Wetland compensation and its impacts on ß-diversity.

The anthropogenic degradation of natural ecological communities can cause biodiversity loss in the form of biotic homogenization (i.e., reduced ß-diversity). Biodiversity offsetting practices, such as compensatory wetland mitigation, may inadvertently cause biotic homogenization if they produce locally homogenous or regionally recurring communities. The fact that compensation wetlands often resemble degraded wetlands suggests that potential impacts to ß-diversity are likely. Yet, it is unknown how high-quality, low-quality (degraded), and compensation wetlands compare in terms of ß-diversity. We compared the ß-diversity of high-quality, low-quality, and compensation wetlands at local and regional scales. ß-diversity was quantified as the average distance to group centroids in multivariate space based on pairwise comparisons of community composition. The local spatial structure of ß-diversity was assessed using species turnover across plots. Indicator species analysis was used to describe compositional differences potentially contributing to differences in ß-diversity. Overall, the ß-diversity of compensation sites did not differ from high-quality or low-quality natural wetlands. However, compensation wetlands had a high degree of internal turnover along the hydrological gradient, which culminated in homogenous zones in the wettest areas. Compared to high-quality wetlands, low-quality wetlands had significantly lower ß-diversity at local scales, but significantly greater ß-diversity at regional scales. Indicator species results showed that compensation wetlands were distinguished by low conservation value species typically found in old fields and waste areas. This analysis also indicated that the invasive grass Phalaris arundinacea was indicative of low-quality and compensation wetlands. This species is likely contributing to differing patterns of ß-diversity between high-quality and low-quality wetlands. These results indicate that conclusions regarding ß-diversity depend on scale and scope of analysis. Particularly, the unique architecture of compensation wetlands makes conclusions regarding within-site ß-diversity dependent on the observer's position along the hydrological gradient. Additionally, while we conclude that compensation wetlands are not contributing to biotic homogenization at the regional scale, these wetlands are distinct from both high-quality and low-quality wetlands in their composition and structure. Therefore, assessments of the overall success of wetland mitigation programs should acknowledge the reality of these differences.

Conquering the beyond Rule of Five Space with an Optimized High-Throughput Caco-2 Assay to Close Gaps in Absorption Prediction.

Current drug development tends towards complex chemical molecules, referred to as "beyond rule of five" (bRo5) compounds, which often exhibit challenging physicochemical properties. Measuring Caco-2 permeability of those compounds is difficult due to technical limitations, including poor recovery and detection sensitivity. We implemented a novel assay, with optimized incubation and analytics, to measure permeability close to equilibrium. In this setup an appropriate characterization of permeability for bRo5 compounds is achievable. This equilibrated Caco-2 assay was verified with respect to data validity, compound recovery, and in vitro to in vivo correlation for human absorption. Compared to a standard assay, it demonstrated comparable performance in predicting the human fraction absorbed (fa) for reference compounds. The equilibrated assay also successfully characterized the permeability of more than 90% of the compounds analyzed, the majority of which were bRo5 (68%). These compounds could not be measured using the standard assay. Permeability and efflux ratio (ER) were highly predictive for in vivo absorption for a large set of internal bRo5 compounds. Reference cut-offs enabled the correct classification of high, moderate, and low absorption. This optimized equilibrated Caco-2 assay closes the gap for a high-throughput cellular permeability method in the bRo5 chemical space.

Beyond Rule of Five and PROTACs in Modern Drug Discovery: Polarity Reducers, Chameleonicity, and the Evolving Physicochemical Landscape.

Developing orally bioavailable drugs demands an understanding of absorption in early drug development. Traditional methods and physicochemical properties optimize absorption for rule of five (Ro5) compounds; beyond rule of five (bRo5) drugs necessitate advanced tools like the experimental measure of exposed polarity (EPSA) and the AbbVie multiparametric score (AB-MPS). Analyzing AB-MPS and EPSA against ∼1000 compounds with human absorption data and ∼10,000 AbbVie tool compounds (∼1000 proteolysis targeting chimeras or PROTACs, ∼7000 Ro5s, and ∼2000 bRo5s) revealed new patterns of physicochemical trends. We introduced a high-throughput "polarity reduction" descriptor: ETR, the EPSA-to-topological polar surface area (TPSA) ratio, highlights unique bRo5 and PROTAC subsets for specialized drug design strategies for effective absorption. Our methods and guidelines refine drug design by providing innovative in vitro approaches, enhancing physicochemical property optimization, and enabling accurate predictions of intestinal absorption in the complex bRo5 domain.

High-Throughput SFC-MS/MS Method to Measure EPSA and Predict Human Permeability.

Permeability is a key factor driving the absorption of orally administered drugs. In early discovery, the efficient evaluation of permeability, particularly for compounds violating Lipinski's Rule of 5, remains challenging. Addressing this, we established a high-throughput method to measure the experimental polar surface area (HT-EPSA) as an in vitro surrogate to measure permeability. Compared to earlier methods, HT-EPSA significantly reduces data acquisition time with enhanced sensitivity, selectivity, and data quality. In the effort of translating EPSA to human in vitro and in vivo passive permeability, we demonstrated the application of EPSA for predicting Caco-2 cell and human intestinal permeability, showing improvements over topological polar surface area and the parallel artificial membrane permeability assay for rank-ordering permeability in a proteolysis targeting chimera case study. The HT-EPSA method is expected to be highly beneficial in guiding early stage compound rank-ordering, faster decision-making, and in predicting in vitro and/or in vivo human intestinal permeability.

Calmodulin kinase II inhibition protects against structural heart disease.

Beta-adrenergic receptor (betaAR) stimulation increases cytosolic Ca(2+) to physiologically augment cardiac contraction, whereas excessive betaAR activation causes adverse cardiac remodeling, including myocardial hypertrophy, dilation and dysfunction, in individuals with myocardial infarction. The Ca(2+)-calmodulin-dependent protein kinase II (CaMKII) is a recently identified downstream element of the betaAR-initiated signaling cascade that is linked to pathological myocardial remodeling and to regulation of key proteins involved in cardiac excitation-contraction coupling. We developed a genetic mouse model of cardiac CaMKII inhibition to test the role of CaMKII in betaAR signaling in vivo. Here we show CaMKII inhibition substantially prevented maladaptive remodeling from excessive betaAR stimulation and myocardial infarction, and induced balanced changes in excitation-contraction coupling that preserved baseline and betaAR-stimulated physiological increases in cardiac function. These findings mark CaMKII as a determinant of clinically important heart disease phenotypes, and suggest CaMKII inhibition can be a highly selective approach for targeting adverse myocardial remodeling linked to betaAR signaling.

Understanding how facilitators adapt to needs of STEM faculty in online learning communities: a case study.

Background: Faculty Learning Communities (FLCs) and Faculty Online Learning Communities (FOLCs) are ways to support STEM faculty implementing research-based curricula. In these communities, faculty facilitators take on the role of sharing expertise and promoting discussion. However, as members gain more experience, their needs change from addressing logistical to pedagogical issues. Hence, facilitators need to change their practices in response. However, there is little research on the mechanisms of faculty facilitator change. In this article, we provide a case study of a specific STEM FOLC facilitator and demonstrate the usefulness of a teacher change model to investigate facilitator change. Results: Guided by our adaptation of the Interconnected Model of Professional Growth (IMPG), we conducted interviews with FOLC facilitators, and selected a case facilitator who reported changes in facilitation goals and strategies over time. The model helped us identify specific areas of change and potential mechanisms for these changes. Using themes of change identified in the case facilitator interview, we developed coding schemes to analyze his FOLC meetings over a 2-year period. We found empirical evidence from multiple data sources, including FOLC meetings and facilitator reflections, that supported the change themes, including: changing his role as an "expert" by sharing his own expertise less and drawing on others' expertise more frequently, changing his response to members' comments by jumping in to answer less frequently and withholding his own responses more often to encourage member sharing, and a change in group discussions towards less logistical and more pedagogical conversations. Conclusions: Our findings suggest that the IMPG can be fruitfully adapted to study facilitator change. A diagrammatic representation of the IMPG provides a description the types of change the case facilitator experienced and the factors that supported those changes. We discuss how the methodology used to analyze facilitator actions in FOLC group meetings may be useful to study other types of professional growth. Finally, because our analytical model allowed us to identify mechanisms of facilitator change, we describe the implications and provide suggestions to support facilitators in other faculty community groups. Supplementary Information: The online version contains supplementary material available at 10.1186/s40594-022-00371-x.

Global Analysis of Models for Predicting Human Absorption: QSAR, In Vitro, and Preclinical Models.

Models intended to predict intestinal absorption are an essential part of the drug development process. Although many models exist for capturing intestinal absorption, many questions still exist around the applicability of these models to drug types like "beyond rule of 5" (bRo5) and low absorption compounds. This presents a challenge as current models have not been rigorously tested to understand intestinal absorption. Here, we assembled a large, structurally diverse dataset of ∼1000 compounds with known in vitro, preclinical, and human permeability and/or absorption data. In silico (quantitative structure-activity relationship), in vitro (Caco-2), and in vivo (rat) models were statistically evaluated for predictive performance against this human intestinal absorption dataset. We expect this evaluation to serve as a resource for DMPK scientists and medicinal/computational chemists to increase their understanding of permeability and absorption model utility and applications for academia and industry.

The Taxonomy of Opportunities to Learn (TxOTL): a tool for understanding the learning potential and substance of interactions in faculty (online) learning community meetings.

BACKGROUND: While many research-based instructional strategies in STEM have been developed, faculty need support in implementing and sustaining use of these strategies. A number of STEM faculty professional development programs aim to provide such pedagogical support, and it is necessary to understand the activity and learning process for faculty in these settings. In this paper, a taxonomy for describing the learning opportunities in faculty (online) learning community meetings is presented. Faculty learning communities, meeting either in-person or (increasingly) online, are a common form of professional development. They aim to develop the pedagogical and reflective skills of participants through regular meetings centered on conversations about teaching and learning. RESULTS: The tool presented in this paper, the Taxonomy of Opportunities to Learn (TxOTL), provides a structured approach to making sense of the dynamic interactions that occur during faculty learning community meetings. The origins and development of the TxOTL are described, followed by a detailed presentation of the constructs that make up the TxOTL: communicative approach used in a conversation, the concepts developed, and the meeting segment category. The TxOTL characterizes the learning opportunities presented by a faculty learning community conversation through describing the content of the conversation as well as how participants engage in the conversation. Examples of the tool in use are provided through an application to a faculty online learning community serving instructors of a physical science curriculum. A visual representation used to compactly display the results of applying the taxonomy to a meeting is detailed as well. These examples serve to illustrate the types of claims the TxOTL facilitates. CONCLUSIONS: The TxOTL allows one to examine learning opportunities available to a faculty learning community group, analyze concept development present in their conversations, track change over time in a given group, and identify patterns between meeting segment categories and communicative approaches. It is useful for researchers as well as facilitators of these STEM faculty professional development groups. The taxonomy is most applicable to faculty (online) learning communities, with limited use for workshops and K-12 professional development contexts. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at (10.1186/s40594-021-00301-3).

Analyzing a faculty online learning community as a mechanism for supporting faculty implementation of a guided-inquiry curriculum.

BACKGROUND: Adoption and use of effective, research-based instructional strategies (RBISs) for STEM education is less widespread than hoped. To promote further use of RBISs, the propagation paradigm suggests that developers work with potential adopters during the development process, and provide ongoing support after adoption. This article investigates the impact of a faculty online learning community (FOLC) as a professional development mechanism for supporting faculty adopting a research-based curriculum. A FOLC uses video conference technology and online platforms to connect geographically dispersed faculty with similar backgrounds (e.g., physics faculty) and supports their teaching development. In the context of a specific FOLC, this article seeks to determine the outcomes the FOLC achieves, and how. RESULTS: Analysis of a FOLC meeting identified opportunities for rich, complex social interaction centered on the research-based curriculum. By functioning as a sounding board for ideas, a space to share experiences, a source of affective support, and a venue for troubleshooting, the FOLC mediates the achievement of a range of outcomes related to implementation of the curriculum. Survey results indicate that members feel a sense of community in the FOLC and that it provides encouragement through teaching challenges. Further results indicate participants' increased confidence in using the curriculum; familiarity with the curriculum structure and content; increased knowledge of pedagogical techniques; reflection on teaching practices in the curriculum; and use of pedagogical techniques aligned with the curriculum's core principles. Emerging evidence supports more distal outcomes, including student learning, persistence in using the curriculum, reflection in teaching practice across courses taught, and use of research-based pedagogy in other courses. CONCLUSIONS: The propagation paradigm emphasizes the need for ongoing support for adopters of RBISs. The FOLC model provides participating faculty with ongoing support through participation in a community and is an effective support mechanism for adopters of a research-based curriculum. In this study, FOLC members are increasing their knowledge and use of pedagogical techniques in the curriculum-specific course and beyond. This is facilitated by the opportunities in the FOLC for troubleshooting, idea sharing, and receiving encouragement through challenges. This model has the potential to support adopters of additional educational innovations. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s40594-020-00268-7.

Animal simulations facilitate smart drug design through prediction of nanomaterial transport to individual tissue cells.

Smart drug design for antibody and nanomaterial-based therapies allows optimization of drug efficacy and more efficient early-stage preclinical trials. The ideal drug must display maximum efficacy at target tissue sites, with transport from tissue vasculature to the cellular environment being critical. Biological simulations, when coupled with in vitro approaches, can predict this exposure in a rapid and efficient manner. As a result, it becomes possible to predict drug biodistribution within single cells of live animal tissue without the need for animal studies. Here, we successfully utilized an in vitro assay and a computational fluid dynamic model to translate in vitro cell kinetics (accounting for cell-induced degradation) to whole-body simulations for multiple species as well as nanomaterial types to predict drug distribution into individual tissue cells. We expect this work to assist in refining, reducing, and replacing animal testing, while providing scientists with a new perspective during the drug development process.

Salt-sensitive hypertension is associated with dysfunctional Cyp4a10 gene and kidney epithelial sodium channel.

Functional and biochemical data have suggested a role for the cytochrome P450 arachidonate monooxygenases in the pathophysiology of hypertension, a leading cause of cardiovascular, cerebral, and renal morbidity and mortality. We show here that disruption of the murine cytochrome P450, family 4, subfamily a, polypeptide 10 (Cyp4a10) gene causes a type of hypertension that is, like most human hypertension, dietary salt sensitive. Cyp4a10-/- mice fed low-salt diets were normotensive but became hypertensive when fed normal or high-salt diets. Hypertensive Cyp4a10-/- mice had a dysfunctional kidney epithelial sodium channel and became normotensive when administered amiloride, a selective inhibitor of this sodium channel. These studies (a) establish a physiological role for the arachidonate monooxygenases in renal sodium reabsorption and blood pressure regulation, (b) demonstrate that a dysfunctional Cyp4a10 gene causes alterations in the gating activity of the kidney epithelial sodium channel, and (c) identify a conceptually novel approach for studies of the molecular basis of human hypertension. It is expected that these results could lead to new strategies for the early diagnosis and clinical management of this devastating disease.

Author Correction: An in vitro assay and artificial intelligence approach to determine rate constants of nanomaterial-cell interactions.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

An in vitro assay and artificial intelligence approach to determine rate constants of nanomaterial-cell interactions.

In vitro assays and simulation technologies are powerful methodologies that can inform scientists of nanomaterial (NM) distribution and fate in humans or pre-clinical species. For small molecules, less animal data is often needed because there are a multitude of in vitro screening tools and simulation-based approaches to quantify uptake and deliver data that makes extrapolation to in vivo studies feasible. Small molecule simulations work because these materials often diffuse quickly and partition after reaching equilibrium shortly after dosing, but this cannot be applied to NMs. NMs interact with cells through energy dependent pathways, often taking hours or days to become fully internalized within the cellular environment. In vitro screening tools must capture these phenomena so that cell simulations built on mechanism-based models can deliver relationships between exposure dose and mechanistic biology, that is biology representative of fundamental processes involved in NM transport by cells (e.g. membrane adsorption and subsequent internalization). Here, we developed, validated, and applied the FORECAST method, a combination of a calibrated fluorescence assay (CF) with an artificial intelligence-based cell simulation to quantify rates descriptive of the time-dependent mechanistic biological interactions between NMs and individual cells. This work is expected to provide a means of extrapolation to pre-clinical or human biodistribution with cellular level resolution for NMs starting only from in vitro data.

Validation of a Smartphone-Based Approach to In Situ Cognitive Fatigue Assessment.

BACKGROUND: Acquired Brain Injuries (ABIs) can result in multiple detrimental cognitive effects, such as reduced memory capability, concentration, and planning. These effects can lead to cognitive fatigue, which can exacerbate the symptoms of ABIs and hinder management and recovery. Assessing cognitive fatigue is difficult due to the largely subjective nature of the condition and existing assessment approaches. Traditional methods of assessment use self-assessment questionnaires delivered in a medical setting, but recent work has attempted to employ more objective cognitive tests as a way of evaluating cognitive fatigue. However, these tests are still predominantly delivered within a medical environment, limiting their utility and efficacy. OBJECTIVE: The aim of this research was to investigate how cognitive fatigue can be accurately assessed in situ, during the quotidian activities of life. It was hypothesized that this assessment could be achieved through the use of mobile assistive technology to assess working memory, sustained attention, information processing speed, reaction time, and cognitive throughput. METHODS: The study used a bespoke smartphone app to track daily cognitive performance, in order to assess potential levels of cognitive fatigue. Twenty-one participants with no prior reported brain injuries took place in a two-week study, resulting in 81 individual testing instances being collected. The smartphone app delivered three cognitive tests on a daily basis: (1) Spatial Span to measure visuospatial working memory; (2) Psychomotor Vigilance Task (PVT) to measure sustained attention, information processing speed, and reaction time; and (3) a Mental Arithmetic Test to measure cognitive throughput. A smartphone-optimized version of the Mental Fatigue Scale (MFS) self-assessment questionnaire was used as a baseline to assess the validity of the three cognitive tests, as the questionnaire has already been validated in multiple peer-reviewed studies. RESULTS: The most highly correlated results were from the PVT, which showed a positive correlation with those from the prevalidated MFS, measuring 0.342 (P<.008). Scores from the cognitive tests were entered into a regression model and showed that only reaction time in the PVT was a significant predictor of fatigue (P=.016, F=2.682, 95% CI 9.0-84.2). Higher scores on the MFS were related to increases in reaction time during our mobile variant of the PVT. CONCLUSIONS: The results show that the PVT mobile cognitive test developed for this study could be used as a valid and reliable method for measuring cognitive fatigue in situ. This test would remove the subjectivity associated with established self-assessment approaches and the need for assessments to be performed in a medical setting. Based on our findings, future work could explore delivering a small set of tests with increased duration to further improve measurement reliability. Moreover, as the smartphone assessment tool can be used as part of everyday life, additional sources of data relating to physiological, psychological, and environmental context could be included within the analysis to improve the nature and precision of the assessment process.

Life cycle assessment needs predictive spatial modelling for biodiversity and ecosystem services.

International corporations in an increasingly globalized economy exert a major influence on the planet's land use and resources through their product design and material sourcing decisions. Many companies use life cycle assessment (LCA) to evaluate their sustainability, yet commonly-used LCA methodologies lack the spatial resolution and predictive ecological information to reveal key impacts on climate, water and biodiversity. We present advances for LCA that integrate spatially explicit modelling of land change and ecosystem services in a Land-Use Change Improved (LUCI)-LCA. Comparing increased demand for bioplastics derived from two alternative feedstock-location scenarios for maize and sugarcane, we find that the LUCI-LCA approach yields results opposite to those of standard LCA for greenhouse gas emissions and water consumption, and of different magnitudes for soil erosion and biodiversity. This approach highlights the importance of including information about where and how land-use change and related impacts will occur in supply chain and innovation decisions.

Targeted deletion of angiotensin II type 2 receptor caused cardiac rupture after acute myocardial infarction.

BACKGROUND: Accumulating evidence has suggested that the cardiac renin-angiotensin system is activated during the remodeling process after myocardial infarction (MI). Although 2 types of angiotensin II receptors (AT(1) and AT(2)) are upregulated in the infarcted tissue, the contribution of AT(2) to the subsequent fibrogenetic phase of wound healing is less certain. This study was conducted to evaluate the role of AT(2) in wound healing after MI using an in vivo intervention study in mice with MI. METHODS AND RESULTS: We examined myocardial hypertrophy, cardiac fibrosis, and morphological evidence of fibrillar collagen accumulation at the infarcted and noninfarcted regions in male mice lacking the AT(2) receptor (Agtr2-/Y) and age-matched wild-type (WT) animals. Of the Agtr2-/Y mice, 63.6% died of cardiac rupture, whereas 23.5% of the WT mice died of the same cause within 1 week. The extent of fibrosis and that of collagen gene expression in Agtr2-/Y mice were significantly reduced compared with WT mice at 1 week after coronary ligation. Furthermore, MI resulted in a marked increase in the prostaglandin E(2) (PGE(2)) level at 4 days after surgery in Agtr2-/Y mice. In WT mice, the PGE(2) level was also elevated after MI but to a significantly lesser extent than in Agtr2-/Y mice. CONCLUSIONS: A chronic loss of AT(2) by gene targeting prevented the collagen deposition and caused cardiac rupture. The markedly elevated PGE(2) may be a mechanism that inhibits collagen synthesis in the infarcted region of Agtr2-/Y mice.

Tetracycline protects myocardium against ischemic injury.

Stress pretreatments protect myocardium from ischemic injury. We hypothesized that tetracycline, an antibiotic, may induce a stress response via the inhibition of mitochondrial translation as it induces the cold stress response by translational inhibition in E. coli. If so, tetracycline may protect myocardium from ischemic injury as stress pretreatments do. Thus, we investigated the effects of tetracycline on myocardial ischemia and its association with stress response. In a dog model of acute ischemia, 4mg/kg tetracycline injected 30 min prior to the occlusion improved the functional recovery from stunning of myocardium caused by ischemia. The same dosage of tetracycline dramatically reduced the size of infarct area in murine hearts analyzed by tetrazolium staining. In HeLa cells, tetracycline induced molecules that were increased by cold stress, which suggests that tetracycline may induce a cold stress-like response in mammalian cells. These molecules were also induced by ischemic stress in murine hearts, suggesting that the stress response caused by translational inhibition in mitochondria may be associated with the cardioprotection by tetracycline. Our results suggest that a subclinical dosage of tetracycline may protect heart from ischemic injury. Therefore, tetracycline may be of great use in suppressing the development of infarction caused by myocardial ischemia. This study is also important for providing new insights into the non-antimicrobial effects of tetracycline and its derivatives.

Non-Cytotoxic Quantum Dot-Chitosan Nanogel Biosensing Probe for Potential Cancer Targeting Agent.

Quantum dot (Qdot) biosensors have consistently provided valuable information to researchers about cellular activity due to their unique fluorescent properties. Many of the most popularly used Qdots contain cadmium, posing the risk of toxicity that could negate their attractive optical properties. The design of a non-cytotoxic probe usually involves multiple components and a complex synthesis process. In this paper, the design and synthesis of a non-cytotoxic Qdot-chitosan nanogel composite using straight-forward cyanogen bromide (CNBr) coupling is reported. The probe was characterized by spectroscopy (UV-Vis, fluorescence), microscopy (Fluorescence, Scanning Electron Microscopy (SEM), Transmission Electron Microscopy (TEM) and Dynamic Light Scattering. This activatable ("OFF"/"ON") probe contains a core-shell Qdot (CdS:Mn/ZnS) capped with dopamine, which acts as a fluorescence quencher and a model drug. Dopamine capped "OFF" Qdots can undergo ligand exchange with intercellular glutathione, which turns the Qdots "ON" to restore fluorescence. These Qdots were then coated with chitosan (natural biocompatible polymer) functionalized with folic acid (targeting motif) and Fluorescein Isothiocyanate (FITC; fluorescent dye). To demonstrate cancer cell targetability, the interaction of the probe with cells that express different folate receptor levels was analyzed, and the cytotoxicity of the probe was evaluated on these cells and was shown to be nontoxic even at concentrations as high as 100 mg/L.

Hyperacute rejection of mouse lung by human blood: characterization of the model and the role of complement.

BACKGROUND: The pathophysiology of hyperacute lung rejection (HALR) is not fully understood. A mouse model of HALR by human blood would be valuable to efficiently dissect the molecular mechanisms underlying this complex process, but it has not been described. METHODS: We developed a xenogenic mouse lung-perfusion model. Perfusion with heparinized autologous blood (n=3) was compared with human blood unmodified (n=7) or pretreated with C1 inhibitor (n=5) or soluble complement receptor type 1 (n=6) at unchanged flow conditions. RESULTS: Perfusion with autologous blood was associated with stable physiologic parameters and no overt evidence of lung injury for up to 2 hr. Pulmonary artery perfusion pressure increased rapidly after introduction of unmodified human blood, plasma anti-Gal(alpha)1,3Gal antibodies declined (90% immunoglobulin [Ig]M, 80% IgG), and lungs reliably met survival endpoints within 11 min (median 10 min, confidence interval [CI]: 9-11). Human Ig and neutrophils were rapidly sequestered in the lung. Survival was significantly prolonged in the soluble complement receptor type 1 group (36 min, CI: 26-46) (P<0.01) and in the C1 inhibitor group (23 min, CI: 21-25) (P<0.05), and pulmonary vascular resistance elevation and complement activation were significantly attenuated but not prevented. CONCLUSIONS: Hyperacute rejection of mouse lung by human blood occurs with kinetics, physiology, and histology closely analogous to the pig-to-human model. In addition, as in that model, neither of two potent soluble-phase complement inhibitors prevented complement activation or HALR. We conclude that the mouse lung model is relevant to dissect the cellular and molecular mechanisms governing HALR.

Calmodulin kinase II inhibition protects against myocardial cell apoptosis in vivo.

Inhibition of the multifunctional Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) or depletion of sarcoplasmic reticulum (SR) Ca(2+) stores protects against apoptosis from excessive isoproterenol (Iso) stimulation in cultured ventricular myocytes, suggesting that CaMKII inhibition could be a novel approach to reducing cell death in conditions of increased adrenergic tone, such as myocardial infarction (MI), in vivo. We used mice with genetic myocardial CaMKII inhibition due to transgenic expression of a highly specific CaMKII inhibitory peptide (AC3-I) to test whether CaMKII was important for apoptosis in vivo. A second line of mice expressed a scrambled, inactive form of AC3-I (AC3-C). AC3-C and wild-type (WT) littermates were used as controls. AC3-I mice have reduced SR Ca(2+) content and are resistant to Iso- and MI-induced apoptosis compared with AC3-C and WT mice. Phospholamban (PLN) is a target for modulation of SR Ca(2+) content by CaMKII. PLN(-/-) mice have increased susceptibility to Iso-induced apoptosis. Verapamil pretreatment prevented Iso-induced apoptosis in PLN(-/-) mice, indicating the involvement of a Ca(2+)-dependent pathway. AC3-I and AC3-C mice were bred into a PLN(-/-) background. Loss of PLN increased and equalized SR Ca(2+) content in AC3-I, AC3-C, and WT mice and abolished the resistance to apoptosis in AC3-I mice after MI. There was a trend (P = 0.07) for increased Iso-induced apoptosis in AC3-I mice lacking PLN compared with AC3-I mice with PLN. These findings indicate CaMKII is proapoptotic in vivo and suggest that regulation of SR Ca(2+) content by PLN contributes to the antiapoptotic mechanism of CaMKII inhibition.