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Truncation mutations in cardiac myosin binding protein C (cMyBP-C) are common causes of hypertrophic cardiomyopathy (HCM). Heterozygous carriers present with classical HCM, while homozygous carriers present with early onset HCM that rapidly progress to heart failure. We used CRISPR-Cas9 to introduce heterozygous (cMyBP-C+/−) and homozygous (cMyBP-C−/−) frame-shift mutations into MYBPC3 in human iPSCs. Cardiomyocytes derived from these isogenic lines were used to generate cardiac micropatterns and engineered cardiac tissue constructs (ECTs) that were characterized for contractile function, Ca2+-handling, and Ca2+-sensitivity. While heterozygous frame shifts did not alter cMyBP-C protein levels in 2-D cardiomyocytes, cMyBP-C+/− ECTs were haploinsufficient. cMyBP-C−/− cardiac micropatterns produced increased strain with normal Ca2+-handling. After 2 wk of culture in ECT, contractile function was similar between the three genotypes; however, Ca2+-release was slower in the setting of reduced or absent cMyBP-C. At 6 wk in ECT culture, the Ca2+-handling abnormalities became more pronounced in both cMyBP-C+/− and cMyBP-C−/− ECTs, and force production became severely depressed in cMyBP-C−/− ECTs. RNA-seq analysis revealed enrichment of differentially expressed hypertrophic, sarcomeric, Ca2+-handling, and metabolic genes in cMyBP-C+/− and cMyBP-C−/− ECTs. Our data suggest a progressive phenotype caused by cMyBP-C haploinsufficiency and ablation that initially is hypercontractile, but progresses to hypocontractility with impaired relaxation. The severity of the phenotype correlates with the amount of cMyBP-C present, with more severe earlier phenotypes observed in cMyBP-C−/− than cMyBP-C+/− ECTs. We propose that while the primary effect of cMyBP-C haploinsufficiency or ablation may relate to myosin crossbridge orientation, the observed contractile phenotype is Ca2+-mediated.

Observation of dispersion in field situations has left three issues that may be better understood by applying advective transport phenomena. (1) In some experiments, the longitudinal dispersivity becomes constant with increasing pathlength and in other cases it remains growing. (2) Dispersivities reported from multiple comprehensive observations at a single site differ at similar pathlength in some cases more than a factor two. (3) The observed difference between the plume fronts and plume tails is not represented in the reported parameters. The analytic equations for advective transport phenomena at macro-scale of (De Lange 2019) describe the thickness of the affected flow-tube and the spread of the plume front and tail. The scale factor defines the size of the averaging domain and so of the initial phase. The new macro-scale correlation coefficient relates the growth of the longitudinal dispersivity beyond the initial phase to the aquifer heterogeneity. Using stochastic parameters for the aquifer heterogeneity, the parameters are quantified at fourteen field experiments in the USA, Canada and Europe enabling the comparison of calculated and reported final dispersivities. Using the quantified parameters, 146 reported and calculated dispersivities along the traveled paths show a good match. A dispersivity derived from the local plume growth may differ a factor of two from the aquifer-representative value. The growths of plume fronts and tails between two plume stages are assessed in fourteen cases and compared to calculated values. Distinctive parameters for the plume front and tail support better understanding of field situations. A user-ready spreadsheet is provided.

Cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CM) may provide an important bridge between animal models and the intact human myocardium. Fulfilling this potential is hampered by their relative immaturity, leading to poor physiological responsiveness. hiPSC-CMs grown in traditional two-dimensional (2D) culture lack a t-tubular system, have only rudimentary intracellular calcium-handling systems, express predominantly embryonic sarcomeric protein isoforms, and preferentially use glucose as an energy substrate. Culturing hiPSC-CM in a variety of three-dimensional (3D) environments and the addition of nutritional, pharmacological, and electromechanical stimuli have proven, to various degrees, to be beneficial for maturation. We present a detailed assessment of a novel model in which hiPSC-CMs and hiPSC-derived cardiac fibroblasts are cocultured in a 3D fibrin matrix to form engineered cardiac tissue constructs (hiPSC-ECTs). The hiPSC-ECTs are responsive to physiological stimuli, including stretch, frequency, and β-adrenergic stimulation, develop a t-tubular system, and demonstrate calcium-handling and contractile kinetics that compare favorably with ventricular human myocardium. Furthermore, transcript levels of various genes involved in calcium-handling and contraction are increased. These markers of maturation become more robust over a relatively short period of time in culture (6 wk vs. 2 wk in hiPSC-ECTs). A comparison of the hiPSC-ECT molecular and performance variables with those of human cardiac tissue and other available engineered tissue platforms is provided to aid selection of the most appropriate platform for the research question at hand. Important and noteworthy aspects of this human cardiac model system are its reliance on “off-the-shelf” equipment, ability to provide detailed physiological performance data, and the ability to achieve a relatively mature cardiac physiology without additional nutritional, pharmacological, and electromechanical stimuli that may elicit unintended effects on function. NEW & NOTEWORTHY This study seeks to provide an in-depth assessment of contractile performance of human iPSC-derived cardiomyocytes cultured together with fibroblasts in a 3-dimensional-engineered tissue and compares performance both over time as cells mature, and with corresponding measures found in the literature using alternative 3D culture configurations. The suitability of 3D-engineered human cardiac tissues to model cardiac function is emphasized, and data provided to assist in the selection of the most appropriate configuration based on the target application.

Significance Hypertrophic cardiomyopathy (HCM) is a common genetic heart disease and a leading cause of sudden cardiac death in young adults. HCM has been linked to mutations in genes encoding sarcomeric proteins, but how different mutations can result in a similar clinical phenotype is unknown. Analysis of surgical heart tissue samples from HCM patients with severe outflow track obstruction using high-resolution mass spectrometry–based top-down proteomics revealed a common pattern of altered sarcomeric proteoforms across HCM tissues compared to non-failing donor heart tissues. Our data suggest that common pathways are associated with clinical phenotypes in patients diagnosed with obstructive HCM, opening the door for the development of interventions that target the HCM phenotype rather than the individual sarcomeric gene mutation., Hypertrophic cardiomyopathy (HCM) is the most common heritable heart disease. Although the genetic cause of HCM has been linked to mutations in genes encoding sarcomeric proteins, the ability to predict clinical outcomes based on specific mutations in HCM patients is limited. Moreover, how mutations in different sarcomeric proteins can result in highly similar clinical phenotypes remains unknown. Posttranslational modifications (PTMs) and alternative splicing regulate the function of sarcomeric proteins; hence, it is critical to study HCM at the level of proteoforms to gain insights into the mechanisms underlying HCM. Herein, we employed high-resolution mass spectrometry–based top-down proteomics to comprehensively characterize sarcomeric proteoforms in septal myectomy tissues from HCM patients exhibiting severe outflow track obstruction (n = 16) compared to nonfailing donor hearts (n = 16). We observed a complex landscape of sarcomeric proteoforms arising from combinatorial PTMs, alternative splicing, and genetic variation in HCM. A coordinated decrease of phosphorylation in important myofilament and Z-disk proteins with a linear correlation suggests PTM cross-talk in the sarcomere and dysregulation of protein kinase A pathways in HCM. Strikingly, we discovered that the sarcomeric proteoform alterations in the myocardium of HCM patients undergoing septal myectomy were remarkably consistent, regardless of the underlying HCM-causing mutations. This study suggests that the manifestation of severe HCM coalesces at the proteoform level despite distinct genotype, which underscores the importance of molecular characterization of HCM phenotype and presents an opportunity to identify broad-spectrum treatments to mitigate the most severe manifestations of this genetically heterogenous disease.

Cardiac myosin binding protein C (cMyBP-C) is a sarcomeric protein important in the regulation of cardiac contraction through its phosphorylation-dependent interactions with myosin heavy chain and actin. MYBPC3, the gene encoding cMyBP-C, contains the largest number of hypertrophic cardiomyopathy (HCM)-causing mutations. Interestingly, the region of MYBPC3 containing the greatest number of HCM-causing missense mutations, the central domains, have an unknown physiological relevance to the overall function of the protein. Intriguingly, central domain C3 contains a positively charged, putative surface binding interface for an as-yet unidentified ligand. Two of the most common HCM-causing mutations, R495Q and R502W, located in C3, appear to alter this charged interface. We have previously shown that these two mutations lead to altered contractile kinetics and hypophosphorylation of cMyBP-C. We hypothesize that this region of C3 contains an important binding region that is disrupted by both the R495Q and R502W mutations. To reveal candidate binding proteins, we performed a pull-down assay using mouse cardiac homogenate and a GST-tagged C2-C4 fragment of cMyBP-C, with or without the R495Q mutation. Mass spectrometry revealed that myomegalin, a 250 kDa phosphodiesterase-4D-interacting protein (PDE4DIP), bound the WT, but not the R495Q C2-C4 fragment. To confirm the interaction between cMyBP-C and myomegalin in situ , we performed Duolink proximity ligase assay (PLA) in cultured neonatal mouse cardiomyocytes which revealed a positive interaction in cMyBP-C +/+ but not in cMyBP-C -/- cells (n>20) (Figure). Our data strongly suggest that a large isoform of myomegalin, separately known to form a protein complex with PKA/AKAP, forms a novel interaction with cMyBP-C. We hypothesize that perturbations in this interaction lead to altered PKA localization and the observed changes in R495Q and R502W cMyBP-C phosphorylation, and may contribute to the HCM phenotype.

Rationale: Human pluripotent stem cell (hPSC)–derived cardiomyocytes exhibit the properties of fetal cardiomyocytes, which limits their applications. Various methods have been used to promote maturation of hPSC-cardiomyocytes; however, there is a lack of an unbiased and comprehensive method for accurate assessment of the maturity of hPSC-cardiomyocytes. Objective: We aim to develop an unbiased proteomics strategy integrating high-throughput top-down targeted proteomics and bottom-up global proteomics for the accurate and comprehensive assessment of hPSC-cardiomyocyte maturation. Methods and Results: Utilizing hPSC-cardiomyocytes from early- and late-stage 2-dimensional monolayer culture and 3-dimensional engineered cardiac tissue, we demonstrated the high reproducibility and reliability of a top-down proteomics method, which enabled simultaneous quantification of contractile protein isoform expression and associated post-translational modifications. This method allowed for the detection of known maturation-associated contractile protein alterations and, for the first time, identified contractile protein post-translational modifications as promising new markers of hPSC-cardiomyocytes maturation. Most notably, decreased phosphorylation of α-tropomyosin was found to be associated with hPSC-cardiomyocyte maturation. By employing a bottom-up global proteomics strategy, we identified candidate maturation-associated markers important for sarcomere organization, cardiac excitability, and Ca 2+ homeostasis. In particular, upregulation of myomesin 1 and transmembrane 65 was associated with hPSC-cardiomyocyte maturation and validated in cardiac development, making these promising markers for assessing maturity of hPSC-cardiomyocytes. We have further validated α-actinin isoforms, phospholamban, dystrophin, αB-crystallin, and calsequestrin 2 as novel maturation-associated markers, in the developing mouse cardiac ventricles. Conclusions: We established an unbiased proteomics method that can provide accurate and specific assessment of the maturity of hPSC-cardiomyocytes and identified new markers of maturation. Furthermore, this integrated proteomics strategy laid a strong foundation for uncovering the molecular pathways involved in cardiac development and disease using hPSC-cardiomyocytes.

While the waste hierarchy promotes the reuse of waste there currently remains limited reuse of mine waste in South Africa. Overburden, waste rock, tailings and slag are stockpiled or disposed leading to environmental liabilities including acid mine drainage and leaching. Globally there are numerous documented uses of mine waste. However, South African waste legislation currently fails to create an enabling environment for mine waste reuse, and consequently hinder the generation of economic value from this waste stream. Mine waste in South Africa is defined as a residue, which is considered a resource of minerals for future re-mining. Being classified as hazardous, the reuse of mining residue is legally challenging in South Africa. Furthermore legislated environmental management process promotes cradle-to-grave approaches, which is not supportive of any waste reuse initiative. Legislative authorisation falls between two government departments’ often with conflicting mandates leaving mine residue becoming trapped between being a waste and being a by-product with economic value.

The absence of recent research on dispersion in engineering applications indicates the need for a description that is more focused on field and modeling practice. Engineers may benefit from simple calculation tools allowing them to understand the processes encountered in the field. Based on a conceptual model for advective transport through an elongated conductivity zone, for example, in fluvial sediments, explicit expressions are presented for macro‐scale phenomena: (1) the different travel distances of water particles traveling in laminar flow through and adjacent to a single zone with conductivity higher or lower than that of the aquifer; (2) the affected thickness of the bundle of flowlines; (3) the distinction of inflow, outflow, and through‐flow sections; (4) the development of a plume front vs. that of a tail; (5) conservation of mass causing water particles to travel both slower and faster than the aquifer average velocity while passing a single zone. The spread derived from a spatial distribution in a field experiment relates to the geometric mean of the spreads of the plume front and tail. The results obtained for a single conductivity zone are expanded for a general aquifer that is characterized by stochastic parameters. A fundamental new expression describes the dispersive mass flux as the product of the advective volume shift and the related local concentration difference. Contrary to Fickian theory, the dispersive mass flux in both the front and tail of a plume in highly heterogeneous aquifers is limited. In modeling, the advective volume shift is proportional to the cell size., Article impact statement: Simple expressions to explain differences in dispersion parameters between field and modeling practice based on advective transport.

Visual Abstract, Highlights • Many cardiomyopathy families have genetic variants whose significance is unknown. We studied a novel (E848G) mutation in MYH7, a sarcomeric protein. • Patient-specific induced pluripotent stem cell–derived cardiomyocytes and engineered heart tissues recapitulated the contractile dysfunction. • Overexpression of the E848G allele in MYH7-null induced pluripotent stem cell–derived cardiomyocytes confirms the causality of the E848G variant. • The E848G allele disrupts the protein–protein interaction between MYH7 and cardiac myosin binding protein C, presenting a potential mechanism of action. • Assessing the pathogenicity of new MYH7 variants by overexpressing them in a null background should accelerate their screening for disease causality., Summary A novel myosin heavy chain 7 mutation (E848G) identified in a familial cardiomyopathy was studied in patient-specific induced pluripotent stem cell–derived cardiomyocytes. The cardiomyopathic human induced pluripotent stem cell–derived cardiomyocytes exhibited reduced contractile function as single cells and engineered heart tissues, and genome-edited isogenic cells confirmed the pathogenic nature of the E848G mutation. Reduced contractility may result from impaired interaction between myosin heavy chain 7 and cardiac myosin binding protein C.

Three-dimensional (3D) human induced pluripotent stem cell-derived engineered cardiac tissues (hiPSC-ECTs) have emerged as a promising alternative to two-dimensional hiPSC-cardiomyocyte monolayer systems because hiPSC-ECTs are a closer representation of endogenous cardiac tissues and more faithfully reflect the relevant cardiac pathophysiology. The ability to perform functional and molecular assessments using the same hiPSC-ECT construct would allow for more reliable correlation between observed functional performance and underlying molecular events, and thus is critically needed. Herein, for the first time, we have established an integrated method that permits sequential assessment of functional properties and top-down proteomics from the same single hiPSC-ECT construct. We quantitatively determined the differences in isometric twitch force and the sarcomeric proteoforms between two groups of hiPSC-ECTs that differed in the duration of time of 3D-ECT culture. Importantly, by using this integrated method we discovered a new and strong correlation between the measured contractile parameters and the phosphorylation levels of alpha-tropomyosin between the two groups of hiPSC-ECTs. The integration of functional assessments together with molecular characterization by top-down proteomics in the same hiPSC-ECT construct enables a holistic analysis of hiPSC-ECTs to accelerate their applications in disease modeling, cardiotoxicity, and drug discovery. Data are available via ProteomeXchange with identifier PXD022814.

Green economic principles call for a broadening of the scope of mine planning and operations to incorporate sectoral diversification as a formal part of the life cycle of mines. In this opinion piece we argue that sectoral integration of mining holds promise to improve the long term business resilience of the mining sector. We explain this notion for the case of diversification to agriculture in South Africa. The challenge is certainly not unique to South Africa and whilst the mining sector has become more socially and environmentally conscious, it remains a challenge world-wide. Ample opportunity remains for innovative interventions that go beyond mitigating risk of the post-mining landscape, towards socially and economically inclusive development solutions. It is hoped that this contribution ignites more debate on finding new ways to develop the complex relationships between sectors into mutually beneficial and cooperative coexistence based on true sectoral integration, so as to improve the long term sustainability of developing economies.

Cardiac myosin-binding protein C (cMyBP-C) is a functional sarcomeric protein that regulates contractility in response to contractile demand, and many mutations in cMyBP-C lead to hypertrophic cardiomyopathy (HCM). To gain insight into the effects of disease-causing cMyBP-C missense mutations on contractile function, we expressed the pathogenic W792R mutation (substitution of a highly conserved tryptophan residue by an arginine residue at position 792) in mouse cardiomyocytes lacking endogenous cMyBP-C and studied the functional effects using three-dimensional engineered cardiac tissue constructs (mECTs). Based on complete conservation of tryptophan at this location in fibronectin type II (FnIII) domains, we hypothesized that the W792R mutation affects folding of the C6 FnIII domain, destabilizing the mutant protein. Adenoviral transduction of wild-type (WT) and W792R cDNA achieved equivalent mRNA transcript abundance, but not equivalent protein levels, with W792R compared with WT controls. mECTs expressing W792R demonstrated abnormal contractile kinetics compared with WT mECTs that were nearly identical to cMyBP-C-deficient mECTs. We studied whether common pathways of protein degradation were responsible for the rapid degradation of W792R cMyBP-C. Inhibition of both ubiquitin-proteasome and lysosomal degradation pathways failed to increase full-length mutant protein abundance to WT equivalence, suggesting rapid cytosolic degradation. Bacterial expression of WT and W792R protein fragments demonstrated decreased mutant stability with altered thermal denaturation and increased susceptibility to trypsin digestion. These data suggest that the W792R mutation destabilizes the C6 FnIII domain of cMyBP-C, resulting in decreased full-length protein expression. This study highlights the vulnerability of FnIII-like domains to mutations that alter domain stability and further indicates that missense mutations in cMyBP-C can cause disease through a mechanism of haploinsufficiency.NEW & NOTEWORTHY This study is one of the first to describe a disease mechanism for a missense mutation in cardiac myosin-binding protein C linked to hypertrophic cardiomyopathy. The mutation decreases stability of the fibronectin type III domain and results in substantially reduced mutant protein expression dissonant to transcript abundance.

Modern day societies and economies are becoming increasingly vulnerable to the continued erosion of the stocks and flows of essential ecosystem services. Thus, the management of complex socio-economic systems to effectively provide these essential services has become a global priority policy and academic research area. Understanding how underlying processes and functions contribute towards the provision of final ecosystem services can facilitate improved dissemination of credible, legitimate and salient information to decision-makers. This paper presents an ecosystem service value chain analysis framework that applies basic system dynamics modelling in the form of causal loop diagrams to facilitate an alternative analysis of ecosystem service value chains. A scoping application of the framework is applied to a case study for flood attenuation services in the Baviaanskloof catchment in South Africa. The framework enables the identification of forward linkages and ripple effects in individual value chains of final ecosystem services as well as the identification and assessment of challenges and opportunities within individual causal pathways. Ultimately, providing the potential to advance strategies for improving the efficiency and effectiveness of final ecosystem service provision.

New and emerging risks create growing uncertainty and unpredictability within enterprise risk management. While ISO 31000:2009 is a progressive risk management framework, it is limited in its guidance on how to contextualise complex risks. The application of systems thinking to risk management provides the opportunity to better understand complexity by viewing risk and the consequence of change as part of overall system behaviour. System modelling tools enable organisations to better contextualise their risk landscape. These tools assist organisations to identify vulnerabilities between social and ecological variables in the system within they exist. Determining drivers of change leading to system vulnerabilities can assist in understanding threshold limits of the system, thus enabling the organisation to build system resilience and organisational sustainability.

The advent of human induced pluripotent stem cell (hiPSC) technology has revolutionized the way we study inherited diseases, as it allows us to study the effect of mutations in a human context. This is particularly true in the heart were species-specific differences has dramatic effects on heart rate, and expression of protein isoforms involved in excitation-contraction coupling. A major drawback of using hiPSCs is, however that cardiomyocytes derived from hiPSC (hiPSC-CM) are relatively immature, limiting their utility as a tool to study adult onset cardiac diseases. We postulated that culturing hiPSC-CM in a 3D environment in engineered cardiac tissue constructs (ECT) would promote hiPSC-CM maturation. ECTs were generated from lactate-selected day 30 hiPSCs that were mixed with isogenic hiPSC derived cardiac fibroblasts in a 10:1 ratio. Cells were mixed with fibrinogen and thrombin and seeded into molds under vacuum in FlexCell dishes to form ECT. ECTs were harvested 14-21 or 42-52 days later and subjected to functional testing, transcriptional analysis and top-down mass-spectrometry of sarcomeric proteins to assess maturation. Functionally, we found that prolonged culture of hiPSCs in ECT resulted in increased calcium transient amplitude (0.856 vs. 0.462; P < 0.01) and an acceleration of calcium kinetics (calcium release time 67.7 ms vs. 109.3 ms; P < 0.01 and calcium decay time 148.3 ms vs. 188.0 ms; P < 0.05). This acceleration of calcium kinetics with time in culture was also true for twitch force kinetics (time to peak twitch force 178.6 ms vs. 208.0 ms; P < 0.05). Furthermore, beta adrenergic stimulation had a much greater effect on twitch kinetics in older ECTs (reduction in contraction time of 26.5% vs. 7.1%; P < 0.01). Transcriptional analysis revealed an increase in expression level of the beta 1 adrenergic receptor in older ECTs, while top-down mass spectrometry showed increased expression and phosphorylation of cardiac troponin I (cTnI) and mono-phosphorylated cTnI, as well as decreased phosphorylation of alpha-tropomyocin, all markers of myocardial maturation. Taken together, these data supports our hypothesis that prolonged culture of hiPSC-CM in ECT promotes maturation of the calcium handling system and the contractile apparatus.

Given the broad (economic, social and environmental) objectives of a green economy, and the limitations associated with mainstream measures of economic performance (such as gross domestic product), an alternative or expanded set of indicators is required for measuring progress toward a green economy. This article develops a composite index for measuring green economic performance, based on 26 indicators across the economic, social and environmental dimensions. The index will enable comparison of a country's green economic performance both over time and relative to other countries. Furthermore, the index is constructed in such a way as to allow for disaggregation (i.e. for scores on individual components to be seen at a glance), such that areas of specific concern can be easily identified and addressed, and progress in each area monitored over time. The index was tested on data from 193 countries, and the resulting country rankings were assessed.

Rationale: With a prevalence of 1 in 200 individuals, hypertrophic cardiomyopathy (HCM) is thought to be the most common genetic cardiac disease, with potential outcomes that include severe hypertrophy, heart failure, and sudden cardiac death (SCD). Though much research has furthered our understanding of how HCM-causing mutations in genes such as cardiac myosin-binding protein C (MYBPC3) impair contractile function, it remains unclear how such dysfunction leads to hypertrophy and/or arrhythmias, which comprise the HCM phenotype. Identification of early response mediators could provide rational therapeutic targets to reduce disease severity. Our goal was to differentiate physiologic and pathophysiologic hypertrophic growth responses and identify early genetic mediators in the development of cardiomegaly in the cardiac myosin-binding protein C-null (cMyBP-C-/-) mouse model of HCM. Methods and Results: We performed microarray analysis on left ventricles of wild-type (WT) and cMyBPC-/- mice (n = 7 each) at postnatal day (PND) 1 and PND 9, before and after the appearance of an overt HCM phenotype. Applying the criteria of ≥2-fold change, we identified genes whose change was exclusive to pathophysiologic growth (n = 61), physiologic growth (n = 30), and genes whose expression changed ≥2-fold in both WT and cMyBP-C-/- hearts (n = 130). Furthermore, we identified genes that were dysregulated in PND1 cMyBP-C-/- hearts prior to hypertrophy, including genes in mechanosensing pathways and potassium channels linked to arrhythmias. One gene of interest, Xirp2, and its protein product, are regulated during growth but also show early, robust prehypertrophic upregulation in cMyBP-C-/- hearts. Additionally, the transcription factor Zbtb16 also shows prehypertrophic upregulation at both gene and protein levels. Conclusion: Our transcriptome analysis generated a comprehensive data set comparing physiologic vs. hypertrophic growth in mice lacking cMyBP-C. It highlights the importance of extracellular matrix pathways in hypertrophic growth and early dysregulation of potassium channels. Prehypertrophic upregulation of Xirp2 in cMyBP-C-/- hearts supports a growing body of evidence suggesting Xirp2 has the capacity to elicit both hypertrophy and arrhythmias in HCM. Dysregulation of Xirp2, as well as Zbtb16, along with other genes associated with mechanosensing regions of the cardiomyocyte implicate stress-sensing in these regions as a potentially important early response in HCM.

Rationale: With a prevalence of 1 in 200 individuals, hypertrophic cardiomyopathy (HCM) is thought to be the most common genetic cardiac disease, with potential outcomes that include severe hypertrophy, heart failure, and sudden cardiac death (SCD). Though much research has furthered our understanding of how HCM-causing mutations in genes such as cardiac myosin-binding protein C (MYBPC3) impair contractile function, it remains unclear how such dysfunction leads to hypertrophy and/or arrhythmias, which comprise the HCM phenotype. Identification of early response mediators could provide rational therapeutic targets to reduce disease severity. Our goal was to differentiate physiologic and pathophysiologic hypertrophic growth responses and identify early genetic mediators in the development of cardiomegaly in the cardiac myosin-binding protein C-null (cMyBP-C-/-) mouse model of HCM. Methods and Results: We performed microarray analysis on left ventricles of wild-type (WT) and cMyBPC-/- mice (n = 7 each) at postnatal day (PND) 1 and PND 9, before and after the appearance of an overt HCM phenotype. Applying the criteria of ≥2-fold change, we identified genes whose change was exclusive to pathophysiologic growth (n = 61), physiologic growth (n = 30), and genes whose expression changed ≥2-fold in both WT and cMyBP-C-/- hearts (n = 130). Furthermore, we identified genes that were dysregulated in PND1 cMyBP-C-/- hearts prior to hypertrophy, including genes in mechanosensing pathways and potassium channels linked to arrhythmias. One gene of interest, Xirp2, and its protein product, are regulated during growth but also show early, robust prehypertrophic upregulation in cMyBP-C-/- hearts. Additionally, the transcription factor Zbtb16 also shows prehypertrophic upregulation at both gene and protein levels. Conclusion: Our transcriptome analysis generated a comprehensive data set comparing physiologic vs. hypertrophic growth in mice lacking cMyBP-C. It highlights the importance of extracellular matrix pathways in hypertrophic growth and early dysregulation of potassium channels. Prehypertrophic upregulation of Xirp2 in cMyBP-C-/- hearts supports a growing body of evidence suggesting Xirp2 has the capacity to elicit both hypertrophy and arrhythmias in HCM. Dysregulation of Xirp2, as well as Zbtb16, along with other genes associated with mechanosensing regions of the cardiomyocyte implicate stress-sensing in these regions as a potentially important early response in HCM.

The economic, social and environmental costs of food waste are being increasingly recognised. Food waste consists of both edible and inedible components. Whilst wastage of edible food is problematic for obvious reasons, there are also costs associated with the disposal of the inedible fraction to landfill. This is the third in a series of papers examining the costs of food waste throughout the value chain in South Africa. The previous papers focused on the edible portion of food waste. In this paper, costs associated with inedible food waste in South Africa are estimated, in terms of the value foregone by not recovering this waste for use in downstream applications, such as energy generation or composting; as well as costs associated with disposal to landfill. Opportunity costs are estimated at R6.4 (US$0.64) billion per annum, or R2668 (US$266) per tonne. Adding this to the previous estimate for edible food waste of R61.5 billion per annum (in 2012 prices; equivalent to R65 billion in 2013 prices) results in a total opportunity cost of food waste in South Africa (in terms of loss of a potentially valuable food source or resource) of R71.4 (US$7.14) billion per annum, or R5667 (US$567) per tonne. Thereafter, estimates of the costs associated with disposal of this food waste to landfill, including both financial costs and externalities (social and environmental costs), are taken into account. These costs amount to R255 (US$25) per tonne, giving rise to a total cost of food waste in South Africa of R75 billion (US$7.5 billion) per annum, or R5922 (US$592) per tonne. This is equivalent to 2.2% of South Africa’s 2013 GDP.

Rationale: Hypertrophic cardiomyopathy (HCM) occurs in ~0.5% of the population and is a leading cause of sudden cardiac death (SCD) in young adults. Cardiomyocyte hypertrophy has been the accepted mechanism for cardiac enlargement in HCM, but the early signaling responsible for initiating hypertrophy is poorly understood. Mutations in cardiac myosin binding protein C (MYBPC3) are among the most common HCM-causing mutations. Ablation of Mybpc3 in an HCM mouse model (cMyBP-C−/−) rapidly leads to cardiomegaly by postnatal day (PND) 9, though hearts are indistinguishable from wild-type (WT) at birth. This model provides a unique opportunity to explore early processes involved in the dramatic postnatal transition to hypertrophy. Methods and Results: We performed microarray analysis, echocardiography, qPCR, immunohistochemistry (IHC), and isolated cardiomyocyte measurements to characterize the perinatal cMyBP-C−/− phenotype before and after overt hypertrophy. cMyBP-C−/− hearts showed elevated cell cycling at PND1 that transitioned to hypertrophy by PND9. An expanded time course revealed that increased cardiomyocyte cycling was associated with elevated heart weight to body weight ratios prior to cellular hypertrophy, suggesting that cell cycling resulted in cardiomyocyte proliferation. Animals heterozygous for the cMyBP-C deletion trended in the direction of the homozygous null, yet did not show increased heart size by PND9. Conclusions: Results indicate that altered regulation of the cell cycling pathway and elevated proliferation precedes hypertrophy in the cMyBP-C−/− HCM model, and suggests that increased cardiomyocyte number contributes to increased heart size in cMyBP-C−/− mice. This pre-hypertrophic period may reflect a unique time during which the commitment to HCM is determined and disease severity is influenced.

Mutations in cardiac myosin binding protein C (cMyBP-C) are prevalent causes of hypertrophic cardiomyopathy (HCM). Although HCM-causing truncation mutations in cMyBP-C are well studied, the growing number of disease-related cMyBP-C missense mutations remain poorly understood. Our objective was to define the primary contractile effect and molecular disease mechanisms of the prevalent cMyBP-C E258K HCM-causing mutation in nonremodeled murine engineered cardiac tissue (mECT). Wild-type and human E258K cMyBP-C were expressed in mECT lacking endogenous mouse cMyBP-C through adenoviral-mediated gene transfer. Expression of E258K cMyBP-C did not affect cardiac cell survival and was appropriately incorporated into the cardiac sarcomere. Functionally, expression of E258K cMyBP-C caused accelerated contractile kinetics and severely compromised twitch force amplitude in mECT. Yeast two-hybrid analysis revealed that E258K cMyBP-C abolished interaction between the N terminal of cMyBP-C and myosin heavy chain sub-fragment 2 (S2). Furthermore, this mutation increased the affinity between the N terminal of cMyBP-C and actin. Assessment of phosphorylation of three serine residues in cMyBP-C showed that aberrant phosphorylation of cMyBP-C is unlikely to be responsible for altering these interactions. We show that the E258K mutation in cMyBP-C abolishes interaction between N-terminal cMyBP-C and myosin S2 by directly disrupting the cMyBP-C–S2 interface, independent of cMyBP-C phosphorylation. Similar to cMyBP-C ablation or phosphorylation, abolition of this inhibitory interaction accelerates contractile kinetics. Additionally, the E258K mutation impaired force production of mECT, which suggests that in addition to the loss of physiological function, this mutation disrupts contractility possibly by tethering the thick and thin filament or acting as an internal load.

Hypertrophic cardiomyopathy (HCM) caused by mutations in cardiac myosin–binding protein-C (cMyBP-C) is a heterogenous disease in which the phenotypic presentation is influenced by genetic, environmental, and developmental factors. Though mouse models have been used extensively to study the contractile effects of cMyBP-C ablation, early postnatal hypertrophic and dilatory remodeling may overshadow primary contractile defects. The use of a murine engineered cardiac tissue (mECT) model of cMyBP-C ablation in the present study permits delineation of the primary contractile kinetic abnormalities in an intact tissue model under mechanical loading conditions in the absence of confounding remodeling events. We generated mechanically integrated mECT using isolated postnatal day 1 mouse cardiac cells from both wild-type (WT) and cMyBP-C–null hearts. After culturing for 1 wk to establish coordinated spontaneous contraction, we measured twitch force and Ca2+ transients at 37°C during pacing at 6 and 9 Hz, with and without dobutamine. Compared with WT, the cMyBP-C–null mECT demonstrated faster late contraction kinetics and significantly faster early relaxation kinetics with no difference in Ca2+ transient kinetics. Strikingly, the ability of cMyBP-C–null mECT to increase contractile kinetics in response to adrenergic stimulation and increased pacing frequency were severely impaired. We conclude that cMyBP-C ablation results in constitutively accelerated contractile kinetics with preserved peak force with minimal contractile kinetic reserve. These functional abnormalities precede the development of the hypertrophic phenotype and do not result from alterations in Ca2+ transient kinetics, suggesting that alterations in contractile velocity may serve as the primary functional trigger for the development of hypertrophy in this model of HCM. Our findings strongly support a mechanism in which cMyBP-C functions as a physiological brake on contraction by positioning myosin heads away from the thin filament, a constraint which is removed upon adrenergic stimulation or cMyBP-C ablation.

Oligomers of homomeric voltage-gated potassium channels associate early in biogenesis as the nascent proteins emerge from the polysome. Less is known about how proteins emerging from different polysomes associate to form hetero-oligomeric channels. Here, we report that alternate mRNA transcripts encoding human ether-a-go-go-related gene (hERG) 1a and 1b subunits, which assemble to produce ion channels mediating cardiac repolarization, are physically associated during translation. We show that shRNA specifically targeting either hERG 1a or 1b transcripts reduced levels of both transcripts, but only when they were coexpressed heterologously. Both transcripts could be copurified with an Ab against the nascent hERG 1a N terminus. This interaction occurred even when translation of 1b was prevented, indicating the transcripts associate independent of their encoded proteins. The association was also demonstrated in cardiomyocytes, where levels of both hERG transcripts were reduced by either 1a or 1b shRNA, but native KCNE1 and ryanodine receptor 2 (RYR2) transcripts were unaffected. Changes in protein levels and membrane currents mirrored changes in transcript levels, indicating the targeted transcripts were undergoing translation. The physical association of transcripts encoding different subunits provides the spatial proximity required for nascent proteins to interact during biogenesis, and may represent a general mechanism facilitating assembly of heteromeric protein complexes involved in a range of biological processes.

Food waste is problematic for a number of reasons, including the loss of a potentially valuable food source or resource for use in other processes (e.g. energy generation or composting), wasted resources and emissions in the food supply chain, and problems associated with the disposal of organic waste to landfill. This paper quantifies the household food waste stream in South Africa, in order to draw attention to the magnitude of the problem. In addition, it estimates the economic (monetary) value of the wasted food, as well as the costs associated with disposing putrescible food waste to landfill, in order to highlight the associated costs to society. Costs associated with the loss of a potentially valuable food source are valued using a weighted average market price of the wasted food. Costs associated with the disposal of food waste to landfill are quantified based on estimates of the financial and external costs associated with landfilling. For household food waste alone, the costs to society associated with these two food-waste related problems are estimated at approximately R21.7 billion (approximately US$2.7 billion) per annum, or 0.82% of South Africa's annual GDP. These costs are therefore significant, particularly considering that household food waste accounts for less than 4% of total food losses across the food supply chain.

The expansion of protected areas is a critical component of strategies to promote the continued existence of biodiversity (i.e., life at all levels of biological organization) as climate changes, but scientific, social, and economic uncertainties associated with climate change are some of the major obstacles preventing such expansion. New models of climate change and species distribution and new methods of conservation planning now make it possible to explore the uncertainties associated with climate changes and species responses. Yet few reliable estimates of the costs of expanding protected areas and methods for determining these costs exist, largely because of the many (and uncertain) determinants of these costs. We developed a cost-accounting model to estimate the range in costs of various options for expanding protected areas and to explore the variables that drive these costs. Model development was informed by an existing plan to expand protected areas in the Cape Floristic Region of South Africa to address species conservation under a scenario of climate change. The 50-year present value of total costs varied from US$260 million ($1077/ha) for an off-reserve option that involves agreements with landowners and no compensation of forgone production and associated revenue to $1020 million ($4228/ha) for an on-reserve option that involves land acquisition and protection. The costs of acquiring land or compensating landowners for forgone production and development opportunities were the major drivers of the total costs across all options because most of the area identified in the protected-area expansion plan consisted of urban and high-quality agricultural lands. Total costs were also affected by changes in protected area extent and discount rate. Model-generated outputs such as these may be useful for informing implementation strategies and the allocation of future efforts in monitoring, data collection, and model development. Resumen: La expansion de areas protegidas es un componente critico de las estrategias para promover la existencia continua de la biodiversidad (i.e., vida en todos los niveles de organizacion biologica) a medida que cambia el clima, pero las incertidumbres cientificas, sociales y economicas asociadas con el cambio climatico son algunos de los principales obstaculos para dicha expansion. Los modelos nuevos de cambio climatico y distribucion de especies y los metodos nuevas para planificar la conservacion hacen posible explorar las incertidumbres asociadas con cambios en el clima y las respuestas de las especies. Sin embargo, existen pocas estimaciones confiables de los costos de la expansion de las areas protegidas y de los metodos para determinar esos costos, debido principalmente a los muchos (e inciertos) determinantes de estos costos. Desarrollamos un metodo que considera costos para estimar el rango de los costos de varias opciones para la expansion de areas protegidas y explorar las variables que influyen en estos costos. El desarrollo del modelos fue informado por un plan existente para expandir las areas protegidas en la Region Floristica del Cabo en Sudafrica para atender la conservacion de especies en un escenario de cambio climatico. El valor actual de los costos totales a 50 anos vario de US$260 millones ($1077/ha) para una opcion que implica acuerdos con propietarios de tierras y sin compensacion por la produccion de oportunidad y los ingresos asociados hasta $1020 millones ($4288/ha) para una opcion que implica la adquisicion y proteccion de tierras. Los costos de adquisicion de tierras o de compensacion a propietarios por produccion de oportunidad y desarrollo de oportunidades fueron los factores principales de los costos totales en todas las opciones debido a que casi toda el area identificada en la expansion de areas protegidas consistio de terrenos urbanos y agricolas de alta calidad. Los costos totales tambien fueron afectados por cambios en la extension de areas protegidas y la tasa de descuento. Los resultados generados por modelos como estos pueden ser utiles para informar la implementacion de estrategias y la asignacion de esfuerzos futuros de monitoreo, recoleccion de datos y desarrollo de modelos.

Global sustainable development depends on the capacity of natural, social and economic systems to adapt to external stimuli. However, building this adaptive capacity in the developing world context of sub-Saharan Africa will require substantial investment in these systems, which most countries in this region simply cannot afford. Given that their social, economic and environmental security depends on developments in developing countries, we argue that developed countries should assume a leading role in making such investments in developing countries, in order to secure their own long-term well-being. This will require cooperative governance between developed and developing countries, and targeted investment in a number of key areas. This calls for a new global contract between rich and poor nations in order to achieve global stability and sustainability. Copyright © 2009 John Wiley & Sons, Ltd and ERP Environment.

This study is a first attempt at a holistic economic evaluation of South African endeavours to manage invasive alien plants using biological control. Our focus was on the delivery of ecosystem services from habitats that are invaded by groups of weeds, rather than by each individual weed species. We established the net present value of the weed biological control efforts, and derived benefit:cost ratios by comparing this value (a cost) to the estimated value of ecosystem services protected by weed biological control. We identified four major functional groupings of invading alien plants, and assessed their impact on water resources, grazing and biodiversity. We estimated the area that remained free of invasions due to all historic control efforts in South Africa, and the proportion that remained free of invasion as a result of biological control (which was initiated in 1913). The estimated value of potential ecosystem services amounted to 152 billion South African rands (ZAR—presently, about US$ 19.7 billion) annually. Although an estimated ZAR 6.5 billion was lost every year due to invading alien plants, this would have amounted to an estimated additional ZAR 41.7 billion had no control been carried out, and 5–75% of this protection was due to biological control. The benefit:cost ratios ranged from 50:1 for invasive sub-tropical shrubs to 3,726:1 for invasive Australian trees. Benefit:cost ratios remained positive and our conclusion, that biological control has brought about a considerable level of protection of ecosystem services, remains robust even when our estimates of the economic impacts of key variables (i.e. sensitivity analyses of indeterminate variables) were substantially reduced.

Development of a conceptualization of a hydrogeologic system serves as the basis of groundwater modeling. While existing groundwater data models are designed to store groundwater system information, none is designed to capture its conceptual view. This study addresses this need by presenting a new object-oriented Conceptualization Groundwater Data Model that represents a groundwater system as a series of aquifer layers with defined aquifer properties and water boundary conditions. A case study is presented that develops the conceptual view of the groundwater system beneath Konza Prairie. This single conceptualization is used to support groundwater models across existing technologies of finite difference, finite element, and analytical element methods. While the models each employ different mathematics, data input files, and formats, all models are founded on the same conceptualization process that is represented using this new data model. The case study illustrates the data model's promise as an effective mechanism for groundwater system conceptualization and data storage, and utility for various groundwater computational models. This conceptualization of a groundwater data model suggests a new focus on incorporating system conceptualization into data model design.

Objective— Drugs that activate peroxisome proliferator–activated receptor (PPAR) γ improve glucose sensitivity and lower blood pressure, whereas dominant-negative mutations in PPARγ cause severe insulin resistance and hypertension. We hypothesize that these PPARγ mutants regulate target genes opposite to those of ligand-mediated activation, and we tested this hypothesis on a genomewide scale. Methods and Results— We integrated gene expression data in aorta specimens from mice treated with the PPARγ ligand rosiglitazone with data from mice containing a globally expressed knockin of the PPARγ P465L dominant-negative mutation. We also integrated our data with publicly available data sets containing the following: (1) gene expression profiles in many human tissues, (2) PPARγ target genes in 3T3-L1 adipocytes, and (3) experimentally validated PPARγ binding sites throughout the genome. Many classic PPARγ target genes were induced by rosiglitazone and repressed by dominant-negative PPARγ. A similar pattern was observed for about 90% of the gene sets regulated by both rosiglitazone and dominant-negative PPARγ. Genes exhibiting this pattern of contrasting regulation were significantly enriched for nearby PPARγ binding sites. Conclusion— These results provide convincing evidence that the PPARγ P465L mutation causes transcriptional effects that are opposite to those mediated by PPARγ ligand, thus validating mice carrying the mutation as a model of PPARγ interference.

Global declines in biodiversity and the widespread degradation of ecosystem services have led to urgent calls to safeguard both. Responses to this urgency include calls to integrate the needs of ecosystem services and biodiversity into the design of conservation interventions. The benefits of such integration are purported to include improvements in the justification and resources available for these interventions. Nevertheless, additional costs and potential trade-offs remain poorly understood in the design of interventions that seek to conserve biodiversity and ecosystem services. We sought to investigate the synergies and trade-offs in safeguarding ecosystem services and biodiversity in South Africa's Little Karoo. We used data on three ecosystem services-carbon storage, water recharge, and fodder provision-and data on biodiversity to examine several conservation planning scenarios. First, we investigated the amount of each ecosystem service captured incidentally by a conservation plan to meet targets for biodiversity only while minimizing opportunity costs. We then examined the costs of adding targets for ecosystem services into this conservation plan. Finally, we explored trade-offs between biodiversity and ecosystem service targets at a fixed cost. At least 30% of each ecosystem service was captured incidentally when all of biodiversity targets were met. By including data on ecosystem services, we increased the amount of services captured by at least 20% for all three services without additional costs. When biodiversity targets were reduced by 8%, an extra 40% of fodder provision and water recharge were obtained and 58% of carbon could be captured for the same cost. The opportunity cost (in terms of forgone production) of safeguarding 100% of the biodiversity targets was about US$500 million. Our results showed that with a small decrease in biodiversity target achievement, substantial gains for the conservation of ecosystem services can be achieved within our biodiversity priority areas for no extra cost.

Tissue-specific knockouts generated through Cre-loxP recombination have become an important tool to manipulate the mouse genome. Normally, two successive rounds of breeding are performed to generate mice carrying two floxed target-gene alleles and a transgene expressing Cre-recombinase tissue-specifically. We show herein that two promoters commonly used to generate endothelium-specific ( Tie2) and smooth muscle-specific [smooth muscle myosin heavy chain ( Smmhc)] knockout mice exhibit activity in the female and male germ lines, respectively. This can result in the inheritance of a null allele in the second generation that is not tissue specific. Careful experimental design is required therefore to ensure that tissue-specific knockouts are indeed tissue specific and that appropriate controls are used to compare strains.

The transcription factor PPARγ is expressed in endothelium and vascular muscle where it may exert antiinflammatory and antioxidant effects. We tested the hypothesis that PPARγ plays a protective role in the vasculature by examining vascular structure and function in heterozygous knockin mice expressing the P465L dominant negative mutation in PPARγ (L/+). In L/+ aorta, responses to the endothelium-dependent agonist acetylcholine (ACh) were not affected, but there was an increase in contraction to serotonin, PGF 2α , and endothelin-1. In cerebral blood vessels both in vitro and in vivo, ACh produced dilation that was markedly impaired in L/+ mice. Superoxide levels were elevated in cerebral arterioles from L/+ mice and responses to ACh were restored to normal with a scavenger of superoxide. Diameter of maximally dilated cerebral arterioles was less, whereas wall thickness and cross-sectional area was greater in L/+ mice, indicating cerebral arterioles underwent hypertrophy and remodeling. Thus, interference with PPARγ signaling produces endothelial dysfunction via a mechanism involving oxidative stress and causes vascular hypertrophy and inward remodeling. These findings indicate that PPARγ has vascular effects which are particularly profound in the cerebral circulation and provide genetic evidence that PPARγ plays a critical role in protecting blood vessels.

Fresh water resources provide a platform for complex and often emotional issues to develop, particularly in resource scarcity situations. Bulk water infrastructure contains elements of a public good and proved vulnerable to failures in market and government driven allocation strategies. Common to both are uncaptured costs and benefits due to shortcomings in cost quantification techniques. Natural ecosystems stands to lose the most since ecosystem services are often not quantifiable in monetary terms and therefore neglected in allocation decision-making. This paper took on the challenge of expanding current decision-support in order to promote more inclusive long-term water management. A case-study approach with the focus on a choice related problem regarding different long-term bulk water resource management options was applied in the Western Cape province. The paper incorporated components of economic valuation theory, a public survey and a modified Delphi expert panel technique. Both spatial and temporal dimensions of the decision-making context were expanded. Two surveys were completed to accommodate these expansions. The first focused on public preference in water allocation management and the relative merit of accommodating public preference in highly specialised decision-making such as long-term water allocation decision-making. The second survey utilized a modified Delphi technique in which an expert panel indicated the relative merit of two alternative long-term allocation strategies. A willingness to pay for "greener" water was observed and may be used to motivate a paradigm shift from management's perspective to consider, without fear of harming their own political position, "greener" water supply options more seriously even if these options imply higher direct costs to public.

In 1985, I attended the first course on analytic ele-ment modeling in the Netherlands, where Professor OttoStrack of the University of Minnesota presented hisnewly conceived analytic element method (AEM; Strack1989) at the Technical University Delft from which hegraduated years before. While he explained the principlesand applications of the method, I started to realize thatthe AEM might be uniquely suited to modeling detailedground water flow systems covering large regions be-cause it enables cutting, pasting, and linking of entiremodels as well as of model parts.In 1987, at the National Institute for Inland Water Man-agement and Waste Water Treatment in the Netherlands(RIZA),therewasmuchinterestinnationalmodelingintheNetherlands because of serious water management prob-lems that first became apparent during the major drought of1976. In fact, there existed a national water managementsystem of models, called PAWN (Policy Analysis for Watermanagement in the Netherlands; Rand Corporation 1982).PAWN is an integrated system of models for simulating thedistribution over the numerous national and regional sur-face waters in this wet country and the effects on agricul-ture, nature (ecology), power plants, shipping, flushing ofcoastal areas against salt water intrusion, and drinkingwater. PAWN was lacking treatment of the ground waterreservoir, which had become apparent in the policy analy-sis of 1985 (Pulles 1985). The NAtional GROundwaterModel (NAGROM) should cover this gap as part ofPAWN.

Infantile-onset Pompe disease is an autosomal recessive disorder caused by the complete loss of lysosomal glycogen-hydrolyzing enzyme acid α-glucosidase (GAA) activity, which results in lysosomal glycogen accumulation and prominent cardiac and skeletal muscle pathology. The mechanism by which loss of GAA activity causes cardiomyopathy is poorly understood. We reprogrammed fibroblasts from patients with infantile-onset Pompe disease to generate induced pluripotent stem (iPS) cells that were differentiated to cardiomyocytes (iPSC-CM). Pompe iPSC-CMs had undetectable GAA activity and pathognomonic glycogen-filled lysosomes. Nonetheless, Pompe and control iPSC-CMs exhibited comparable contractile properties in engineered cardiac tissue. Impaired autophagy has been implicated in Pompe skeletal muscle; however, control and Pompe iPSC-CMs had comparable clearance rates of LC3-II-detected autophagosomes. Unexpectedly, the lysosome-associated membrane proteins, LAMP1 and LAMP2, from Pompe iPSC-CMs demonstrated higher electrophoretic mobility compared with control iPSC-CMs. Brefeldin A induced disruption of the Golgi in control iPSC-CMs reproduced the higher mobility forms of the LAMPs, suggesting that Pompe iPSC-CMs produce LAMPs lacking appropriate glycosylation. Isoelectric focusing studies revealed that LAMP2 has a more alkaline pI in Pompe compared with control iPSC-CMs due largely to hyposialylation. MALDI-TOF-MS analysis of N-linked glycans demonstrated reduced diversity of multiantennary structures and the major presence of a trimannose complex glycan precursor in Pompe iPSC-CMs. These data suggest that Pompe cardiomyopathy has a glycan processing abnormality and thus shares features with hypertrophic cardiomyopathies observed in the congenital disorders of glycosylation.

Aging is associated with declining cardiac contractile function as well as changes in metabolism and mitochondrial function. The relationship between age‐related changes in cardiac metabolism and declining cardiac contractile function has not been determined. In order to define the role energetics play in changes in contractile function, we measured mitochondrial NADH, [NADH] m , during continuous contractions of isolated left ventricular myocytes from young (Y) and old (O) FBN rats. Second, we explored the role of metabolic disruption with rotenone and increased workload with isoproterenol (ISO) had on age‐related changes in myocytes shortening. Single, intact myocytes were stimulated for 10 min of continuous contraction at either 2 Hz or 4 Hz while being perfused with Ringer9s solution. Properties of shortening (peak shortening and rate of shortening) were measured at the onset (T0) and after 10 min (T10) of continuous contraction, and the decline in shortening over time (T10/T0) was determined. Although young and old myocytes had similar contractile function under resting conditions, old myocytes demonstrated decrements in [NADH] m during continuous stimulation, while young myocytes maintained constant [NADH]m over this time. In addition, old myocytes exhibited impaired contractile function to a workload (ISO) and metabolic (rotenone) stress compared to young myocytes. Taken together, these results demonstrated that old myocytes are susceptible to stress‐induced contractile dysfunction which may be related to altered cellular energetics.

Mouse engineered cardiac tissue constructs (mECTs) are a new tool available to study human forms of genetic heart disease within the laboratory. The cultured strips of cardiac cells generate physiologic calcium transients and twitch force, and respond to electrical pacing and adrenergic stimulation. The mECT can be made using cells from existing mouse models of cardiac disease, providing a robust readout of contractile performance and allowing a rapid assessment of genotype-phenotype correlations and responses to therapies. mECT represents an efficient and economical extension to the existing tools for studying cardiac physiology. Human ECTs generated from iPSCMs represent the next logical step for this technology and offer significant promise of an integrated, fully human, cardiac tissue model.

We assess the monetary value of forage provisioning services for honeybees as provided by an alien tree species in the Western Cape province of South Africa. Although Eucalyptus cladocalyx is not an officially declared invader, it is cleared on a regular basis along with other invasive Eucalyptus species such as Eucalyptus camaldulensis, and Eucalyptus conferruminata (which have been prioritised for eradication in South Africa). We present some of the trade-offs associated with the clearing of E. cladocalyx by means of a practical example that illustrates a situation where the benefits of the species to certain stakeholders could support the containment of the species in demarcated areas, while allowing clearing outside such areas. Given the absence of market prices for such forage provisioning services, the replacement cost is used to present the value of the loss in forage as provided by E. cladocalyx if the alien tree species is cleared along with invasive alien tree species. Two replacement scenarios formed the basis for our calculations. The first scenario was an artificial diet as replacement for the forage provisioning service, which yielded a direct cost estimate of US$7.5 m per year. The second was based on a Fynbos cultivation/restoration initiative aimed at substituting the forage provisioning service of E. cladocalyx, which yielded a direct cost of US$20.2 m per year. These figures provide estimates of the potential additional cost burden on the beekeeping industry if E. cladocalyx is completely eradicated from the Western Cape. The cost estimates should be balanced against the negative impacts of E. cladocalyx on ecosystem services in order to make an informed decision with regard to appropriate management strategies for this species. The findings therefore serve as useful inputs to balance trade-offs for alien species that are considered as beneficial to some, but harmful to other.

SummaryDominant-negative (DN) mutations in the nuclear hormone receptor peroxisome proliferator-activated receptor-γ (PPARγ) cause hypertension by an unknown mechanism. Hypertension and vascular dysfunction are recapitulated by expression of DN PPARγ specifically in vascular smooth muscle of transgenic mice. DN PPARγ increases RhoA and Rho-kinase activity, and inhibition of Rho-kinase restores normal reactivity and reduces arterial pressure. RhoBTB1, a component of the Cullin-3 RING E3 ubiquitin ligase complex, is a PPARγ target gene. Decreased RhoBTB1, Cullin-3, and neddylated Cullin-3 correlated with increased levels of the Cullin-3 substrate RhoA. Knockdown of Cullin-3 or inhibition of cullin-RING ligase activity in aortic smooth muscle cells increased RhoA. Cullin-RING ligase inhibition enhanced agonist-mediated contraction in aortic rings from normal mice by a Rho-kinase-dependent mechanism, and it increased arterial pressure in vivo. We conclude that Cullin-3 regulates vascular function and arterial pressure, thus providing a mechanistic link between mutations in Cullin-3 and hypertension in humans.

SummaryThe renin-angiotensin system (RAS), in addition to its endocrine functions, plays a role within individual tissues such as the brain. The brain RAS is thought to control blood pressure through effects on fluid intake, vasopressin release, and sympathetic nerve activity (SNA), and may regulate metabolism through mechanisms which remain undefined. We used a double-transgenic mouse model that exhibits brain-specific RAS activity to examine mechanisms contributing to fluid and energy homeostasis. The mice exhibit high fluid turnover through increased adrenal steroids, which is corrected by adrenalectomy and attenuated by mineralocorticoid receptor blockade. They are also hyperphagic but lean because of a marked increase in body temperature and metabolic rate, mediated by increased SNA and suppression of the circulating RAS. β-adrenergic blockade or restoration of circulating angiotensin-II, but not adrenalectomy, normalized metabolic rate. Our data point to contrasting mechanisms by which the brain RAS regulates fluid intake and energy expenditure.

Bridging the science–policy gap has been of major concern to the European Commission since the beginning of this century and has led to several activities to support the issue. For instance, in the FP5 project Harmoni-CA (Chapter 2.4) workshops have been held for water managers and policy makers to ...

Improving research integration into the policy-making and implementation process remains one of the major challenges in managing complex environmental problems. Owing to its high ambition to achieve ‘good ecological status’ of Europe's fresh water resources by 2015, the adoption of the Water Framewo...

Making good use of relevant science has always been and always will be a requirement of successful policy making. However, while interaction between scientists and policy makers takes place at various levels in various ways to a greater or lesser extent, there is very rarely an explicit mechanism by...