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Over the last decades, the role of remote sensing gained in importance for monitoring applications in precision agriculture. A key factor for assessing the development of crops during the growing period is the actual biomass. As non-destructive methods of directly measuring biomass do not exist, parameters like plant height are considered as estimators. In this contribution, first results of multitemporal surveys on a maize field with a terrestrial laser scanner are shown. The achieved point clouds are interpolated to generate Crop Surface Models (CSM) that represent the top canopy. These CSMs are used for visualizing the spatial distribution of plant height differences within the field and calculating plant height above ground with a high resolution of 1 cm. In addition, manual measurements of plant height were carried out corresponding to each TLS campaign to verify the results. The high coefficient of determination (R² = 0.93) between both measurement methods shows the applicability of the presented approach. The established regression model between CSM-derived plant height and destructively measured biomass shows a varying performance depending on the considered time frame during the growing period. This study shows that TLS is a suitable and promising method for measuring plant height of maize. Moreover, it shows the potential of plant height as a non-destructive estimator for biomass in the early growing period. However, challenges are the non-linear development of plant height and biomass over the whole growing period. [ABSTRACT FROM AUTHOR]

While several models have proven to result in accurate estimations when measuring cardiac output using indicator dilution, the mono-exponential model has primarily been chosen for deriving coronary blood/plasma volume. In this study, we compared four models to derive coronary plasma volume using indicator dilution; the mono-exponential, power-law, gamma-variate, and local density random walk (LDRW) model. In anesthetized goats (N = 14), we determined the distribution volume of high molecular weight (2,000 kDa) dextrans. A bolus injection (1.0 ml, 0.65 mg/ml) was given intracoronary and coronary venous blood samples were taken every 0.5-1.0 s; outflow curves were analyzed using the four aforementioned models. Measurements were done at baseline and during adenosine infusion. Absolute coronary plasma volume estimates varied by ~25% between models, while the relative volume increase during adenosine infusion was similar for all models. The gamma-variate, LDRW, and mono-exponential model resulted in volumes corresponding with literature, whereas the power-model seemed to overestimate the coronary plasma volume. The gamma-variate and LDRW model appear to be suitable alternative models to the mono-exponential model to analyze coronary indicator-dilution curves, particularly since these models are minimally influenced by outliers and do not depend on data of the descending slope of the curve only. [ABSTRACT FROM AUTHOR]

Let (Xm)∞1 be a sequence of independent and identically distributed random variables. We give sufficient conditions for the fractional part of rnax (X1., Xn) to converge in distribution, as n←∞ to a random variable with a uniform distribution on [0, 1). [ABSTRACT FROM AUTHOR]

A long-held tenet in inositol-lipid signaling is that cleavage of membrane phosphoinositides by phospholipase Cβ (PLCβ) isozymes to increase cytosolic Ca 2+ in living cells is exclusive to Gq- and Gi-sensitive G protein-coupled receptors (GPCRs). Here we extend this central tenet and show that Gs-GPCRs also partake in inositol-lipid signaling and thereby increase cytosolic Ca 2+ . By combining CRISPR/Cas9 genome editing to delete Gα s , the adenylyl cyclase isoforms 3 and 6, or the PLCβ1-4 isozymes, with pharmacological and genetic inhibition of Gq and G11, we pin down Gs-derived Gβγ as driver of a PLCβ2/3-mediated cytosolic Ca 2+ release module. This module does not require but crosstalks with Gα s -dependent cAMP, demands Gα q to release PLCβ3 autoinhibition, but becomes Gq-independent with mutational disruption of the PLCβ3 autoinhibited state. Our findings uncover the key steps of a previously unappreciated mechanism utilized by mammalian cells to finetune their calcium signaling regulation through Gs-GPCRs., (© 2024. The Author(s).)

This paper examines how humans judge the capabilities of artificial intelligence (AI) to evaluate human attributes, specifically focusing on two key dimensions of human social evaluation: morality and competence. Furthermore, it investigates the impact of exposure to advanced Large Language Models on these perceptions. In three studies (combined N = 200), we tested the hypothesis that people will find it less plausible that AI is capable of judging the morality conveyed by a behavior compared to judging its competence. Participants estimated the plausibility of AI origin for a set of written impressions of positive and negative behaviors related to morality and competence. Studies 1 and 3 supported our hypothesis that people would be more inclined to attribute AI origin to competence-related impressions compared to morality-related ones. In Study 2, we found this effect only for impressions of positive behaviors. Additional exploratory analyses clarified that the differentiation between the AI origin of competence and morality judgments persisted throughout the first half year after the public launch of popular AI chatbot (i.e., ChatGPT) and could not be explained by participants' general attitudes toward AI, or the actual source of the impressions (i.e., AI or human). These findings suggest an enduring belief that AI is less adept at assessing the morality compared to the competence of human behavior, even as AI capabilities continued to advance., (© 2024. The Author(s).)

Background: Hypertensive disorders of pregnancy (HDP) are associated with subsequent adverse cardiac remodeling and cardiovascular disease. The role of myocardial microvascular disease among individuals with HDP and left ventricular (LV) remodeling as a potential link to cardiovascular disease is unknown. We aimed to determine whether individuals with HDP history have coronary microvascular dysfunction measured by coronary flow reserve 8 to 10 years after delivery and whether microvascular dysfunction correlates with LV remodeling., Methods: Individuals with pregnancies delivered from 2008 to 2010 underwent burst-replenishment myocardial contrast echocardiography (2017-2020) to quantify myocardial perfusion at rest and during dobutamine stress. Video intensity versus time data were used to derive β, the rate of rise of video intensity, a correlate for myocardial blood flow. Coronary flow reserve was calculated as the ratio of β at peak stress to β at rest, averaged across LV myocardial regions of interest., Results: We studied 91 individuals (aged 38±6 and 9.1±0.9 years postdelivery) and 19 with a history of HDP. Individuals with coronary microvascular dysfunction (coronary flow reserve <2.0; n=13) had a higher proportion of HDP (46.2% versus 16.7%; P =0.026) and higher prepregnancy body mass index, baseline heart rate, and hemoglobin A1c compared with those without microvascular dysfunction. The association of coronary flow reserve and HDP was attenuated after adjusting for cardiometabolic factors ( P =0.133). In exploratory subgroup analyses, individuals with both LV remodeling (relative wall thickness >0.42) and HDP (n=12) had the highest proportion of microvascular dysfunction (41.7% versus +HDP-LV remodeling [n=7] 14.3%; -HDP+LV remodeling [n=26] 7.7%; P =0.0498)., Conclusions: In this small study, HDP history is associated with coronary microvascular dysfunction 1 decade after delivery, findings that may, in part, be driven by metabolic factors including obesity and diabetes. Microvascular dysfunction may contribute to cardiovascular disease among individuals with a history of HDP., Competing Interests: Disclosures Dr Straub is a consultant and a stockholder in Creegh Pharmaceuticals. The other authors report no conflicts.

Competing Interests: Competing interests: AMS and JR are board members of the not-for-profit Metro Mapping Foundation (no honorarium involved). JB is managing director of Design Studio Panton Deventer, who provide consultation in the use of Metro Mapping. The other authors declare no conflict of interest.

Background: Virus infections drive COPD exacerbations and progression. Antiviral immunity centres on the activation of virus-specific CD8 + T-cells by viral epitopes presented on major histocompatibility complex (MHC) class I molecules of infected cells. These epitopes are generated by the immunoproteasome, a specialised intracellular protein degradation machine, which is induced by antiviral cytokines in infected cells., Methods: We analysed the effects of cigarette smoke on cytokine- and virus-mediated induction of the immunoproteasome in vitro , ex vivo and in vivo using RNA and Western blot analyses. CD8 + T-cell activation was determined in co-culture assays with cigarette smoke-exposed influenza A virus (IAV)-infected cells. Mass-spectrometry-based analysis of MHC class I-bound peptides uncovered the effects of cigarette smoke on inflammatory antigen presentation in lung cells. IAV-specific CD8 + T-cell numbers were determined in patients' peripheral blood using tetramer technology., Results: Cigarette smoke impaired the induction of the immunoproteasome by cytokine signalling and viral infection in lung cells in vitro , ex vivo and in vivo . In addition, cigarette smoke altered the peptide repertoire of antigens presented on MHC class I molecules under inflammatory conditions. Importantly, MHC class I-mediated activation of IAV-specific CD8 + T-cells was dampened by cigarette smoke. COPD patients exhibited reduced numbers of circulating IAV-specific CD8 + T-cells compared to healthy controls and asthmatics., Conclusion: Our data indicate that cigarette smoke interferes with MHC class I antigen generation and presentation and thereby contributes to impaired activation of CD8 + T-cells upon virus infection. This adds important mechanistic insight on how cigarette smoke mediates increased susceptibility of smokers and COPD patients to viral infections., Competing Interests: Conflict of Interest: H. Ma reports support for the present manuscript from the German Center for Lung Research (DZL). M. Nakayama reports overseas grant from Uehara Memorial Foundation (Japan) and overseas grant from Shiga university of Medical Science, outside the submitted work. A.L. Mora reports support for the present manuscript from NIH (NIH U01 HL1455550-01 and NIH NHLBI R01 HL149825). J.S. Lee reports participation on clinical adjudication committee with Janssen R&D, outside the submitted work. S. Krauss-Etschmann reports support for the present manuscript from the German Center for Lung Research. K. Milger reports consulting fees and lecture honoraria from AstraZeneca, GSK, Janssen, Novartis and Sanofi, outside the submitted work. C.A. Staab-Weijnitz reports support for the present manuscript from Helmholtz Association, German Center for Lung Research (DZL) and Deutsche Forschungsgemeinschaft (DFG) within the Research Training Group GRK2338. K.I. Gaede reports support for the present manuscript from Research Center Borstel – Leibniz Lung Center – BioMaterialBank North, Airway Research Center North, German Center for Lung Research (DZL), PopGen 2.0 Network (P2N). K.I. Gaede also holds a leadership role as member of the Board of Directors of the TMF (www.tmf-ev.de), outside the submitted work. I.E. Kammerl reports support for the present manuscript from ERS (Short Term Fellowship). All other authors have no potential conflicts of interest to declare., (Copyright ©The authors 2023.)

Type II alveolar epithelial cell (AECII) redox imbalance contributes to the pathogenesis of idiopathic pulmonary fibrosis (IPF), a deadly disease with limited treatment options. Here, we show that expression of membrane-bound cytochrome B5 reductase 3 (CYB5R3), an enzyme critical for maintaining cellular redox homeostasis and soluble guanylate cyclase (sGC) heme iron redox state, is diminished in IPF AECIIs. Deficiency of CYB5R3 in AECIIs led to sustained activation of the pro-fibrotic factor TGF-β1 and increased susceptibility to lung fibrosis. We further show that CYB5R3 is a critical regulator of ERK1/2 phosphorylation and the sGC/cGMP/protein kinase G axis that modulates activation of the TGF-β1 signaling pathway. We demonstrate that sGC agonists (BAY 41-8543 and BAY 54-6544) are effective in reducing the pulmonary fibrotic outcomes of in vivo deficiency of CYB5R3 in AECIIs. Taken together, these results show that CYB5R3 in AECIIs is required to maintain resilience after lung injury and fibrosis and that therapeutic manipulation of the sGC redox state could provide a basis for treating fibrotic conditions in the lung and beyond.

Women with adverse pregnancy outcomes later experience excess hypertension and cardiovascular disease, but how the events are linked is unknown. Examination of the placenta may provide clues to vascular impairments after delivery. Maternal vascular malperfusion lesions (MVMs) were abstracted from clinical reports, validated and characterized using clinical guidelines and severity score. A total of 492 women (170 with MVMs and 322 without MVMs) participated in a study visit 8 to 10 years after delivery to assess blood pressure, cardiometabolic factors, and sublingual microvascular features using sidestream dark field imaging. Covariates included age, race, adverse pregnancy outcomes (preeclampsia, small for gestational age, and preterm birth), and health behaviors. Women with versus without MVM had a distinct sublingual microvascular profile comprised of (1) lower microvascular density (-410 μm/mm 2 , P =0.015), (2) higher red blood cell filling as a marker of perfusion (2%, P =0.004), and (3) smaller perfused boundary region (-0.07 µm, P =0.025) as a measure of glycocalyx integrity, adjusted for covariates including adverse pregnancy outcomes. Women with MVM also had higher adjusted diastolic blood pressure (+2.6 mm Hg, P =0.021), total and LDL (low-density lipoprotein)-cholesterol (+11.2 mg/dL, P =0.016; +8.7 mg/dL, P =0.031). MVM associations with subsequent cardiovascular measures did not vary by type of adverse pregnancy outcome, except among women with preterm births where blood pressure was higher only among those with MVM. Results were similar when evaluated as MVM severity. A decade after delivery, women with placental vascular lesions had an adverse cardiovascular profile comprised of microvascular rarefaction, higher blood pressure and more atherogenic lipids. Placental histopathology may reveal a woman's early trajectory toward subsequent vascular disease.

Preeclampsia is a spontaneously occurring pregnancy complication diagnosed by new-onset hypertension and end-organ dysfunction with or without proteinuria. This pregnancy-specific syndrome contributes to maternal morbidity and mortality and can have detrimental effects on fetal outcomes. Preeclampsia is also linked to increased risk of maternal cardiovascular disease throughout life. Despite intense investigation of this disorder, few treatment options are available. The aim of this study was to investigate the potential therapeutic effects of maternal l-citrulline supplementation on pregnancy-specific vascular dysfunction in the male C57BL/6J × female C57BL/6J C1q -/- preeclampsia-like mouse model. l-Citrulline is a nonessential amino acid that is converted to l-arginine to promote smooth muscle and blood vessel relaxation and improve nitric oxide (NO)-mediated vascular function. To model a preeclampsia-like pregnancy, female C57BL/6J mice were mated to C1q -/- male mice, and a subset of dams was supplemented with l-citrulline throughout pregnancy. Blood pressure, systemic vascular glycocalyx, and ex vivo vascular function were investigated in late pregnancy, and postpartum at 6 and 10 mo of age. Main findings show that l-citrulline reduced blood pressure, increased vascular glycocalyx volume, and rescued ex-vivo vascular function at gestation day 17.5 in this preeclampsia-like model. The vascular benefit of l-citrulline also extended postpartum, with improved vascular function and glycocalyx measures at 6 and 10 mo of age. l-Citrulline-mediated vascular improvements appear, in part, attributable to NO pathway signaling. Taken together, l-citrulline supplementation during pregnancy appears to have beneficial effects on maternal vascular health, which may have translational implications for improved maternal cardiovascular health.

Background: Collagen VI related myopathies are congenital diseases of variable phenotype. The severe phenotype is referred to as Ullrich congenital muscular dystrophy. In this study, we describe analoguos clinical signs and histopathological alterations in Landseer dogs., Materials: We collected clinical data from two affected dogs and investigated the neuromuscular changes in five dogs from two different litters with immunohistochemistry and immunofluorescence. All affected dogs were homozygous for the p.Glu97* nonsense variant in the COL6A1 gene encoding the alpha-1 chain of collagen VI., Results: Muscle biopsies revealed alterations similar to those in human patients with Ullrich congenital muscular dystrophy including the virtual absence of collagen VI in skeletal muscles., Conclusions: The clinical and pathological characterization of the affected Landseer dogs enhances the value of this animal model for human Ullrich congenital muscular dystrophy., (© 2020 Wiley Periodicals LLC.)

Mechanisms that control mobilization of cytosolic calcium [Ca 2+ ] i are key for regulation of numerous eukaryotic cell functions. One such paradigmatic mechanism involves activation of phospholipase Cβ (PLCβ) enzymes by G protein βγ subunits from activated Gα i -Gβγ heterotrimers. Here, we report identification of a master switch to enable this control for PLCβ enzymes in living cells. We find that the Gα i -Gβγ-PLCβ-Ca 2+ signaling module is entirely dependent on the presence of active Gα q . If Gα q is pharmacologically inhibited or genetically ablated, Gβγ can bind to PLCβ but does not elicit Ca 2+ signals. Removal of an auto-inhibitory linker that occludes the active site of the enzyme is required and sufficient to empower "stand-alone control" of PLCβ by Gβγ. This dependence of Gi-Gβγ-Ca 2+ on Gα q places an entire signaling branch of G-protein-coupled receptors (GPCRs) under hierarchical control of Gq and changes our understanding of how Gi-GPCRs trigger [Ca 2+ ] i via PLCβ enzymes., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Preeclampsia is a spontaneously occurring, pregnancy-specific syndrome that is clinically diagnosed by new onset hypertension and proteinuria. Epidemiological evidence describes an association between a history of preeclampsia and increased risk for cardiovascular disease in later life; however, the mechanism(s) driving this relationship are unclear. Our study aims to leverage a novel preeclampsia-like mouse model, the C1q -/- model, to help elucidate the acute and persistent vascular changes during and following a preeclampsia-like pregnancy. Female C57BL/6J mice were mated to C1q -/- male mice to model a preeclampsia-like pregnancy ("PE-like"), and the maternal cardiovascular phenotype (blood pressure, renal function, systemic glycocalyx, and ex vivo vascular function) was assessed in late pregnancy and postpartum at 6 and 10 mo of age. Uncomplicated, normotensive pregnancies (female C57BL/6J bred to male C57BL/6J mice) served as age-matched controls. In pregnancy, PE-like dams exhibited increased systolic and diastolic pressure during mid- and late gestation, renal dysfunction, fetal growth restriction, and reduced placental efficiency. Ex vivo wire myography studies of mesenteric arteries revealed severe pregnancy-specific endothelial-dependent and -independent vascular dysfunction. At 3 and 7 mo postpartum (6 and 10 mo old, respectively), hypertension resolved in PE-like dams, whereas mild vascular dysfunction persisted at 3 mo postpartum. In conclusion, the female C57BL/6J-by-male C57BL/6J C1q -/- model recapitulates many aspects of the human preeclampsia syndrome in a low-risk, wild-type female mouse. The pregnancy-specific phenotype results in systemic maternal endothelial-dependent and -independent vascular dysfunction that persists postpartum.

Carbon dioxide (CO 2 ), the major product of metabolism, has a strong impact on cerebral blood vessels, a phenomenon known as cerebrovascular reactivity. Several vascular risk factors such as hypertension or diabetes dampen this response, making cerebrovascular reactivity a useful diagnostic marker for incipient vascular pathology, but its functional relevance, if any, is still unclear. Here, we found that GPR4, an endothelial H + receptor, and endothelial Gα q/11 proteins mediate the CO 2 /H + effect on cerebrovascular reactivity in mice. CO 2 /H + leads to constriction of vessels in the brainstem area that controls respiration. The consequential washout of CO 2 , if cerebrovascular reactivity is impaired, reduces respiration. In contrast, CO 2 dilates vessels in other brain areas such as the amygdala. Hence, an impaired cerebrovascular reactivity amplifies the CO 2 effect on anxiety. Even at atmospheric CO 2 concentrations, impaired cerebrovascular reactivity caused longer apneic episodes and more anxiety, indicating that cerebrovascular reactivity is essential for normal brain function. The site-specific reactivity of vessels to CO 2 is reflected by regional differences in their gene expression and the release of vasoactive factors from endothelial cells. Our data suggest the central nervous system (CNS) endothelium as a target to treat respiratory and affective disorders associated with vascular diseases., Competing Interests: The authors declare no competing interest.

Background: Risk factors for coronary artery disease (CAD) have been associated with endothelial dysfunction and degradation of the endothelial glycocalyx. This study was designed to compare sublingual microvascular perfusion and glycocalyx barrier properties in CAD patients and controls using noninvasive side stream darkfield imaging., Methods: Imaging of the sublingual microvasculature was performed in 52 case subjects (CAD confirmed by left heart catheterization) and 63 controls (low Framingham risk score). Red blood cell (RBC) filling percentage and functional microvascular density, measures of microvascular perfusion, and perfused boundary region (PBR), an index of glycocalyx barrier function, were measured in microvessels with a diameter ranging from 5-25 µm., Results: RBC filling percentage was lower in patients with CAD compared to controls (p < .001). Functional microvascular density did not differ between groups. The overall PBR was marginally greater in the CAD group compared to the control group (p = .08). PBR did not differ between male CAD cases and controls (p = .17). However, PBR was greater in females with CAD compared with female controls (p = .04), indicating reduced glycocalyx barrier function. This difference became more pronounced after adjusting for potential confounders., Conclusions: Our data suggest that patients with CAD are characterized by a reduction in percentage of time microvessels are occupied by RBCs. In addition, CAD is significantly associated with impaired sublingual microvascular glycocalyx barrier function in women but not men. More research is needed to determine the significance of peripheral microvascular dysfunction in the pathophysiology of CAD, and how this may differ by sex., (© 2020 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.)

Objectives: To examine barriers and facilitators to sustaining a sexual health continuous quality improvement (CQI) programme in clinics serving remote Aboriginal communities in Australia., Design: Qualitative study., Setting: Primary health care services serving remote Aboriginal communities in the Northern Territory, Australia., Participants: Seven of the 11 regional sexual health coordinators responsible for supporting the Northern Territory Government Remote Sexual Health Program., Methods: Semi-structured in-depth interviews conducted in person or by telephone; data were analysed using an inductive and deductive thematic approach., Results: Despite uniform availability of CQI tools and activities, sexual health CQI implementation varied across the Northern Territory. Participant narratives identified five factors enhancing the uptake and sustainability of sexual health CQI. At clinic level, these included adaptation of existing CQI tools for use in specific clinic contexts and risk environments (eg, a syphilis outbreak), local ownership of CQI processes and management support for CQI. At a regional level, factors included the positive framing of CQI as a tool to identify and act on areas for improvement, and regional facilitation of clinic level CQI activities. Three barriers were identified, including the significant workload associated with acute and chronic care in Aboriginal primary care services, high staff turnover and lack of Aboriginal staff. Considerations affecting the future sustainability of sexual health CQI included the need to reduce the burden on clinics from multiple CQI programmes, the contribution of regional sexual health coordinators and support structures, and access to and use of high-quality information systems., Conclusions: This study contributes to the growing evidence on how CQI approaches may improve sexual health in remote Australian Aboriginal communities. Enhancing sustainability of sexual health CQI in this context will require ongoing regional facilitation, efforts to build local ownership of CQI processes and management of competing demands on health service staff., Competing Interests: Competing interests: The authors have ongoing involvement in Aboriginal health, sexual health and CQI in Australia, as researchers, clinicians and policymakers., (© Author(s) (or their employer(s)) 2019. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

The development of mature natural killer (NK) cells expressing killer cell immunoglobulin-like receptors (KIRs) depends on cell contact-dependent signals from nonhematopoietic cells. So far, detailed studies of this process have been hampered by the lack of an appropriate in vitro model. Here, human bone marrow-derived mesenchymal stem cells (MSCs), generated under good manufacturing practice (GMP) conditions, are established as a supportive niche for in vitro NK cell differentiation. In the presence of MSCs, cord blood and bone marrow-derived hematopoietic stem and progenitor cells (HSPCs) effectively and reproducibly differentiated into mature KIR-expressing NK cells. Notably, the novel in vitro differentiation assay enabled us to analyze the impact of HLA class I ligands on KIR repertoire development. To this end, a panel of MSC lines divergent for expression of the major KIR ligands C1, C2, and Bw4 was used for NK cell differentiation. The resulting NK cell repertoires were independent of the presence of specific KIR ligands on MSCs and were, in fact, invariably dominated by expression of the C1-specific inhibitory KIR2DL3. Similarly, short hairpin RNA-mediated knockdown of HLA class I ligands on MSCs did not delay or change the course of KIR expression. Our data suggest that the initial acquisition of KIRs during NK cell development is biased toward recognition of C1 ligands, irrespective of the presence of self-ligands. Altogether, the MSC/HSPC model constitutes a novel platform to study NK cell development in a human stem cell niche. Moreover, the system constitutes a promising GMP-compliant platform to develop clinical-grade NK cell products from cord blood HSPCs., (© 2018 by The American Society of Hematology.)

Indigenous Australians experience a substantially higher cancer mortality rate than non-Indigenous Australians. While cancer outcomes are improving for non-Indigenous Australians, they are worsening for Indigenous Australians. Reducing this disparity requires evidence-based and culturally-appropriate guidance. The purpose of this paper is to describe an initiative by Cancer Australia and Menzies School of Health Research (Menzies) to develop Australia&rsquo;s first National Aboriginal and Torres Strait Islander Cancer Framework using a process of co-design with relevant stakeholders. The initiative was guided by three core principles: achieving policy-relevant evidence-based outcomes; engaging and maintaining trust with Indigenous Australians at every phase; and employing best-practice and appropriate research methods. Four components of research comprised the Framework development: evidence review; multifaceted stakeholder consultation and input; triangulation of findings; and direct stakeholder input in drafting and refining the Framework. The evidence review confirmed the increasing burden of cancer on Indigenous Australians, while stakeholder consultations facilitated comprehensive input from those with lived experience. The consultations revealed issues not identified in existing literature, and gave different emphases of priority, thus reinforcing the value of including stakeholder perspectives. This paper focuses primarily on documenting the methods used; findings are presented only in order to illustrate the results of the process. The published Framework is available at www.canceraustralia.gov.au; further description and analyses of findings from the consultations will be published elsewhere. The logistics inherent in large-scale consultation are considerable. However, the quality of data and the foundation for sustained partnership with stakeholders and knowledge translation vastly outweighed the challenges. The process of wide-ranging stakeholder consultation described in this paper offers a model for other areas of national and international Indigenous priority setting and policy and practice development that meets the needs of those most affected. The Framework, through the establishment of an agreed, shared and evidence-based agenda, provides guidance for jurisdictional cancer plans, optimal care pathways, and program and service planning for the multiple players across all levels of the health system.

PINK1 (PTEN-induced putative kinase 1) is a key regulator of mitochondrial homeostasis that is relatively depleted in aging lungs and in lung epithelial cells from patients with idiopathic pulmonary fibrosis (IPF), a disease linked with aging. Impaired PINK1 expression and accumulation of damaged mitochondria in lung epithelial cells from fibrotic lungs were associated with the presence of ER stress. Here, we show that ATF3 (activating transcription factor 3), a member of the integrated stress response (ISR), negatively regulates transcription of the PINK1 gene. An ATF3 binding site within the human PINK1 promoter is located in the first 150 bp upstream of the transcription start site. Induction of ER stress or overexpression of ATF3 inhibited the activity of the PINK1 promoter. Importantly, overexpression of ATF3 causes accumulation of depolarized mitochondria, increased production of mitochondrial ROS, and loss of cell viability. Furthermore, conditional deletion of ATF3 in type II lung epithelial cells protects mice from bleomycin-induced lung fibrosis. Finally, we observed that ATF3 expression increases in the lung with age and, specially, in lung epithelial cells from IPF lungs. These data provide a unique link between ATF3 and PINK1 expression suggesting that persistent stress, driven by ATF3, can dysregulate mitochondrial homeostasis by repression of PINK1 mRNA synthesis., (© 2018 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.)

Purpose: Objective assessment of visual acuity (VA) is possible with VEP methodology, but established with sufficient precision only for vision better than about 1.0 logMAR. We here explore whether this can be extended down to 2.0 logMAR, highly desirable for low-vision evaluations., Methods: Based on the stepwise sweep algorithm (Bach et al. in Br J Ophthalmol 92:396-403, 2008) VEPs to monocular steady-state brief onset pattern stimulation (7.5-Hz checkerboards, 40% contrast, 40 ms on, 93 ms off) were recorded for eight different check sizes, from 0.5° to 9.0°, for two runs with three occipital electrodes in a Laplace-approximating montage. We examined 22 visually normal participants where acuity was reduced to ≈ 2.0 logMAR with frosted transparencies. With the established heuristic algorithm the "VEP acuity" was extracted and compared to psychophysical VA, both obtained at 57 cm distance., Results: In 20 of the 22 participants with artificially reduced acuity the automatic analysis indicated a valid result (1.80 logMAR on average) in at least one of the two runs. 95% test-retest limits of agreement on average were ± 0.09 logMAR for psychophysical, and ± 0.21 logMAR for VEP-derived acuity. For 15 participants we obtained results in both runs and averaged them. In 12 of these 15 the low-acuity results stayed within the 95% confidence interval (± 0.3 logMAR) as established by Bach et al. (2008)., Conclusions: The fully automated analysis yielded good agreement of psychophysical and electrophysiological VAs in 12 of 15 cases (80%) in the low-vision range down to 2.0 logMAR. This encourages us to further pursue this methodology and assess its value in patients.

Brain endothelial cells are the major building block of the blood-brain barrier. To study the role of brain endothelial cells in vitro , the isolation of primary cells is of critical value. Here, we describe a protocol in which vessel fragments are isolated from adult mice. After density centrifugation and mild digestion of the fragments, outgrowing endothelial cells are selected by puromycin treatment and grown to confluence within one week.

A novel canine muscular dystrophy in Landseer dogs was observed. We had access to five affected dogs from two litters. The clinical signs started at a few weeks of age, and the severe progressive muscle weakness led to euthanasia between 5 and 15 months of age. The pedigrees of the affected dogs suggested a monogenic autosomal-recessive inheritance of the trait. Linkage and homozygosity mapping indicated two potential genome segments for the causative variant on chromosomes 10 and 31 harboring a total of 4.8 Mb of DNA or 0.2% of the canine genome. Using the Illumina sequencing technology, we obtained a whole-genome sequence from one affected Landseer. Variants were called with respect to the dog reference genome and compared with the genetic variants of 170 control dogs from other breeds. The affected Landseer dog was homozygous for a single, private nonsynonymous variant in the critical intervals, a nonsense variant in the COL6A1 gene (Chr31:39,303,964G>T; COL6A1:c.289G>T; p.E97*). Genotypes at this variant showed perfect concordance with the muscular dystrophy phenotype in all five cases and more than 1000 control dogs. Variants in the human COL6A1 gene cause Bethlem myopathy or Ullrich congenital muscular dystrophy. We therefore conclude that the identified canine COL6A1 variant is most likely causative for the observed muscular dystrophy in Landseer dogs. On the basis of the nature of the genetic variant in Landseer dogs and their severe clinical phenotype these dogs represent a model for human Ullrich congenital muscular dystrophy., (Copyright © 2015 Steffen et al.)

Human hematopoietic stem and progenitor cells (HSPCs) from umbilical cord blood exhibit higher differentiation potential and repopulation capacity compared to adult HSPCs. The molecular basis for these functional differences is currently unknown. Upon screening for epigenetic effector genes being differentially expressed in neonatal and adult HSPC subpopulations, the Polycomb Repressive Complex 2 (PRC2) member EED was identified. Even though EED is expressed at comparable amounts in neonatal and adult multipotent HSPCs, early adult lineage committed progenitors of the lymphomyeloid (LM) and erythromyeloid lineages expressed higher EED amounts than neonatal HPCs. We demonstrate that EED overexpression directly leads to higher H3K27me3 levels, a repressive histone modification that is mediated by the PRC2 complex. Quantitative analysis of H3K27me3 levels by FPLC-based ELISA revealed elevated levels in primary blood cells from adults. Besides quantitative changes, gene ontology analysis of the genome-wide H3K27me3 distribution revealed qualitative changes in adult HSPCs with elevated levels in genes associated with nonhematopoietic development pathways. In contrast, H3K4me3 which labels active chromatin was enriched on hematopoietic genes. In vitro differentiation of EED-transfected neonatal HSPCs revealed aberrant expression of the myelopoietic marker CD14, suggesting that EED affects the lymphoid versus myeloid decision processes within the lymphomyeloid lineage. This is in line with LM progenitors having the most pronounced differences in EED expression. Highlighting the dynamic roles of epigenetic modifications in human hematopoiesis, the present data demonstrate shifts in the PRC2-associated histone modification H3K27me3 from birth to adulthood.

Despite epicardial coronary artery reperfusion by percutaneous coronary intervention, distal micro-embolization into the coronary microcirculation limits myocardial salvage during acute myocardial infarction. Thrombolysis using ultrasound and microbubbles (sonothrombolysis) is an approach that induces microbubble oscillations to cause clot disruption and restore perfusion. We sought to determine whether this technique could restore impaired tissue perfusion caused by thrombotic microvascular obstruction. In 16 rats, an imaging transducer was placed on the biceps femoris muscle, perpendicular to a single-element 1-MHz treatment transducer. Ultrasound contrast perfusion imaging was performed at baseline and after micro-embolization. Therapeutic ultrasound (5000 cycles, pulse repetition frequency = 0.33 Hz, 1.5 MPa) was delivered to nine rats for two 10-min sessions during intra-arterial infusion of lipid-encapsulated microbubbles; seven control rats received no ultrasound-microbubble therapy. Ultrasound contrast perfusion imaging was repeated after each treatment or control period, and microvascular volume was measured as peak video intensity. There was a 90% decrease in video intensity after micro-embolization (from 8.6 ± 4.8 to 0.7 ± 0.8 dB, p < 0.01). The first and second ultrasound-microbubble sessions were respectively followed by video intensity increases of 5.8 ± 5.1 and 8.7 ± 5.7 dB (p < 0.01, compared with micro-embolization). The first and second control sessions, respectively, resulted in no significant increase in video intensity (2.4 ± 2.3 and 3.6 ± 4.9) compared with micro-embolization (0.6 ± 0.7 dB). We have developed an in vivo model that simulates the distal thrombotic microvascular obstruction that occurs after primary percutaneous coronary intervention. Long-pulse-length ultrasound with microbubbles has a therapeutic effect on microvascular perfusion and may be a valuable adjunct to reperfusion therapy for acute myocardial infarction., (Copyright © 2015 World Federation for Ultrasound in Medicine & Biology. Published by Elsevier Inc. All rights reserved.)

Although aging is a known risk factor for idiopathic pulmonary fibrosis (IPF), the pathogenic mechanisms that underlie the effects of advancing age remain largely unexplained. Some age-related neurodegenerative diseases have an etiology that is related to mitochondrial dysfunction. Here, we found that alveolar type II cells (AECIIs) in the lungs of IPF patients exhibit marked accumulation of dysmorphic and dysfunctional mitochondria. These mitochondrial abnormalities in AECIIs of IPF lungs were associated with upregulation of ER stress markers and were recapitulated in normal mice with advancing age in response to stimulation of ER stress. We found that impaired mitochondria in IPF and aging lungs were associated with low expression of PTEN-induced putative kinase 1 (PINK1). Knockdown of PINK1 expression in lung epithelial cells resulted in mitochondria depolarization and expression of profibrotic factors. Moreover, young PINK1-deficient mice developed similarly dysmorphic, dysfunctional mitochondria in the AECIIs and were vulnerable to apoptosis and development of lung fibrosis. Our data indicate that PINK1 deficiency results in swollen, dysfunctional mitochondria and defective mitophagy, and promotes fibrosis in the aging lung.

Background: Health promotion by and with Aboriginal and Torres Strait Islander (hereafter Indigenous) Australians is critically important given a wide gap in health parity compared to other Australians. The development and implementation of step-by-step guides, instruments, packages, frameworks or resources has provided a feasible and low-resource strategy for strengthening evidence-informed health promotion practice. Yet there has been little assessment of where and how these tools are implemented or their effectiveness. This paper reviews the characteristics, implementation and effects of Indigenous health promotion tools., Methods: Indigenous health promotion tools were identified through a systematic literature search including a prior scoping study, eight databases, references of other reviews and the authors' knowledge (n = 1494). Documents in the peer reviewed and grey literature were included if they described or evaluated tools designed, recommended or used for strengthening Indigenous Australian health promotion. Eligible publications were entered into an Excel spreadsheet and documented tools classified according to their characteristics, implementation and effects. Quality was appraised using the Dictionary for Effective Public Health Practice Project (EPHPP) and Critical Appraisal Skills Program (CASP) tools for quantitative and qualitative studies respectively., Results: The review found that Indigenous health promotion tools were widely available. Of 74 publications that met inclusion criteria, sixty (81%) documented tools developed specifically for the Indigenous Australian population. All tools had been developed in reference to evidence; but only 22/74 (30%) publications specified intended or actual implementation, and only 11/74 (15%) publications evaluated impacts of the implemented tools. Impacts included health, environmental, community, organisational and health care improvements. The quality of impact evaluations was strong for only five (7%) studies., Conclusions: The small number and generally moderate quality of implementation and evaluation studies means that little is known about how tools work to strengthen Indigenous health promotion practice. The findings suggest that rather than continuing to invest in tool development, practitioners, policy makers and researchers could evaluate the implementation and effects of existing tools and publish the results. There is a need for long-term investment in research to review the current use of health promotion tools and the factors that are likely to enhance their implementation.

Background: Strengthening primary healthcare systems is vital to improving health outcomes and reducing inequity. However, there are few tools and models available in published literature showing how primary care system strengthening can be achieved on a large scale. Challenges to strengthening primary healthcare (PHC) systems include the dispersion, diversity and relative independence of primary care providers; the scope and complexity of PHC; limited infrastructure available to support population health approaches; and the generally poor and fragmented state of PHC information systems.Drawing on concepts of comprehensive PHC, integrated quality improvement (IQI) methods, system-based research networks, and system-based participatory action research, we describe a learning model for strengthening PHC that addresses these challenges. We describe the evolution of this model within the Australian Aboriginal and Torres Strait Islander primary healthcare context, successes and challenges in its application, and key issues for further research., Discussion: IQI approaches combined with system-based participatory action research and system-based research networks offer potential to support program implementation and ongoing learning across a wide scope of primary healthcare practice and on a large scale. The Partnership Learning Model (PLM) can be seen as an integrated model for large-scale knowledge translation across the scope of priority aspects of PHC. With appropriate engagement of relevant stakeholders, the model may be applicable to a wide range of settings. In IQI, and in the PLM specifically, there is a clear role for research in contributing to refining and evaluating existing tools and processes, and in developing and trialling innovations. Achieving an appropriate balance between funding IQI activity as part of routine service delivery and funding IQI related research will be vital to developing and sustaining this type of PLM., Summary: This paper draws together several different previously described concepts and extends the understanding of how PHC systems can be strengthened through systematic and partnership-based approaches. We describe a model developed from these concepts and its application in the Australian Indigenous primary healthcare context, and raise questions about sustainability and wider relevance of the model.

Background: Variation in effectiveness of continuous quality improvement (CQI) interventions between services is commonly reported, but with little explanation of how contextual and other factors may interact to produce this variation. Therefore, there is scant information available on which policy makers can draw to inform effective implementation in different settings. In this paper, we explore how patterns of change in delivery of services may have been achieved in a diverse range of health centers participating in a wide-scale program to achieve improvements in quality of care for Indigenous Australians., Methods: We elicited key informants' interpretations of factors explaining patterns of change in delivery of guideline-scheduled services over three or more years of a wide-scale CQI project, and inductively analyzed these interpretations to propose fine-grained realist hypotheses about what works for whom and in what circumstances. Data were derived from annual clinical audits from 36 health centers operating in diverse settings, quarterly project monitoring reports, and workshops with 12 key informants who had key roles in project implementation. We abstracted potential context-mechanism-outcome configurations from the data, and based on these, identified potential program-strengthening strategies., Results: Several context-specific, mechanism-based explanations for effectiveness of this CQI project were identified. These were collective valuing of clinical data for improvement purposes; collective efficacy; and organizational change towards a population health orientation. Health centers with strong central management of CQI, and those in which CQI efforts were more dependent on local health center initiative and were adapted to resonate with local priorities were both favorable contexts for collective valuing of clinical data. Where health centers had prior positive experiences of collaboration, effects appeared to be achieved at least partly through the mechanism of collective efficacy. Strong community linkages, staff ability to identify with patients, and staff having the skills and support to take broad ranging action, were favorable contexts for the mechanism of increased population health orientation., Conclusions: Our study provides evidence to support strategies for program strengthening described in the literature, and extends the understanding of mechanisms through which strategies may be effective in achieving particular outcomes in different contexts.

Drag-reducing polymers (DRPs) significantly increase blood flow, tissue perfusion, and tissue oxygenation in various animal models. In rectangular channel microfluidic systems, DRPs were found to significantly reduce the near-wall cell-free layer (CFL) as well as modify traffic of red blood cells (RBC) into microchannel branches. In the current study we further investigated the mechanism by which DRP enhances microvascular perfusion. We studied the effect of various concentrations of DRP on RBC distribution in more relevant round microchannels and the effect of DRP on CFL in the rat cremaster muscle in vivo. In round microchannels hematocrit was measured in parent and daughter branch at baseline and after addition of DRP. At DRP concentrations of 5 and 10 ppm, the plasma skimming effect in the daughter branch was eliminated, as parent and daughter branch hematocrit were equivalent, compared to a significantly lowered hematocrit in the daughter branch without DRPs. In anesthetized rats (N=11) CFL was measured in the cremaster muscle tissue in arterioles with a diameter of 32.6 ± 1.7 µm. In the control group (saline, N=6) there was a significant increase in CFL in time compared to corresponding baseline. Addition of DRP at 1 ppm (N=5) reduced CFL significantly compared to corresponding baseline and the control group. After DRP administration the CFL reduced to about 85% of baseline at 5, 15, 25 and 35 minutes after DRP infusion was complete. These in vivo and in vitro findings demonstrate that DRPs induce a reduction in CFL width and plasma skimming in the microvasculature. This may lead to an increase of RBC flux into the capillary bed, and thus explain previous observations of a DRP mediated enhancement of capillary perfusion.

Adenosine-mediated recruitment of microvascular volume in heart and muscle has been suggested to include, in addition to vasodilation of resistance vessels, an increased accessibility of the endothelial glycocalyx for flowing plasma as a result of an impairment of its barrier properties. The aim of the current study was to investigate the effect of systemic intravenous administration of adenosine on the glycocalyx-dependent exclusion of circulating blood at a whole-body level. In anesthetized goats (N = 6), systemic blood-excluded glycocalyx volume was measured by comparing the intravascular distribution volume of the suggested glycocalyx accessible tracer dextrans with a molecular weight of 40 kDa (Dex-40) to that of circulating plasma, derived from the dilution of labeled red blood cells and large vessel hematocrit. Systemic glycocalyx volume was determined at baseline and during intravenous infusion of adenosine (157 ± 11.6 μg/kg min(-1)). Blood-inaccessible glycocalyx volume decreased from 458.1 ± 95.5 to 18.1 ± 62.2 mL (P < 0.01) during adenosine administration. While circulating plasma volume did not change significantly (617.1 ± 48.5 vs. 759.2 ± 47.9 mL, NS), the decrease in blood-excluded glycocalyx volume was associated with a decrease in Dex-40 distribution volume (from 1075.2 ± 71.0 to 777.3 ± 60.0 mL, P < 0.01). Intravenous administration of adenosine is associated with a robust impairment of whole-body glycocalyx barrier properties, reflected by a greatly reduced exclusion of circulating blood compared to small dextrans. The observed decrease in Dex-40 distribution volume suggests that the reduction in glycocalyx volume coincides with a reduction in tracer-accessible vascular volume.

The endothelial glycocalyx forms a hyaluronan-containing interface between the flowing blood and the endothelium throughout the body. By comparing the systemic distribution of a small glycocalyx-accessible tracer vs. a large circulating plasma tracer, the size-selective barrier properties of the glycocalyx have recently been utilized to estimate whole body glycocalyx volumes in humans and animals, but a comprehensive validation of this approach has been lacking at the moment. In the present study, we compared, in anesthetized, ventilated C57Bl/6 mice, the whole body distribution of small (40 kDa) dextrans (Texas Red labeled; Dex40) vs. that of intermediate (70 kDa) and large (500 kDa) dextrans (both FITC labeled; Dex70 and Dex500, respectively) using tracer dilution and vs. that of circulating plasma, as derived from the dilution of fluorescein-labeled red blood cells and large-vessel hematocrit. The contribution of the glycocalyx was evaluated by intravenous infusion of a bolus of the enzyme hyaluronidase. In saline-treated control mice, distribution volume (in ml) differed between tracers (P < 0.05; ANOVA) in the following order: Dex40 (0.97 ± 0.04) > Dex70 (0.90 ± 0.04) > Dex500 (0.81 ± 0.10) > plasma (0.71 ± 0.02), resulting in an inaccessible vascular volume, i.e., compared with the distribution volume of Dex40, of 0.03 ± 0.01, 0.15 ± 0.04, and 0.31 ± 0.05 ml for Dex70, Dex500, and plasma, respectively. In hyaluronidase-treated mice, Dex70 and Dex40 volumes were not different from each other, and inaccessible vascular volumes for Dex500 (0.03 ± 0.03) and plasma (0.14 ± 0.05) were smaller (P < 0.05) than those in control animals. Clearance of Dex70 and Dex500 from the circulation was enhanced (P < 0.05) in hyaluronidase-treated vs. control mice. These results indicate that the glycocalyx contributes to size-dependent differences in whole body vascular distribution of plasma solutes in mice. Whole body vascular volume measurements based on the differential distribution of glycocalyx-selective tracers appear appropriate for the detection of generalized glycocalyx degradation in experimental animals and humans.

Objective: We have shown that drag-reducing polymers (DRP) enhance capillary perfusion during severe coronary stenosis and increase red blood cell velocity in capillaries, through uncertain mechanisms. We hypothesize that DRP decreases pressure loss from the aorta to the arteriolar compartment., Methods: Intravital microscopy of the rat cremaster muscle and measurement of pressure in arterioles (diameters 20-132 μm) was performed in 24 rats. DRP (polyethylene oxide, 1 ppm) was infused i.v. and measurements were made at baseline and 20 minutes after completion of DRP infusion. In a 10-rat subset, additional measurements were made three minutes after the start, and one to five and 10 minutes after completion of DRP., Results: Twenty minutes after the completion of DRP, mean arteriolar pressure was 22% higher than baseline (from 42 ± 3 to 49 ± 3 mmHg, p < 0.005, n = 24). DRP decreased the pressure loss from the aorta to the arterioles by 24% (from 35 ± 6 to 27 ± 5 mmHg, p = 0.001, n = 10). In addition, there was a strong trend toward an increase in pressure at 10 minutes after the completion of DRP (n = 10)., Conclusions: Drag-reducing polymers diminish pressure loss between the aorta and the arterioles. This results in a higher pre-capillary pressure and probably explains the observed DRP enhancement in capillary perfusion., (© 2012 John Wiley & Sons Ltd.)

Chemotherapy can be a life-prolonging treatment for many cancer patients, but it is often associated with profound nausea and vomiting that is so distressing that patients may delay or decline treatment to avoid these side effects. EMEND (aprepitant) is the first and only neurokinin-1 (NK-1) receptor antagonist available on the market for the prevention of acute and delayed chemotherapy-induced nausea and vomiting (CINV). Aprepitant acts centrally at NK-1 receptors in vomiting centers within the central nervous system to block their activation by substance P released as an unwanted consequence of chemotherapy. By controlling nausea and vomiting, EMEND helps improve patients' daily living and their ability to complete multiple cycles of chemotherapy. The development of aprepitant included a novel nanoparticle formulation to optimize oral absorption and innovative chemistry to discover a prodrug form suitable for intravenous administration to improve compliance and convenience for healthcare professionals and cancer patients., (© 2011 New York Academy of Sciences.)

Unrestricted somatic stem cells (USSC) from human cord blood display a broad differentiation potential for ectodermal, mesodermal, and endodermal cell types. The molecular basis for these stem cell properties is unclear and unlike embryonic stem cells (ESC) none of the major stem cell factors OCT4, SOX2, and NANOG exhibits significant expression in USSC. Here, we report that these key stem cell genes hold an epigenetic state in between that of an ESC and a terminally differentiated cell type. DNA methylation analysis exhibits partial demethylation of the regulatory region of OCT4 and a demethylated state of the NANOG and SOX2 promoter/enhancer regions. Further genome-wide DNA methylation profiling identified a partially demethylated state of the telomerase gene hTERT. Moreover, none of the pluripotency factors exhibited a repressive histone signature. Notably, SOX2 exhibits a bivalent histone signature consisting of the opposing histone marks dimeH3K4 and trimeH3K27, which is typically found on genes that are "poised" for transcription. Consequently, ectopic expression of OCT4 in USSC led to rapid induction of expression of its known target gene SOX2. Our data suggest that incomplete epigenetic repression and a "poised" epigenetic status of pluripotency genes preserves the USSC potential to be able to react adequately to distinct differentiation and reprogramming cues., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Pharmaceutical synthesis can benefit greatly from the selectivity gains associated with enzymatic catalysis. Here, we report an efficient biocatalytic process to replace a recently implemented rhodium-catalyzed asymmetric enamine hydrogenation for the large-scale manufacture of the antidiabetic compound sitagliptin. Starting from an enzyme that had the catalytic machinery to perform the desired chemistry but lacked any activity toward the prositagliptin ketone, we applied a substrate walking, modeling, and mutation approach to create a transaminase with marginal activity for the synthesis of the chiral amine; this variant was then further engineered via directed evolution for practical application in a manufacturing setting. The resultant biocatalysts showed broad applicability toward the synthesis of chiral amines that previously were accessible only via resolution. This work underscores the maturation of biocatalysis to enable efficient, economical, and environmentally benign processes for the manufacture of pharmaceuticals.

The endothelial glycocalyx is the negatively charged, gel-like mesh residing at the luminal side of the vascular endothelium and forming the interface between the flowing blood and the vessel wall. The vast majority of glycocalyx volume resides in the microcirculation, particularly in the capillaries. Intravital microscopic observations of capillaries in striated muscle preparations illustrate that under resting conditions, the glycocalyx is not accessible for flowing red blood cells and greatly hinders plasma flow in the axial direction, causing a significant reduction in functionally perfused capillary volume. Glycocalyx exclusion properties have been shown to be reduced by adenosine and other vasoactive substances. A diminished exclusion of circulating blood by the glycocalyx may facilitate nutrient exchange since it is associated with an increase in functionally perfused blood volume and surface area in the capillaries. Our recent studies have focused on the effect of adenosine on glycocalyx exclusion in the coronary circulation and demonstrate an important role for this mechanism in the increase in circulating coronary blood volume during administration of this vasodilator. The current review elaborates on the glycocalyx as a blood-excluding intravascular layer and how it can be modulated by various agonists. Further, the potential role of adenosine-induced modulation of glycocalyx exclusion properties in coupling increases in blood flow and circulating blood volume in the coronary circulation is discussed. Finally, we consider how degradation of the glycocalyx may impact on coronary blood volume regulation, thereby providing new opportunities to diagnose glycocalyx damage in the clinical setting.

Vascular endothelium is covered with an extensive mesh of glycocalyx constituents, which acts like an effective barrier up to several micrometers thick that shields the luminal surface of the vasculature from direct exposure to flowing blood. Many studies report that various enzymatic and pharmaceutical challenges are able to increase glycocalyx porosity, resulting in farther permeation of plasma macromolecules and greater access of red blood cells into glycocalyx domain. Attenuation of glycocalyx barrier properties therefore potentially increases the amount of blood that effectively occupies available microvascular volume. We tested in the present study whether attenuation of coronary glycocalyx barrier properties actually increases coronary blood volume and whether such changes would be noticeable during measurements of coronary flow reserve using adenosine. In anesthetized goats (n = 6) with cannulated left main coronary artery that were perfused under controlled pressure, coronary blood volume was measured via the indicator-dilution technique using high-molecular-weight (2,000 kDa) dextrans as plasma tracer and labeled red blood cells as red blood cell tracer. Coronary blood volume was determined at baseline and during intracoronary infusion of adenosine causing maximal vasodilation (0.2-0.6 mg.kg(-1).h(-1)) before and after intracoronary hyaluronidase treatment (170,000 units) of the glycocalyx. With an intact glycocalyx, coronary blood volume was 18.9 +/- 1.1 ml/100 g heart tissue at baseline, which increased to 26.3 +/- 2.7 ml/100 g after hyaluronidase treatment of the coronary glycocalyx. Maximal vasodilation by administration of adenosine further increased coronary blood volume to 33.9 +/- 6.8 ml/100 g, a value not different from the maximal coronary blood volume of 33.2 +/- 5.3 ml/100 g obtained by administration of adenosine in the absence of hyaluronidase treatment. Adenosine-induced increases in coronary conductance were not affected by hyaluronidase treatment. We conclude that acute attenuation of glycocalyx barrier properties increases coronary blood volume by approximately 40%, which is of similar magnitude as additional changes in coronary blood volume during subsequent maximal vasodilation with adenosine. Furthermore, maximal coronary blood volume following administration of adenosine was similar with and without prior hyaluronidase degradation of the glycocalyx, suggesting that adenosine and hyaluronidase potentially increase glycocalyx porosity to a similar extent. Hyaluronidase-mediated changes in coronary blood volume did not affect baseline and adenosine-induced increases in coronary conductance, demonstrating that measurements of coronary flow reserve are insufficient to detect impairment of coronary blood volume recruitment in conditions of damaged glycocalyx.

Previous studies have suggested that agonists may increase functionally perfused capillary volume by modulation of blood-excluding glycocalyx volume, but direct evidence for this association is lacking at the moment. Using intravital microscopic visualization of mouse cremaster muscle, we determined the effects of bradykinin (10(-5) M) and sodium nitroprusside (10(-6) M) on capillary tube haematocrit and glycocalyx barrier properties. In control C57Bl/6 mice (n = 10), tube haematocrit in capillaries (n = 71) increased (P < 0.05) from 8.7 +/- 0.3% during baseline to 21.2 +/- 1.2 and 22.2 +/- 0.9% during superfusion with bradykinin and nitroprusside, respectively. In parallel, the exclusion zone of FITC-labelled 70 kDa dextrans decreased (P < 0.05) from 0.37 +/- 0.01 microm during baseline to 0.17 +/- 0.01 microm with bradykinin and 0.15 +/- 0.01 microm with nitroprusside. Bradykinin and nitroprusside had no effect on dextran exclusion and tube haematocrit in capillaries (n = 55) of hyperlipidemic ApoE3-Leiden mice, which showed impaired exclusion of 70 kDa dextrans (0.05 +/- 0.02 microm; P < 0.05 versus C57Bl/6) and increased capillary tube haematocrit (23 +/- 0.8%; P < 0.05 versus C57Bl/6) under baseline conditions, indicating glycocalyx degradation. Our data show that vasodilator substances increase functionally perfused capillary volume and that this effect is associated with a reduction in glycocalyx exclusion of 70 kDa dextrans. Modulation of glycocalyx volume might represent a novel mechanism of perfusion control at the capillary level.

The endothelial glycocalyx is a hydrated mesh of polysaccharides and adsorbed plasma proteins that forms the true interface between the flowing blood and the endothelium. We hypothesized in the present study that competitive binding of heparin to glycocalyx-associated proteins would affect glycocalyx barrier properties and mechanotransduction of shear stress to the endothelium. In anesthetized mice, the clearance of 70-kDa dextrans from the circulation was increased (P<0.05 versus saline) 1 hour after heparin (1.25 U) and glycocalyx degradation with hyaluronidase (35 U; amount cleared in 30 minutes after saline: 11+/-5%; after heparin: 45+/-8%; after hyaluronidase: 30+/-3%). Clearance of 40-kDa dextrans increased (P<0.05 versus saline) to a lesser extent after both treatments (saline: 46+/-3%; heparin: 60+/-5%; hyaluronidase: 60+/-2%). The dilator response of second-order arterioles in cremaster muscle during reactive hyperemia was reduced for < or =90 minutes after heparin as reflected by a decrease (P=0.008) in t(50) of diameter recovery, and this effect was associated with a diminished NO bioavailability. Infusion of hyaluronidase resulted in reductions (P<0.05) in baseline and peak reactive hyperemic diameter, whereas, despite an increase in wall shear rate at the beginning of reactive hyperemia, t(50) of diameter recovery was not affected. In conclusion, our data in mice show that a heparin challenge is associated with increased vascular leakage of dextrans and impaired arteriolar vasodilation during reactive hyperemia. Our data suggest that protein-heparan sulfate interactions are important for a functional glycocalyx.