin-like domains. We evaluate these structural models and place them within current understanding of the structural basis for viral immune evasion of T cell and natural killer cell recognition.

Supplemental Digital Content is available in the text., Background: Complex molecular programs in specific cell lineages govern human heart development. Hypoplastic left heart syndrome (HLHS) is the most common and severe manifestation within the spectrum of left ventricular outflow tract obstruction defects occurring in association with ventricular hypoplasia. The pathogenesis of HLHS is unknown, but hemodynamic disturbances are assumed to play a prominent role. Methods: To identify perturbations in gene programs controlling ventricular muscle lineage development in HLHS, we performed whole-exome sequencing of 87 HLHS parent–offspring trios, nuclear transcriptomics of cardiomyocytes from ventricles of 4 patients with HLHS and 15 controls at different stages of heart development, single cell RNA sequencing, and 3D modeling in induced pluripotent stem cells from 3 patients with HLHS and 3 controls. Results: Gene set enrichment and protein network analyses of damaging de novo mutations and dysregulated genes from ventricles of patients with HLHS suggested alterations in specific gene programs and cellular processes critical during fetal ventricular cardiogenesis, including cell cycle and cardiomyocyte maturation. Single-cell and 3D modeling with induced pluripotent stem cells demonstrated intrinsic defects in the cell cycle/unfolded protein response/autophagy hub resulting in disrupted differentiation of early cardiac progenitor lineages leading to defective cardiomyocyte subtype differentiation/maturation in HLHS. Premature cell cycle exit of ventricular cardiomyocytes from patients with HLHS prevented normal tissue responses to developmental signals for growth, leading to multinucleation/polyploidy, accumulation of DNA damage, and exacerbated apoptosis, all potential drivers of left ventricular hypoplasia in absence of hemodynamic cues. Conclusions: Our results highlight that despite genetic heterogeneity in HLHS, many mutations converge on sequential cellular processes primarily driving cardiac myogenesis, suggesting novel therapeutic approaches.

SUMMARYHuman heart regeneration is one of the most critical unmet clinical needs at a global level1. Muscular regeneration is hampered both by the limited renewing capacity of adult cardiomyocytes2-4 and the onset of cardiac fibrosis5,6, resulting in reduced compliance of the tissue. Primate have proven to be ideal models for pluripotent stem cell strategies for heart regeneration, but unravelling specific approaches to drive cell migration to the site of injury and inhibition of subsequent fibrosis have been elusive. Herein, by combining human cardiac progenitor lineage tracing and single-cell transcriptomics in injured non-human primate heart bio-mimics, we uncover the coordinated muscular regeneration of the primate heart via directed migration of human ventricular progenitors to sites of injury, subsequent fibroblast repulsion targeting fibrosis, and ultimate functional replacement of damaged cardiac muscle by differentiation and electromechanical integration. Single-cell RNAseq captured distinct modes of action, uncovering chemoattraction mediated by CXCL12/CXCR4 signalling and fibroblast repulsion regulated by SLIT2/ROBO1 guidance in organizing cytoskeletal dynamics. Moreover, transplantation of human cardiac progenitors into hypo-immunogenic CAG-LEA29Y transgenic porcine hearts following injury proved their chemotactic response and their ability to generate a remuscularized scar without the risk of arrhythmogenesis in vivo. Our study demonstrates that inherent developmental programs within cardiac progenitors are sequentially activated in the context of disease, allowing the cells to sense and counteract injury. As such, they may represent an ideal bio-therapeutic for functional heart rejuvenation.

Alzheimer’s Disease (AD) is the leading cause of age-related neurodegeneration and is characterized neuropathologically by the accumulation of insoluble beta-amyloid (Aß) peptides. In AD brains, plaque-associated myeloid (PAM) cells cluster around Aß plaques but fail to effectively clear Aß by phagocytosis. PAM cells were originally thought to be brain-resident microglia. However, several studies have also suggested that Aß-induced inflammation causes peripheral monocytes to enter the otherwise immune-privileged brain. The relationship between AD progression and inflammation in the brain remains ambiguous because microglia and monocyte-derived macrophages are extremely difficult to distinguish from one another in an inflamed brain. Whether PAM cells are microglia, peripheral macrophages, or a mixture of both remains unclear. CD11a is a component of the ß2 integrin LFA1. We have determined that CD11a is highly expressed on peripheral immune cells, including macrophages, but is not expressed by mouse microglia. These expression patterns remain consistent in LPS-treated inflamed mice, as well as in two mouse models of AD. Thus, CD11a can be used as a marker to distinguish murine microglia from infiltrating peripheral immune cells. Using CD11a, we show that PAM cells in AD transgenic brains are comprised entirely of microglia. We also demonstrate a novel Fluorescence-Assisted Quantification Technique (FAQT), which reveals a significant increase in T lymphocytes, especially in the brains of female AD mice. Our findings support the notion that microglia are the lead myeloid players in AD and that rejuvenating their phagocytic potential may be an important therapeutic strategy.

OBJECTIVES We systematically evaluated changes in availability, price, and quality of perishable food items from the beginning to the end of the month in lowincome, urban neighborhoods. METHODS The sample included grocery stores or supermarkets in Cleveland, Ohio, within neighborhoods with >30% of population receiving food assistance. We collected data for 2 sequential months during the first and fourth weeks of each month. Two coders evaluated stores, collecting measures of availability, price, and quality for 50 items. We examined difference in number and proportion of items available at the beginning of the month (BOM) to items remaining available at the end of the month (EOM), as well as quality and price of those items. RESULTS Across 48 stores, availability at EOM was lower than BOM; as store size increased, reduction in availability (ie, food melt) was significantly (p < .01) less pronounced. Overall, items became less expensive at the EOM whereas quality remained consistent; we noted no statistically significant differences by store type for price or quality. CONCLUSIONS Food melt differentially affects individuals in neighborhoods without grocery stores. Findings reveal composition of food environments is dynamic rather than static, influencing food-purchasing choices among lowincome consumers.

IL-1β is produced by myeloid cells and acts as a critical mediator of host defense during infection and injury. We found that the intracellular protozoan parasite Toxoplasma gondii induced an early IL-1β response (within 4 h) in primary human peripheral blood monocytes isolated from healthy donors. This process involved upregulation of IL-1β, IL-1RN (IL-1R antagonist), and NLRP3 transcripts, de novo protein synthesis, and the release of pro- and mature IL-1β from infected primary monocytes. The released pro–IL-1β was cleavable to mature bioactive IL-1β in the extracellular space by the protease caspase-1. Treatment of primary monocytes with the NLRP3 inhibitor MCC950 or with extracellular potassium significantly reduced IL-1β cleavage and release in response to T. gondii infection, without affecting the release of TNF-α, and indicated a role for the inflammasome sensor NLRP3 and for potassium efflux in T. gondii–induced IL-1β production. Interestingly, T. gondii infection did not induce an IL-1β response in primary human macrophages derived from the same blood donors as the monocytes. Consistent with this finding, NLRP3 was downregulated during the differentiation of monocytes to macrophages and was not induced in macrophages during T. gondii infection. To our knowledge, these findings are the first to identify NLRP3 as an inflammasome sensor for T. gondii in primary human peripheral blood cells and to define an upstream regulator of its activation through the release of intracellular potassium.

Brain infection by the parasite Toxoplasma gondii in mice is thought to generate vulnerability to predation by mechanisms that remain elusive. Monocytes play a key role in host defense and inflammation and are critical for controlling T. gondii . However, the dynamic and regional relationship between brain-infiltrating monocytes and parasites is unknown. We report the mobilization of inflammatory (CCR2 + Ly6C hi ) and patrolling (CX3CR1 + Ly6C lo ) monocytes into the blood and brain during T. gondii infection of C57BL/6J and CCR2 RFP/+ CX3CR1 GFP/+ mice. Longitudinal analysis of mice using 2-photon intravital imaging of the brain through cranial windows revealed that CCR2-RFP monocytes were recruited to the blood–brain barrier (BBB) within 2 wk of T. gondii infection, exhibited distinct rolling and crawling behavior, and accumulated within the vessel lumen before entering the parenchyma. Optical clearing of intact T. gondii -infected brains using iDISCO + and light-sheet microscopy enabled global 3D detection of monocytes. Clusters of T. gondii and individual monocytes across the brain were identified using an automated cell segmentation pipeline, and monocytes were found to be significantly correlated with sites of T. gondii clusters. Computational alignment of brains to the Allen annotated reference atlas [E. S. Lein et al., Nature 445:168–176 (2007)] indicated a consistent pattern of monocyte infiltration during T. gondii infection to the olfactory tubercle, in contrast to LPS treatment of mice, which resulted in a diffuse distribution of monocytes across multiple brain regions. These data provide insights into the dynamics of monocyte recruitment to the BBB and the highly regionalized localization of monocytes in the brain during T. gondii CNS infection.

Arthropod-borne viruses, referred to collectively as arboviruses, infect millions of people worldwide each year and have the potential to cause severe disease. They are predominately transmitted to humans through blood-feeding behavior of three main groups of biting arthropods: ticks, mosquitoes, and sandflies. The pathogens harbored by these blood-feeding arthropods (BFA) are transferred to animal hosts through deposition of virus-rich saliva into the skin. Sometimes these infections become systemic and can lead to neuro-invasion and life-threatening viral encephalitis. Factors intrinsic to the arboviral vectors can greatly influence the pathogenicity and virulence of infections, with mounting evidence that BFA saliva and salivary proteins can shift the trajectory of viral infection in the host. This review provides an overview of arbovirus infection and ways in which vectors influence viral pathogenesis. In particular, we focus on how saliva and salivary gland extracts from the three dominant arbovirus vectors impact the trajectory of the cellular immune response to arbovirus infection in the skin.

Since their independent discovery by Frederick Twort in 1915 and Felix d’Herelle in 1917, bacteriophages have captured the attention of scientists for more than a century. They are the most abundant organisms on the planet, often outnumbering their bacterial hosts by tenfold in a given environment, and they constitute a vast reservoir of unexplored genetic information. The increased prevalence of antibiotic resistant pathogens has renewed interest in the use of naturally obtained phages to combat bacterial infections, aka phage therapy. The development of tools to modify phages, genetically or chemically, combined with their structural flexibility, cargo capacity, ease of propagation, and overall safety in humans has opened the door to a myriad of applications. This review article will introduce readers to many of the varied and ingenious ways in which researchers are modifying phages to move them well beyond their innate ability to target and kill bacteria.

Background Recent evidence indicates a widening gap in fruit and vegetable (F/V) consumption between high- and low-income Americans. This gap is related, in part, to decreased access to food retailers that sell fresh F/V in low-income communities. Farmers' markets are identified as a strategy for improving F/V consumption by increasing access to these foods. Objectives The aim of this systematic review was to examine literature published from 1994 to 2014 to identify facilitators and barriers of farmers' markets use, particularly among low-income consumers. Design Peer-reviewed literature was identified in Ebsco Host (Academic Search Complete). Inclusion criteria for abstract review was primary research focused on farmers' market use identifying 87 studies for full-text review. Full-text review identified articles focused on facilitators and/or barriers of farmers' market use resulting in 49 articles. At least two reviewers completed review of all articles. Results Of the 49 articles, 39% specified inclusion of low-income consumers and fewer than 15% focused on racial and ethnic minorities. Few studies were guided by theory and/or used standardized metrics. Results indicate farmers' market use is influenced by multiple economic, service delivery, spatial-temporal, social, and personal factors. Among studies that included low-income populations (n=19), key barriers to farmers' market use were perceptions that food assistance benefits were not accepted, belief that food variety at farmers' markets was limited, lack of access to transportation, lack of racial/ethnic diversity in the market space, and mismatch between markets and personal lifestyles. There is wide variation in study design and reporting standards and infrequent use of standardized measures limiting comparisons across studies. Conclusions There is a need to establish valid and reliable metrics and reporting standards for evaluating farmers' markets. Findings may inform interventions, programs, and policies to promote farmers' market use.

Introduction Human herpesvirus-8-associated B-cell lymphoma is a common disease entity in immunocompromised individuals, particularly in patients with chronic HIV-infection or AIDS. However, cardiac manifestations are extremely rare. Tissue for histopathology of left cardiac tumours is most commonly obtained by open surgery. Case presentation In this report, we present a case of a solitary left atrial manifestation of an HHV8+ B-cell lymphoma in a 59-year-old patient presenting with B symptoms and a cardiac mass on echocardiography. Due to the high operative risk of the patient, a transcatheter/trans-septal biopsy was performed to establish the diagnosis. Discussion In the era of routine trans-septal catheter interventions, this approach may represent a straight-forward, minimally invasive alternative for patients at high risk for surgery.

The long coevolution of β-herpesviruses with humans has resulted in the acquisition of a large variety of immune evasion strategies by these viruses. Presumably immune evasion genes are necessary for each herpesvirus to establish a life-long infection in their host. Human herpesvirus 7 (HHV-7) is a relatively understudied virus, likely because it does not typically pose significant health concerns. HHV-7 establishes a life-long infection in approximately 90% of the population before the age of 3 suggesting it likely possesses significant immune evasion strategies. Several genes found in a cluster near a known immune evasion gene, U21, may possibly encode novel immune evasion proteins. In an attempt to determine the function of one of these genes, U20, we previously expressed the protein in a variety of cell types. As predicted, U20 is a type I membrane protein targeted to the secretory system through a predicted N-terminal signal sequence. Interestingly, although U20 does not possess any predicted endoplasmic reticulum (ER) retention motifs, the exogenously expressed protein remained localized to the ER in all cell types examined. Here we describe a cloning project to generate constructs that will be used to examine the role of each domain (lumenal, transmembrane, or cytoplasmic) of U20 in ER retention.

A central issue for adoptive cellular immunotherapy is overcoming immunosuppressive signals to achieve tumor clearance. While γδ T cells are known to be potent cytolytic effectors that can kill a variety of cancers, it is not clear whether they are inhibited by suppressive ligands expressed in tumor microenvironments. Here, we have used a powerful preclinical model where EBV infection drives the de novo generation of human B cell lymphomas in vivo, and autologous T lymphocytes are held in check by PD-1/CTLA-4-mediated inhibition. We show that a single dose of adoptively transferred Vδ2+ T cells has potent antitumor effects, even in the absence of checkpoint blockade or activating compounds. Vδ2+ T cell immunotherapy given within the first 5 days of EBV infection almost completely prevented the outgrowth of tumors. Vδ2+ T cell immunotherapy given more than 3 weeks after infection (after neoplastic transformation is evident) resulted in a dramatic reduction in tumor burden. The immunotherapeutic Vδ2+ T cells maintained low cell surface expression of PD-1 in vivo, and their recruitment to tumors was followed by a decrease in B cells expressing PD-L1 and PD-L2 inhibitory ligands. These results suggest that adoptively transferred PD-1lo Vδ2+ T cells circumvent the tumor checkpoint environment in vivo.

NKG2D is an activating receptor expressed on several types of human lymphocytes. NKG2D ligands can be induced upon cell stress and are frequently targeted post-translationally in infected or transformed cells to avoid immune recognition. Virus infection and inflammation alter protein N-glycosylation, and we have previously shown that changes in cellular N-glycosylation are involved in regulation of NKG2D ligand surface expression. The specific mode of regulation through N-glycosylation is, however, unknown. Here we investigated whether direct N-glycosylation of the NKG2D ligand MICA itself is critical for cell surface expression and sought to identify the essential residues. We found that a single N-glycosylation site (Asn(8)) was important for MICA018 surface expression. The frequently expressed MICA allele 008, with an altered transmembrane and intracellular domain, was not affected by mutation of this N-glycosylation site. Mutational analysis revealed that a single amino acid (Thr(24)) in the extracellular domain of MICA018 was essential for the N-glycosylation dependence, whereas the intracellular domain was not involved. The HHV7 immunoevasin, U21, was found to inhibit MICA018 surface expression by affecting N-glycosylation, and the retention was rescued by T24A substitution. Our study reveals N-glycosylation as an allele-specific regulatory mechanism important for regulation of surface expression of MICA018, and we pinpoint the residues essential for this N-glycosylation dependence. In addition, we show that this regulatory mechanism of MICA surface expression is likely targeted during different pathological conditions.

IL-1β is produced by myeloid cells and acts as a critical mediator of host defense during infection and injury. We found that the intracellular protozoan parasite Toxoplasma gondii induced an early IL-1β response (within 4 hr) in primary human peripheral blood monocytes isolated from healthy donors. This process involved up-regulation of IL-1β, IL-1RN (IL-1 receptor antagonist), and NLRP3 transcripts, de novo protein synthesis, and the release of both pro- and mature-IL-1β from infected primary monocytes. The released pro-IL-1β was cleavable to mature, bioactive IL-1β in the extracellular space by the protease caspase-1. Treatment of primary monocytes with the NLRP3 inhibitor MCC950 or with extracellular potassium significantly reduced IL-1β cleavage and release in response to T. gondii infection, without affecting the release of TNF-α, and indicated a role for the inflammasome sensor NLRP3 and for potassium efflux in T. gondii-induced IL-1β production. Interestingly, T. gondii infection did not induce an IL-1β response in primary human macrophages derived from the same blood donors as the monocytes. Consistent with this finding, NLRP3 was down-regulated during the differentiation of monocytes to macrophages and was not induced in macrophages during T. gondii infection. To our knowledge, these findings are the first to identify NLRP3 as an inflammasome sensor for T. gondii in primary human peripheral blood cells and to define an upstream regulator of its activation through the release of intracellular potassium.

Translational perspective Cardiomyocytes (CMs) generated from human induced pluripotent stem cells are an evolving platform to understand molecular disease mechanism and evaluate cardiovascular drugs. A major limitation of this system is that they represent a heterogeneous mix of ventricular-, atrial-, and nodal-like CMs. By expressing a voltage-sensitive fluorescent protein under the control of lineage-specific promoters, we developed a convenient system allowing high-throughput subtype-specific optical action potential (AP) imaging in these cells. This enables not only quantification of electrical phenotypes in patient-specific CMs but also subtype-specific investigation of drug effects, which may aid both drug development and safety pharmacology in the cardiovascular field., Aims Cardiomyocytes (CMs) generated from human induced pluripotent stem cells (hiPSCs) are increasingly used in disease modelling and drug evaluation. However, they are typically a heterogeneous mix of ventricular-, atrial-, and nodal-like cells based on action potentials (APs) and gene expression. This heterogeneity and the paucity of methods for high-throughput functional phenotyping hinder the full exploitation of their potential. We aimed at developing a method for rapid, sequential, and subtype-specific phenotyping of hiPSC-CMs with respect to AP morphology and single-cell arrhythmias. Methods and results We used cardiac lineage-specific promoters to drive the expression of a voltage-sensitive fluorescent protein (VSFP-CR) in hiPSC-CMs, enabling subtype-specific optical AP recordings. In a patient-specific hiPSC model of long-QT syndrome type 1, AP prolongation and frequent early afterdepolarizations were evident in mutant ventricular- and atrial like, but not in nodal-like hiPSC-CMs compared with their isogenic controls, consistent with the selective expression of the disease-causing gene. Furthermore, we demonstrate the feasibility of sequentially probing a cell over several days to investigate genetic rescue of the disease phenotype and to discern CM subtype-specific drug effects. Conclusion By combining a genetically encoded membrane voltage sensor with promoters that drive its expression in the major subtypes of hiPSC-CMs, we developed a convenient system for disease modelling and drug evaluation in the relevant cell type, which has the potential to advance the emerging utility of hiPSCs in cardiovascular medicine.

In viral infection, morbidity and mortality often result from extrahepatic disease manifestations such as vasculitis. We hereby show that human microvascular endothelial cells express viral receptors of the innate immune system which are induced upon ligand engagement. Furthermore, stimulation of endothelial cells with the synthetic analog of viral DNA, poly (dA:dT), human DNA and hepatitis B virus-containing immunoprecipitates from a patient with polyarteritis nodosa induces an inflammatory response including the upregulation of adhesion molecules, which is mediated exclusively by TLR9 and involves an IRF3-dependent pathway. Thus, endothelial cells are able to actively participate in immune mediated vascular inflammation caused by viral infections. Furthermore, we provide evidence for the ability of LL37 to bind and internalize viral or endogenous DNA into non-immune cells. DNA nucleotides internalized by LL37 suppress the production of proinflammatory mediators suggesting a protective effect against direct responses to viral infection or circulating DNA-fragments of endogenous origin.

We simulated nests constructed in an area heavily used by ovipositing Diamond-backed Terrapins (Malaclemys terrapin) and foraging raccoons (Procyon lotor), to investigate the cues used by raccoons to locate terrapin nests. Marking nests with flags did not increase predation rates, and human scent decreased predation rates. Raccoons seemed to locate nests based on soil disturbance, ocean-water scent, or a combination of these cues.

Objective—To determine analgesic effects of intraneural injection of ethyl alcohol or formaldehyde in the palmar digital nerves of horses. Animals—6 horses. Procedures—Ethyl alcohol was injected in the medial palmar digital nerve of 1 forelimb, and formaldehyde was injected in the contralateral nerve. The lateral palmar digital nerve in 1 forelimb was surgically exposed, but not injected, and the contralateral lateral palmar digital nerve was not treated. For each heel, severity of lameness in response to experimentally induced heel pain (lameness score and peak vertical force), thermal reaction time, and heel skin sensitivity scores were recorded. Heel pain was experimentally induced by advancing a threaded bolt through a custom-made horseshoe to apply pressure to the palmar horned aspect of the hoof. Horses were followed up for 112 days, when a subset of nerves was sampled for histologic analysis. Results—Alcohol and formaldehyde significantly reduced all measures of heel pain, and analgesia was evident over the 112 days of the study. Pastern circumference was significantly greater for formaldehyde-treated than for alcohol-treated limbs. Histologic evaluation showed preservation of nerve fiber alignment with an intact epineurium, loss of axons, axon degeneration, fibrosis, and inflammation in alcohol-treated and formaldehyde-treated nerves. Conclusions and Clinical Relevance—Results suggested that intraneural injection of either ethyl alcohol or formaldehyde in the palmar digital nerves of horses resulted in substantial, but partial, heel analgesia that persisted for at least 112 days. No advantage of formaldehyde over alcohol was found, and formaldehyde resulted in greater soft tissue inflammation.

Signal-induced activation of caspases, the critical protease effectors of apoptosis, requires proteolytic processing of their inactive proenzymes. Consequently, regulation of procaspase processing is critical to apoptotic execution. We report here that baculovirus pancaspase inhibitor P35 and inhibitor of apoptosis Op-IAP prevent caspase activation in vivo, but at different steps. By monitoring proteolytic processing of endogenous Sf-caspase-1, an insect group II effector caspase, we show that Op-IAP blocked the first activation cleavage at TETD downward arrowG between the large and small caspase subunits. In contrast, P35 failed to affect this cleavage, but functioned downstream to block maturation cleavages (DXXD downward arrow(G/A)) of the large subunit. Substitution of P35's reactive site residues with TETDG failed to increase its effectiveness for blocking TETD downward arrowG processing of pro-Sf-caspase-1, despite wild-type function for suppressing apoptosis. These data are consistent with the involvement of a novel initiator caspase that is resistant to P35, but directly or indirectly inhibitable by Op-IAP. The conservation of TETD downward arrowG processing sites among insect effector caspases, including Drosophila drICE and DCP-1, suggests that in vivo activation of these group II caspases involves a P35-insensitive caspase and supports a model wherein apical and effector caspases function through a proteolytic cascade to execute apoptosis in insects.

The aspartate-specific caspases are critical protease effectors of programmed cell death and consequently represent important targets for apoptotic intervention. Baculovirus P35 is a potent substrate inhibitor of metazoan caspases, a property that accounts for its unique effectiveness in preventing apoptosis in phylogenetically diverse organisms. Here we report the 2.2 A resolution crystal structure of P35, the first structure of a protein inhibitor of the death caspases. The P35 monomer possesses a solvent-exposed loop that projects from the protein's main beta-sheet core and positions the requisite aspartate cleavage site at the loop's apex. Distortion or destabilization of this reactive site loop by site-directed mutagenesis converted P35 to an efficient substrate which, unlike wild-type P35, failed to interact stably with the target caspase or block protease activity. Thus, cleavage alone is insufficient for caspase inhibition. These data are consistent with a new model wherein the P35 reactive site loop participates in a unique multi-step mechanism in which the spatial orientation of the loop with respect to the P35 core determines post-cleavage association and stoichiometric inhibition of target caspases.

Henkel’s social commitment is firmly embedded in its corporate values and its corporate history. Launched in 2005, the Henkel Smile program brings together all aspects of Henkel’s social commitment – internationally known as corporate citizenship – that go beyond its business activities. Henkel Smile unites four modules, one of which is the MIT (Make an Impact on Tomorrow) Initiative, which support the volunteer work of Henkel employees and pensioners. The MIT Initiative focuses in particular on the involvement of employees and pensioners who identify social challenges, develop innovative solutions to these challenges and make them known within the Company. As corporate volunteers, they are active ambassadors of the company at all Henkel sites throughout the world.

Recent scholars have dismissed the utility of self-esteem as well as programs designed to improve it. The authors challenge these contentions on conceptual, methodological, and empirical grounds. They begin by proposing that the scope of recent analyses has been overly narrow and should be broadened to include specific as well as global self-views. Using this conceptualization, the authors place recent critiques in historical context, recalling that similarly skeptical commentaries on global attitudes and traits inspired theorizing and empirical research that subsequently restored faith in the value of both constructs. Specifically, they point to 3 strategies for attaining more optimistic assessments of the predictive validity of self-views: recognizing the utility of incorporating additional variables in predictive schemes, matching the specificity of predictors and criteria, and using theoretically informed standards for evaluating predictor- criterion relationships. The authors conclude that self-views do matter and that it is worthwhile and important to develop and implement theoretically informed programs to improve them.

In their thoughtful commentary on our article (Swann, Chang-Schneider, & McClarty, February–March 2007), Krueger, Vohs, and Baumeister (2008, this issue) brought up many points with which we agree. Nevertheless, as they noted these points of agreement, we focus instead on several points of continued disagreement. In addition, we comment on a few new twists that they have added to their argument. Krueger et al. (2008) began by disputing our claim that they “have violated the specificity matching principle by focusing on the capacity of global measures of selfesteem to predict specific outcomes” (Swann et al., 2007, p. 87). They protested that they specifically drew attention to the specificity matching principle, reminding the reader that in their original article (Baumeister, Campbell, Krueger, & Vohs, 2003) they indicated that “it is difficult to detect a correspondence between a global attitude and specific behaviors” (p. 6). It is true that Baumeister et al. (2003) acknowledged the specificity matching principle in their original article. Nevertheless, as Krueger et al. (2008) themselves allowed, after acknowledging the principle, Baumeister et al. focused their review on the relative incapacity of measures of global self-esteem to predict specific outcomes, which is to say they violated the principle repeatedly. If Baumeister et al. had faithfully followed the implications of the specificity matching principle, they would have likely reached the same conclusion we reached, which is that most of the research conducted on self-esteem offers little insight into the capacity of selfknowledge to predict important outcomes because it violates the specificity matching principle. Furthermore, they also would have acknowledged (as we did) that when researchers have conformed to the specificity matching principle, they have discovered that the relationship between selfviews and outcome variables improves considerably. For example, we cited evidence that specific academic self-concepts offered better predictions of academic ability than did global self-esteem (Hansford & Hattie, 1982). Krueger et al. (2008) introduced a novel argument into their comment, suggesting that the alleged predictive impotence of self-esteem stems from a tendency for responses to measures of self-esteem to have no motivational implications. To make their point, they singled out an item from the Rosenberg (1965) Self-Esteem Scale: “I feel that I have a number of good qualities.” We were startled by Krueger et al.’s (2008) attempt to discredit one of psychology’s most venerable constructs by questioning the properties of this single item of a single self-esteem scale. But even if the viability of the self-esteem construct could be imagined to rest on the validity of a single item, the research literature suggests that believing that one has lots of good qualities does indeed have motivational implications. Indeed, Baumeister himself (McFarlin, Baumeister, & Blascovich, 1984) has published evidence that people with high self-esteem persist longer in the wake of failure than do people with low self-esteem. Also, there is growing evidence that people who feel that they lack good qualities will be surprised and upset by positive treatment and that such reactions guide subsequent behavior. In fact, in our article we cited evidence that people with positive self-views withdraw from their marriage partners (either psychologically or through divorce/separation) insofar as their partners perceive them negatively and that people with negative self-views withdraw from their marriage partners insofar as their partners perceive them positively (e.g., Cast & Burke, 2002; Swann, De La Ronde, & Hixon, 1994). Moreover, in a recent series of four studies, Wiesenfeld, Swann, Brockner, and Bartel (2007) discovered that selfesteem moderated people’s reactions to “procedural justice” (how fairly one is treated by one’s organization). For people with high self-esteem, being treated more fairly by their work organization increased emotional and behavioral commitment to the organization, but people with low selfesteem showed no such preference for fair treatment. In short, there is growing evidence that believing that one has good qualities and is worthwhile has profound motivational implications, influencing behaviors ranging from task persistence and relationship longevity to the frequency with which people show up for work. Krueger et al. (2008) also reinforced one of their key assertions in their original article (Baumeister et al., 2003), which was that self-esteem and narcissism are closely allied. From their vantage point, self-esteem is, by association, guilty of all the negative qualities that have been empirically linked to narcissism. This association, in turn, supposedly explains why success in maintaining high self-esteem is a nasty, competitive process in which one person’s success requires another person’s failure. Although it is true that measures of selfesteem and narcissism are related, the relation is modest. More important, narcissism is a multifaceted construct, and only the socially benign components of narcissism (e.g., vanity, authority) covary with selfesteem; the socially noxious aspects of self-esteem (e.g., entitlement, aggressiveness) are largely independent of self-esteem (Trzesniewski et al., 2006). Given this, it is not surprising that just as narcissism predicts negative behaviors such as defensiveness, self-esteem predicts a wide array of happy, prosocial outcomes; see p. 87 of Swann et al. (2007) for citations to six papers that report evidence that supports this conclusion. We urge readers to examine these articles and reach their own judgment about the viability of Krueger et al.’s continued insistence that conflating self-esteem and narcissism represents a scientific advance. Krueger et al. (2008) strove to buttress their conviction that self-esteem has deleterious consequences by pointing to a press release reporting the findings of Jean Twenge and Keith Campbell (Associated Press, 2007). The press release contended that these researchers found that narcissism has increased in recent years among young Americans. The researchers did not explain precisely why narcissism appears to have increased but instead implied that it is linked to self-enhancement, which is, in turn, related to high self-esteem. The wisdom of using data summarized in a press release to buttress a scientific argument aside, we find ourselves persuaded by a recent study that challenges the premise of this press release. On the basis of a careful