ATF6 protects against protein misfolding during cardiac hypertrophy.

Cardiomyocytes activate the unfolded protein response (UPR) transcription factor ATF6 during pressure overload-induced hypertrophic growth. The UPR is thought to increase ER protein folding capacity and maintain proteostasis. ATF6 deficiency during pressure overload leads to heart failure, suggesting that ATF6 protects against myocardial dysfunction by preventing protein misfolding. However, conclusive evidence that ATF6 prevents toxic protein misfolding during cardiac hypertrophy is still pending. Here, we found that activation of the UPR, including ATF6, is a common response to pathological cardiac hypertrophy in mice. ATF6 KO mice failed to induce sufficient levels of UPR target genes in response to chronic isoproterenol infusion or transverse aortic constriction (TAC), resulting in impaired cardiac growth. To investigate the effects of ATF6 on protein folding, the accumulation of poly-ubiquitinated proteins as well as soluble amyloid oligomers were directly quantified in hypertrophied hearts of WT and ATF6 KO mice. Whereas only low levels of protein misfolding was observed in WT hearts after TAC, ATF6 KO mice accumulated increased quantities of misfolded protein, which was associated with impaired myocardial function. Collectively, the data suggest that ATF6 plays a critical adaptive role during cardiac hypertrophy by protecting against protein misfolding.

Investigating foundations for hominin fire exploitation: Savanna-dwelling chimpanzees (Pan troglodytes verus) in fire-altered landscapes.

Humans' extensive use of fire is one behavior that sets us apart from all other animals. However, our ancestors' reliance on controlled forms of fire-i.e., for cooking-was likely preceded by a long familiarity with fire beginning with passive exploitation of naturally burned landscapes and followed by intermediate steps including active ecological modification via intentional burning. Here we explore our pyrophilic beginnings using observational data from savanna-dwelling chimpanzees. These data highlight the extent to which anthropogenic burning impacts the behavior and ecology of sympatric primates and provides an opportunity to study the ways in which apes living in a fire-altered world exploit opportunities presented by burning. Using monthly burn scar data and daily range use data we quantify the impact of burning episodes on chimpanzee habitat. Over the course of one dry season, approximately 74% of the total estimated range of the Fongoli community of savanna-dwelling chimpanzees (Pan troglodytes verus) was impacted by fire. We combine fire occurrences with behavioral data to test for relationships between burning and rate of encounter with food items and duration of subsequent patch residence time. Results show more frequent encounters and shorter patch residence times in burned areas. These data can be leveraged as a frame of reference for conceptualizing our extinct relatives' behavior around fire.

CRISPR-Mediated Activation of Endogenous Gene Expression in the Postnatal Heart.

RATIONALE: Genome editing by CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 is evolving rapidly. Recently, second-generation CRISPR/Cas9 activation systems based on nuclease inactive dead (d)Cas9 fused to transcriptional transactivation domains were developed for directing specific guide (g)RNAs to regulatory regions of any gene of interest, to enhance transcription. The application of dCas9 to activate cardiomyocyte transcription in targeted genomic loci in vivo has not been demonstrated so far. OBJECTIVE: We aimed to develop a mouse model for cardiomyocyte-specific, CRISPR-mediated transcriptional modulation, and to demonstrate its versatility by targeting Mef2d and Klf15 loci (2 well-characterized genes implicated in cardiac hypertrophy and homeostasis) for enhanced transcription. METHODS AND RESULTS: A mouse model expressing dCas9 with the VPR transcriptional transactivation domains under the control of the Myh (myosin heavy chain) 6 promoter was generated. These mice innocuously expressed dCas9 exclusively in cardiomyocytes. For initial proof-of-concept, we selected Mef2d, which when overexpressed, led to hypertrophy and heart failure, and Klf15, which is lowly expressed in the neonatal heart. The most effective gRNAs were first identified in fibroblast (C3H/10T1/2) and myoblast (C2C12) cell lines. Using an improved triple gRNA expression system (TRISPR [triple gRNA expression construct]), up to 3 different gRNAs were transduced simultaneously to identify optimal conditions for transcriptional activation. For in vivo delivery of the validated gRNA combinations, we employed systemic administration via adeno-associated virus serotype 9. On gRNA delivery targeting Mef2d expression, we recapitulated the anticipated cardiac hypertrophy phenotype. Using gRNA targeting Klf15, we could enhance its transcription significantly, although Klf15 is physiologically silenced at that time point. We further confirmed specific and robust dCas9VPR on-target effects. CONCLUSIONS: The developed mouse model permits enhancement of gene expression by using endogenous regulatory genomic elements. Proof-of-concept in 2 independent genomic loci suggests versatile applications in controlling transcription in cardiomyocytes of the postnatal heart.

Fire's impact on threat detection and risk perception among vervet monkeys: Implications for hominin evolution.

The spatial behavior of primates is shaped by many factors including predation risk, the distribution of food sources, and access to water. In fire-prone settings, burning is a catalyst of change, altering the distribution of both plants and animals. Recent research has shown that primates alter their behavior in response to this change. Here, we study primates' perceived threat of predation in fire-modified landscapes. We focus on the predator-related behaviors of vervet monkeys (Chlorocebus pygerythrus) after controlled burning events. We compare the occurrence of vigilance and predator-deterrent behaviors, including alarm calls, scanning, and flight across different habitats and burn conditions to test the hypothesis that subjects exhibit fewer predator-specific vigilance and predator-deterrent behaviors in burned areas. The results demonstrate that predator-related behaviors occur less often in burned habitats, suggesting that predators are less common in these areas. These results provide foundations for examining hypotheses about the use of fire-altered landscapes among extinct hominins. We set these data in the context of increasing aridity, changes in burning regimes, and the emergence of pyrophilia in the human lineage.

Functional Metalloblock Copolymers for the Preparation and In Situ Functionalization of Porous Silica Films.

Stimuli-responsive mesoporous silica films were prepared by evaporation-induced self-assembly through the physical entrapment of a functional metalloblock copolymer structuring agent, which simultaneously served to functionalize the mesopore. After end-functionalization with a silane group, the applied functional metalloblock copolymers were covalently integrated into the silica mesopore wall. In addition, they were partly degraded after the formation of the mesoporous film, which enabled the precise design of accessible mesopores. These polymer-silica hybrid materials exhibited remarkable and gating ionic permselectivity and offer the potential for highly precise pore filling design and combination with high-throughput printing techniques. This in situ functionalization strategy of mesoporous silica using responsive metalloblock copolymers has the potential to improve how we approach the design of complex architectures at the nanoscale for tailored transport. This functionalization strategy paves the way for a variety of technologies based on molecular transport in nanoscale pores, including separation, sensing, catalysis, and energy conversion.

Fluid Flow Programming in Paper-Derived Silica-Polymer Hybrids.

In paper-based devices, capillary fluid flow is based on length-scale selective functional control within a hierarchical porous system. The fluid flow can be tuned by altering the paper preparation process, which controls parameters such as the paper grammage. Interestingly, the fiber morphology and nanoporosity are often neglected. In this work, porous voids are incorporated into paper by the combination of dense or mesoporous ceramic silica coatings with hierarchically porous cotton linter paper. Varying the silica coating leads to significant changes in the fluid flow characteristics, up to the complete water exclusion without any further fiber surface hydrophobization, providing new approaches to control fluid flow. Additionally, functionalization with redox-responsive polymers leads to reversible, dynamic gating of fluid flow in these hybrid paper materials, demonstrating the potential of length scale specific, dynamic, and external transport control.

The pyrophilic primate hypothesis.

Members of genus Homo are the only animals known to create and control fire. The adaptive significance of this unique behavior is broadly recognized, but the steps by which our ancestors evolved pyrotechnic abilities remain unknown. Many hypotheses attempting to answer this question attribute hominin fire to serendipitous, even accidental, discovery. Using recent paleoenvironmental reconstructions, we present an alternative scenario in which, 2 to 3 million years ago in tropical Africa, human fire dependence was the result of adapting to progressively fire-prone environments. The extreme and rapid fluctuations between closed canopy forests, woodland, and grasslands that occurred in tropical Africa during that time, in conjunction with reductions in atmospheric carbon dioxide levels, changed the fire regime of the region, increasing the occurrence of natural fires. We use models from optimal foraging theory to hypothesize benefits that this fire-altered landscape provided to ancestral hominins and link these benefits to steps that transformed our ancestors into a genus of active pyrophiles whose dependence on fire for survival contributed to its rapid expansion out of Africa.

What's burning got to do with it? Primate foraging opportunities in fire-modified landscapes.

OBJECTIVES: Anecdotal and formal evidence indicate that primates take advantage of burned landscapes. However, little work has been done to quantify the costs and benefits of this behavior. Using systematic behavioral observations from a population of South African vervet monkeys (Chlorocebus aethiops pygerythrus), we evaluate differences in food availability and energetics before and after controlled burns altered vegetation near their home range. We aim to determine whether burned habitats offer improved foraging opportunities. METHODS: We collected feeding data from foraging individuals and analyzed common plant foods for their energetic content. We then used the feeding and energetic data to calculate postencounter profitabilities and encounter rates for food types. Using negative binomial and mixed linear regression models we compared data from burned and unburned habitats. RESULTS: Our results show significantly improved encounter rates in burned landscapes for two prey items, invertebrates and grasses. However, postencounter profitabilities in burned areas were not significantly different than those achieved in unburned areas. CONCLUSIONS: Results suggest that improved encounters alone can motivate changes in foraging behavior. These foraging benefits enable the exploitation of burned savanna habitats, likely driving postburn range expansions observed among populations of vervet monkeys. Thus quantified, these results may serve as a foundation for hypotheses regarding the evolution of fire-use in our own lineage.

Fire and home range expansion: a behavioral response to burning among savanna dwelling vervet monkeys (Chlorocebus aethiops).

The behavioral adaptations of primates to fire-modified landscapes are of considerable interest to anthropologists because fire is fundamental to life in the African savanna-the setting in which genus Homo evolved. Here we report the behavioral responses of a savanna-dwelling primate, vervet monkeys (Chlorocebus aethiops), to fire-induced ecological change. Using behavioral and spatial data to characterize ranging patterns prior to and postburn and between burn and nonburn years, we show that these primates inhabiting small, spatially bound, riverine habitats take advantage of newly burned savanna landscapes. When subjects encountered controlled fires, they did not flee but instead avoided the path of the fire seemingly unbothered by its approach. After fire, the primates' home range expanded into newly burned but previously unused areas. These results contribute to understanding the response of non-human primates to fire-modified landscapes and can shed light on the nature and scope of opportunities and constraints posed by the emergence of fire-affected landscapes in the past. Results also expose deficiencies in our knowledge of fire-related behavioral responses in the primate lineage and highlight the need for further investigation of these responses as they relate to foraging opportunities, migration, resource use, and especially fire-centric adaptations in our own genus.

The inotropic peptide ßARKct improves ßAR responsiveness in normal and failing cardiomyocytes through G(ßγ)-mediated L-type calcium current disinhibition.

RATIONALE: The G(ßγ)-sequestering peptide ß-adrenergic receptor kinase (ßARK)ct derived from the G-protein-coupled receptor kinase (GRK)2 carboxyl terminus has emerged as a promising target for gene-based heart failure therapy. Enhanced downstream cAMP signaling has been proposed as the underlying mechanism for increased ß-adrenergic receptor (ßAR) responsiveness. However, molecular targets mediating improved cardiac contractile performance by ßARKct and its impact on G(ßγ)-mediated signaling have yet to be fully elucidated. OBJECTIVE: We sought to identify G(ßγ)-regulated targets and signaling mechanisms conveying ßARKct-mediated enhanced ßAR responsiveness in normal (NC) and failing (FC) adult rat ventricular cardiomyocytes. METHODS AND RESULTS: Assessing viral-based ßARKct gene delivery with electrophysiological techniques, analysis of contractile performance, subcellular Ca²(+) handling, and site-specific protein phosphorylation, we demonstrate that ßARKct enhances the cardiac L-type Ca²(+) channel (LCC) current (I(Ca)) both in NCs and FCs on ßAR stimulation. Mechanistically, ßARKct augments I(Ca) by preventing enhanced inhibitory interaction between the α1-LCC subunit (Cav1.2α) and liberated G(ßγ) subunits downstream of activated ßARs. Despite improved ßAR contractile responsiveness, ßARKct neither increased nor restored cAMP-dependent protein kinase (PKA) and calmodulin-dependent kinase II signaling including unchanged protein kinase (PK)Cε, extracellular signal-regulated kinase (ERK)1/2, Akt, ERK5, and p38 activation both in NCs and FCs. Accordingly, although ßARKct significantly increases I(Ca) and Ca²(+) transients, being susceptible to suppression by recombinant G(ßγ) protein and use-dependent LCC blocker, ßARKct-expressing cardiomyocytes exhibit equal basal and ßAR-stimulated sarcoplasmic reticulum Ca²(+) load, spontaneous diastolic Ca²(+) leakage, and survival rates and were less susceptible to field-stimulated Ca²(+) waves compared with controls. CONCLUSION: Our study identifies a G(ßγ)-dependent signaling pathway attenuating cardiomyocyte I(Ca) on ßAR as molecular target for the G(ßγ)-sequestering peptide ßARKct. Targeted interruption of this inhibitory signaling pathway by ßARKct confers improved ßAR contractile responsiveness through increased I(Ca) without enhancing regular or restoring abnormal cAMP-signaling. ßARKct-mediated improvement of I(Ca) rendered cardiomyocytes neither susceptible to ßAR-induced damage nor arrhythmogenic sarcoplasmic reticulum Ca²(+) leakage.

Orai1 and Stim1 regulate normal and hypertrophic growth in cardiomyocytes.

Cardiac hypertrophy is an independent risk for heart failure (HF) and sudden death. Deciphering signalling pathways dependent on extracellular calcium (Ca(2+)) influx that control normal and pathological cardiac growth may enable identification of novel therapeutic targets. The objective of the present study is to determine the role of the Ca(2+) release-activated Ca(2+) (CRAC) channel Orai1 and stromal interaction molecule 1 (Stim1) in postnatal cardiomyocyte store operated Ca(2+) entry (SOCE) and impact on normal and hypertrophic postnatal cardiomyocyte growth. Employing a combination of siRNA-mediated gene silencing, cultured neonatal rat ventricular cardiomyocytes together with indirect immunofluorescence, epifluorescent Ca(2+) imaging and site-specific protein phosphorylation and real-time mRNA expression analysis, we show for the first time that both Orai1 and Stim1 are present in cardiomyocytes and required for SOCE due to intracellular Ca(2+) store depletion by thapsigargin. Stim1-KD but not Orai1-KD significantly decreased diastolic Ca(2+) levels and caffeine-releasable Ca(2+) from the sarcoplasmic reticulum (SR). Conversely, Orai1-KD but not Stim1-KD significantly diminished basal NRCM cell size, anp and bnp mRNA levels and activity of the calcineurin (CnA) signalling pathway although diminishing both Orai1 and Stim1 proteins similarly attenuated calmodulin kinase II (CamKII) and ERK1/2 activity under basal conditions. Both Orai1- and Stim1-KD completely abrogated phenylephrine (PE) mediated hypertrophic NRCM growth and enhanced natriuretic factor expression by inhibiting G(q)-protein conveyed activation of the CamKII and ERK1/2 signalling pathway. Interestingly, only Orai1-KD but not Stim1-KD prevented Gq-mediated CaN-dependent prohypertrophic signalling. This study shows for the first time that both Orai1 and Stim1 have a key role in cardiomyocyte SOCE regulating both normal and hypertrophic postnatal cardiac growth in vitro.

Gravure printing for mesoporous film preparation.

This study presents gravure printing as a new strategy for rapid printing of ceramic mesoporous films and highlights its advantages over conventional mesoporous film preparation using evaporation induced self-assembly together with dip-coating. By varying the printing process parameters, the mesoporous coating thicknesses can be adjusted between 20 and 200 nm while maintaining a very high film homogeneity allowing the printing of ultrathin mesoporous films. Step gradients in film composition are accessible by consecutively printing two different "inks". Thereby, gravure printing is a much faster process than mesoporous single- and multilayer preparation using conventional dip-coating because lower amounts of solution are transferred and dissolution of previously deposited layers is avoided. The effect of printing process parameters on resulting film characteristics as well as the resulting mesoporous film's ionic accessibility is systematically investigated.

Remote preconditioning by infrarenal occlusion of the aorta protects the heart from infarction: a newly identified non-neuronal but PKC-dependent pathway.

BACKGROUND: Ischemic preconditioning is a powerful mechanism in reducing infarct size of the heart. Protection can be performed either by an ischemic stimulus of the heart itself or by ischemia of an organ distant to the heart. To address the question whether this remote preconditioning is transduced by neuronal or humoral factors an in situ model of infrarenal occlusion of the aorta (IOA) in the rat was developed. Furthermore, the signal transduction pathways of classical and remote preconditioning regarding protein kinase C, which is one of the key enzymes in classical preconditioning, were compared. METHODS AND RESULTS: Controls (30 min regional ischemia followed by 2 h of reperfusion) had an infarct size of 62+/-5% whereas classical preconditioning reduced it to 10+/-3% of the risk zone (P< or =0.001). Fifteen minutes IOA without reperfusion of the aorta had no influence on infarct size (52+/-4%). When, however, IOA was performed for 15, 10, or 5 min, respectively, followed by a 10-min reperfusion period the size of myocardial infarction decreased significantly. This decrease was dependent on the duration of IOA (18+/-3%, 37+/-8%, 42+/-2%, respectively; P< or =0.001 for the time-dependent linear trend in decrease of infarct size). Fifteen minutes IOA showed the strongest protection which was comparable to classical preconditioning (18+/-3%, P< or =0.001 vs. control). Blockade of the nervous pathway by 20 mg/kg hexamethonium could not inhibit the protection afforded by IOA (14+/-4%). Using chelerythrine, a selective protein kinase C-inhibitor, at a dose of 5 mg/kg body weight, protection from remote (68+/-4%, P< or =0.001 vs. 15 min IOA followed by 10 min of reperfusion without chelerythrine) as well as from classical preconditioning (56+/-5%, P< or =0.001) was completely blocked. CONCLUSION: Protection of the heart by remote preconditioning using IOA is as powerful as classical preconditioning. Both protection methods share protein kinase C as a common element in their signal transduction pathways. Since hexamethonium could not block the protection from IOA and a reperfusion period has to be necessarily interspaced between the IOA and the infarct inducing ischemia of the heart, a neuronal signal transmission from the remote area to the heart can be excluded with certainty. A humoral factor must be responsible for the remote protection. Interestingly the production of the protecting factor is dependent on the duration of the ischemia of the lower limb. The protecting substance, which must be upstream of protein kinase C, remains to be identified.

Surface plasmon & visible light for polymer functionalization of mesopores and manipulation of ionic permselectivity.

The near-field of surface plasmons is locally confined to nanometer dimensions. Here, we use surface plasmons to initiate polymer-functionalization of mesoporous silica films allowing subsequent ionic permselectivity gating based on polymer charge. We expect this functionalization approach to open a new dimension of functional miniaturization e.g. in nanofluidics.

Effects of previous influenza vaccination on subsequent readmission and mortality in elderly patients hospitalized with pneumonia.

PURPOSE: To determine the effect of influenza vaccination on mortality and hospital readmission rates following discharge of elderly patients admitted with pneumonia. METHODS: We reviewed the medical records of 12,566 randomly selected Medicare beneficiaries hospitalized for pneumonia from October 1 through December 31, 1998, to assess mortality and hospital readmission rates from the date of discharge through the influenza season, May 1, 1999. Patients were grouped based on vaccination status: before hospitalization, during hospitalization, or unknown (no evidence of vaccination). RESULTS: Severity-adjusted mortality rates were 22.4% (95% confidence interval [CI]: 14.4% to 29.7%) for the vaccination before hospitalization group, 26.4% (95% CI: 20.4% to 31.9%) for the in-hospital vaccination group, and 29.4% (95% CI: 28.1% to 30.6%) for the unknown vaccination status group. Patients vaccinated before hospitalization had significantly lower mortality than did patients with unknown vaccination status (hazard ratio [HR] = 0.65; 95% CI: 0.59 to 0.70; P <0.0001). Adjusted readmission rates were 42.6% (95% CI: 40.0% to 45.1%) for the vaccination before hospitalization group, 40.0% (95% CI: 33.2% to 46.1%) for the in-hospital vaccination group, and 44.8% (95% CI: 43.3% to 46.4%) for the unknown vaccination status group. Patients vaccinated before hospitalization had significantly lower readmission rates than patients with unknown vaccination status (HR = 0.92; 95% CI: 0.87 to 0.98; P = 0.009). CONCLUSION: Influenza vaccination before hospitalization was effective in decreasing subsequent mortality and hospital readmission in elderly patients with pneumonia.

Positron emission tomography is not useful in detecting metastasis in the sentinel lymph node in patients with primary malignant melanoma stage I and II.

The most powerful predictor for recurrence and survival in patients with primary malignant melanoma is the presence or absence of lymph node metastases. In the present study, 18-fluoro-2-deoxyglucose positron emission tomography (FDG-PET) findings were compared with histopathological results of sentinel lymph node biopsy (SNB). The purpose was to determine the value of FDG-PET in predicting regional lymph node involvement in patients with primary malignant melanoma stage I and II. Forty-eight consecutive patients with primary cutaneous melanoma stage I (Breslow thickness > 1 mm) and II underwent FDG-PET scans, preoperative lymphoscintigraphy, and SNB. The FDG-PET and SNB results were interpreted independently of each other and then compared. Of the 48 patients included in the study, eight (16.7%) had a positive SNB. PET was positive in only one patient with a positive SNB, yielding a sensitivity of 13%. All other positive sentinel nodes could not be detected by metabolic FDG-PET imaging. Our study revealed that FDG-PET is obviously not an adequate screening test for subclinical and sonographically inconspicuous lymph node metastases in patients with malignant melanoma stage I and II. The low sensitivity is probably due to the small size of metastatic deposits in sentinel nodes. Therefore, SNB remains the technique of choice for evaluating the histological status of lymph node basins in patients with early-stage cutaneous melanoma.

S100A1 is released from ischemic cardiomyocytes and signals myocardial damage via Toll-like receptor 4.

Members of the S100 protein family have been reported to function as endogenous danger signals (alarmins) playing an active role in tissue inflammation and repair when released from necrotic cells. Here, we investigated the role of S100A1, the S100 isoform with highest abundance in cardiomyocytes, when released from damaged cardiomyocytes during myocardial infarction (MI). Patients with acute MI showed significantly increased S100A1 serum levels. Experimental MI in mice induced comparable S100A1 release. S100A1 internalization was observed in cardiac fibroblasts (CFs) adjacent to damaged cardiomyocytes. In vitro analyses revealed exclusive S100A1 endocytosis by CFs, followed by Toll-like receptor 4 (TLR4)-dependent activation of MAP kinases and NF-κB. CFs exposed to S100A1 assumed an immunomodulatory and anti-fibrotic phenotype characterized i.e. by enhanced intercellular adhesion molecule-1 (ICAM1) and decreased collagen levels. In mice, intracardiac S100A1 injection recapitulated these transcriptional changes. Moreover, antibody-mediated neutralization of S100A1 enlarged infarct size and worsened left ventricular functional performance post-MI. Our study demonstrates alarmin properties for S100A1 from necrotic cardiomyocytes. However, the potentially beneficial role of extracellular S100A1 in MI-related inflammation and repair warrants further investigation.

S100A1 genetically targeted therapy reverses dysfunction of human failing cardiomyocytes.

OBJECTIVES: This study investigated the hypothesis whether S100A1 gene therapy can improve pathological key features in human failing ventricular cardiomyocytes (HFCMs). BACKGROUND: Depletion of the Ca²âº-sensor protein S100A1 drives deterioration of cardiac performance toward heart failure (HF) in experimental animal models. Targeted repair of this molecular defect by cardiac-specific S100A1 gene therapy rescued cardiac performance, raising the immanent question of its effects in human failing myocardium. METHODS: Enzymatically isolated HFCMs from hearts with severe systolic HF were subjected to S100A1 and control adenoviral gene transfer and contractile performance, calcium handling, signaling, and energy homeostasis were analyzed by video-edge-detection, FURA2-based epifluorescent microscopy, phosphorylation site-specific antibodies, and mitochondrial assays, respectively. RESULTS: Genetically targeted therapy employing the human S100A1 cDNA normalized decreased S100A1 protein levels in HFCMs, reversed both contractile dysfunction and negative force-frequency relationship, and improved contractile reserve under beta-adrenergic receptor (ß-AR) stimulation independent of cAMP-dependent (PKA) and calmodulin-dependent (CaMKII) kinase activity. S100A1 reversed underlying Ca²âº handling abnormalities basally and under ß-AR stimulation shown by improved SR Ca²âº handling, intracellular Ca²âº transients, diastolic Ca²âº overload, and diminished susceptibility to arrhythmogenic SR Ca²âº leak, respectively. Moreover, S100A1 ameliorated compromised mitochondrial function and restored the phosphocreatine/adenosine-triphosphate ratio. CONCLUSIONS: Our results demonstrate for the first time the therapeutic efficacy of genetically reconstituted S100A1 protein levels in HFCMs by reversing pathophysiological features that characterize human failing myocardium. Our findings close a gap in our understanding of S100A1's effects in human cardiomyocytes and strengthen the rationale for future molecular-guided therapy of human HF.

BMP-2 and FGF-2 synergistically facilitate adoption of a cardiac phenotype in somatic bone marrow c-kit+/Sca-1+ stem cells.

The aim of this study was to explore the effect of bone morphogenetic protein-2 (BMP-2) and fibroblast growth factor-2 (FGF-2)- paracrine factors implicated in both cardiac embryogenesis and cardiac repair following myocardial infarction (MI)-on murine bone marrow stem cell (mBMSC) differentiation in an ex vivo cardiac microenvironment. For this purpose, green fluorescent protein (GFP) expressing hematopoietic lineage negative (lin-) c-kit ligand (c-kit) and stem cell antigen-1 (Sca-1) positive (GFP-lin-/c-kit+/sca+) mBMSC were co-cultured with neonatal rat ventricular cardiomyocytes (NVCMs). GFP+ mBMSC significantly induced the expression of BMP-2 and FGF-2 in NVCMs, and approximately 4% GFP+ mBMSCs could be recovered from the co-culture at day 10. The addition of BMP-2 in concert with FGF-2 significantly enhanced the amount of integrated GFP+ mBMSCs by 5-fold ( approximately 20%), whereas the addition of anti-BMP-2 and/or anti-FGF-2 antibodies completely abolished this effect. An analysis of calcium cycling revealed robust calcium transients in GFP+ mBMSCs treated with BMP-2/FGF-2 compared to untreated co-cultures. BMP-2 and FGF-2 addition led to a significant induction of early (NK2 transcription factor related, locus 5; Nkx2.5, GATA binding protein 4; GATA-4) and late (myosin light chain kinase [MLC-2v], connexin 43 [Cx43]) cardiac marker mRNA expression in mBMSCs following co-culture. In addition, re-cultured fluorescence-activated cell sorting (FACS)-purified BMP-2/FGF-2-treated mBMSCs revealed robust calcium transients in response to electrical field stimulation which were inhibited by the L-type calcium channel (LTCC) inhibitor, nifedipine, and displayed caffeine-sensitive intracellular calcium stores. In summary, our results show that mBMSCs can adopt a functional cardiac phenotype through treatment with factors essential to embryonic cardiogenesis that are induced after cardiac ischemia. This study provides the first evidence that mBMSCs with long-term self-renewal potential possess the capability to serve as a functional cardiomyocyte precursor through the appropriate paracrine input and cross-talk within an appropriate cardiac microenvironment.

Prevention of pediatric medication errors by hospital pharmacists and the potential benefit of computerized physician order entry.

OBJECTIVES: The purpose of this work was to characterize medication errors and adverse drug events intercepted by a system of pediatric clinical pharmacists and to determine whether the addition of a computerized physician order entry system would improve medication safety. METHODS: The study included 16,938 medication orders for 678 admissions to the pediatric units of a large academic community hospital. Pediatric clinical pharmacists reviewed medication orders and monitored subsequent medication use. Medication errors and adverse drug events were identified by daily review of documentation, voluntary reporting, and solicitation. Each potentially harmful medication error was judged whether or not it was intercepted and, if not, whether it would have been captured by a computerized physician order entry system. RESULTS: Overall, 865 medication errors occurred, corresponding with a rate of 5.2 per 100 medication orders. A near-miss rate of 0.96% and a preventable adverse drug event rate of 0.09% were observed. Overall, 78% of potentially harmful prescribing errors were intercepted; however, none of the potentially harmful errors occurring at administration was intercepted and accounted for 50% of preventable adverse drug events. A computerized physician order entry system could capture additional potentially harmful prescribing and transcription errors (54%-73%) but not administration errors (0% vs 6%). CONCLUSIONS: A system of pediatric clinical pharmacists effectively intercepted inpatient prescribing errors but did not capture potentially harmful medication administration errors. The addition of a computerized physician order entry system to pharmacists is unlikely to prevent administration errors, which pose the highest risk of patient injury.