Better Respiratory Function in Heart Failure Patients With Use of Central-Acting Therapeutics.

Background: Diaphragm atrophy can contribute to dyspnea in patients with heart failure (HF) with its link to central neurohormonal overactivation. HF medications that cross the blood-brain barrier could act centrally and improve respiratory function, potentially alleviating diaphragmatic atrophy. Therefore, we compared the benefit of central- vs peripheral-acting HF drugs on respiratory function, as assessed by a single cardiopulmonary exercise test (CPET) and outcomes in HF patients. Methods: A retrospective study was conducted of 624 ambulatory adult HF patients (80% male) with reduced left ventricular ejection fraction ≤ 40% and a complete CPET, followed at a single institution between 2001 and 2017. CPET parameters, and the outcomes all-cause death, a composite endpoint (all-cause death, need for left ventricular assist device, heart transplantation), and all-cause and/or HF hospitalizations, were compared in patients receiving central-acting (n = 550) vs peripheral-acting (n = 74) drugs. Results: Compared to patients who receive peripheral-acting drugs, patients who receive central-acting drugs had better respiratory function (peak breath-by breath oxygen uptake [VO2], P = 0.020; forced expiratory volume in 1 second [FEV1], P = 0.007), and ventilatory efficiency (minute ventilation / carbon dioxide production [VE/VCO2], P < 0.001; end-tidal carbon dioxide tension [PETCO2], P = 0.015; and trend for forced vital capacity [FVC], P = 0.056). Many of the associations between the CPET parameters and drug type remained significant after multivariate adjustment. Moreover, patients receiving central-acting drugs had fewer composite events (P = 0.023), and HF hospitalizations (P = 0.044), although significance after multivariant correction was not achieved, despite the hazard ratio being 0.664 and 0.757, respectively. Conclusions: Central-acting drugs were associated with better respiratory function as measured by CPET parameters in HF patients. This could extend to clinically meaningful composite outcomes and hospitalizations but required more power to be definitive in linking to drug effect. Central-acting HF drugs show a role in mitigating diaphragm weakness.

Contexte: L'atrophie du diaphragme peut contribuer à la dyspnée chez les personnes atteintes d'insuffisance cardiaque (IC), compte tenu de son lien avec la suractivation neuro-hormonale centrale. Or, les médicaments contre l'IC qui franchissent la barrière hématoencéphalique pourraient exercer une action centrale, améliorer la respiration et ainsi éventuellement atténuer l'atrophie du diaphragme. C'est pourquoi nous avons voulu comparer, au moyen d'une seule épreuve d'effort cardiopulmonaire (EECP), les effets bénéfiques exercés par des médicaments à action périphérique et des médicaments à action centrale sur la fonction respiratoire, de même que l'issue des patients atteints d'IC auxquels ils ont été administrés. Méthodologie: Nous avons réalisé une étude rétrospective auprès de 624 adultes ambulatoires atteints d'IC (80 % d'hommes) dont la fraction d'éjection ventriculaire gauche était réduite (≤ 40 %), qui se sont prêtés à une EECP complète et qui ont été suivis dans le même établissement entre 2001 et 2017. Les paramètres de l'EECP et la mortalité toutes causes confondues, un critère d'évaluation composé (décès toutes causes confondues, nécessité de recourir à un dispositif d'assistance ventriculaire gauche, transplantation cardiaque), et les hospitalisations toutes causes confondues et/ou liées à l'IC ont été comparés entre les patients qui recevaient des médicaments à action centrale (n = 550) et ceux qui recevaient des médicaments à action périphérique (n = 74). Résultats: Comparativement aux patients ayant reçu des médicaments à action périphérique, ceux qui ont reçu des médicaments à action centrale ont bénéficié d'une meilleure fonction respiratoire (consommation maximale d'oxygène [VO2], p = 0,020; volume expiratoire maximal par seconde [VEMS], p = 0,007) et d'une meilleure efficacité ventilatoire (ventilation minute/production de dioxyde de carbone [VE/VCO2], p < 0,001; pression partielle de dioxyde de carbone en fin d'expiration [PETCO2], p = 0,015; et tendance de la capacité vitale forcée [CVF], p = 0,056). De plus, bon nombre des associations entre les paramètres de l'EECP et le type de médicament sont demeurées significatives après ajustement multivarié. Les patients qui ont reçu des médicaments à action centrale ont également présenté moins d'événements faisant partie du critère d'évaluation composé (p = 0,023) et moins d'hospitalisations liées à l'IC (p = 0,044), même si la différence après correction multivariée n'a pas été significative et que les rapports de risques étaient respectivement de 0,664 et de 0,757. Conclusions: Les médicaments à action centrale ont été associés à une meilleure fonction respiratoire, mesurée à l'aide des paramètres d'une EECP, chez les patients atteints d'IC. Ce résultat pourrait également s'appliquer au critère d'évaluation composé et aux hospitalisations, mais une étude plus puissante est nécessaire pour établir un lien cliniquement significatif avec l'effet des médicaments. Les médicaments à action centrale contre l'IC ont donc un rôle à jouer dans la correction de la faiblesse du diaphragme.

Muscle weakness and mitochondrial stress occur before metastasis in a novel mouse model of ovarian cancer cachexia.

Objectives: A high proportion of women with advanced epithelial ovarian cancer (EOC) experience weakness and cachexia. This relationship is associated with increased morbidity and mortality. EOC is the most lethal gynecological cancer, yet no preclinical cachexia model has demonstrated the combined hallmark features of metastasis, ascites development, muscle loss and weakness in adult immunocompetent mice. Methods: Here, we evaluated a new model of ovarian cancer-induced cachexia with the advantages of inducing cancer in adult immunocompetent C57BL/6J mice through orthotopic injections of EOC cells in the ovarian bursa. We characterized the development of metastasis, ascites, muscle atrophy, muscle weakness, markers of inflammation, and mitochondrial stress in the tibialis anterior (TA) and diaphragm ~45, ~75 and ~90 days after EOC injection. Results: Primary ovarian tumour sizes were progressively larger at each time point while robust metastasis, ascites development, and reductions in body, fat and muscle weights occurred by 90 Days. There were no changes in certain inflammatory (TNFα), atrogene (MURF1 and Atrogin) or GDF15 markers within both muscles whereas IL-6 was increased at 45 and 90 Day groups in the diaphragm. TA weakness in 45 Day preceded atrophy and metastasis that were observed later (75 and 90 Day, respectively). The diaphragm demonstrated both weakness and atrophy in 45 Day. In both muscles, this pre-metastatic muscle weakness corresponded with considerable reprogramming of gene pathways related to mitochondrial bioenergetics as well as reduced functional measures of mitochondrial pyruvate oxidation and creatine-dependent ADP/ATP cycling as well as increased reactive oxygen species emission (hydrogen peroxide). Remarkably, muscle force per unit mass at 90 days was partially restored in the TA despite the presence of atrophy and metastasis. In contrast, the diaphragm demonstrated progressive weakness. At this advanced stage, mitochondrial pyruvate oxidation in both muscles exceeded control mice suggesting an apparent metabolic super-compensation corresponding with restored indices of creatine-dependent adenylate cycling. Conclusion: This mouse model demonstrates the concurrent development of cachexia and metastasis that occurs in women with EOC. The model provides physiologically relevant advantages of inducing tumour development within the ovarian bursa in immunocompetent adult mice. Moreover, the model reveals that muscle weakness in both TA and diaphragm precedes metastasis while weakness also precedes atrophy in the TA. An underlying mitochondrial bioenergetic stress corresponded with this early weakness. Collectively, these discoveries can direct new research towards the development of therapies that target pre-atrophy and pre-metastatic weakness during EOC in addition to therapies targeting cachexia.

Therapy-naïve malignancy causes cardiovascular disease: a state-of-the-art cardio-oncology perspective.

Cardiovascular disease (CVD) and cancer are the leading causes of mortality worldwide. Although generally thought of as distinct diseases, the intersectional overlap between CVD and cancer is increasingly evident in both causal and mechanistic relationships. The field of cardio-oncology is largely focused on the cardiotoxic effects of cancer therapies (e.g., chemotherapy, radiation). Furthermore, the cumulative effects of cardiotoxic therapy exposure and the prevalence of CVD risk factors in patients with cancer lead to long-term morbidity and poor quality of life in this patient population, even when patients are cancer-free. Evidence from patients with cancer and animal models demonstrates that the presence of malignancy itself, independent of cardiotoxic therapy exposure or CVD risk factors, negatively impacts cardiac structure and function. As such, the primary focus of this review is the cardiac pathophysiological and molecular features of therapy-naïve cancer. We also summarize the strengths and limitations of preclinical cancer models for cardio-oncology research and discuss therapeutic strategies that have been tested experimentally for the treatment of cancer-induced cardiac atrophy and dysfunction. Finally, we explore an adjacent area of interest, called "reverse cardio-oncology," where the sequelae of heart failure augment cancer progression. Here, we emphasize the cross-disease communication between malignancy and the injured heart and discuss the importance of chronic low-grade inflammation and endocrine factors in the progression of both diseases.

Comprehensive Proteomics Analysis of Polyhydroxyalkanoate (PHA) Biology in Pseudomonas putida KT2440: The Outer Membrane Lipoprotein OprL is a Newly Identified Phasin.

Pseudomonas putida KT2440 is an important bioplastic-producing industrial microorganism capable of synthesizing the polymeric carbon-rich storage material, polyhydroxyalkanoate (PHA). PHA is sequestered in discrete PHA granules, or carbonosomes, and accumulates under conditions of stress, for example, low levels of available nitrogen. The pha locus responsible for PHA metabolism encodes both anabolic and catabolic enzymes, a transcription factor, and carbonosome-localized proteins termed phasins. The functions of phasins are incompletely understood but genetic disruption of their function causes PHA-related phenotypes. To improve our understanding of these proteins, we investigated the PHA pathways of P.putida KT2440 using three types of experiments. First, we profiled cells grown in nitrogen-limited and nitrogen-excess media using global expression proteomics, identifying sets of proteins found to coordinately increase or decrease within clustered pathways. Next, we analyzed the protein composition of isolated carbonosomes, identifying two new putative components. We carried out physical interaction screens focused on PHA-related proteins, generating a protein-protein network comprising 434 connected proteins. Finally, we confirmed that the outer membrane protein OprL (the Pal component of the Pal-Tol system) localizes to the carbonosome and shows a PHA-related phenotype and therefore is a novel phasin. The combined datasets represent a valuable overview of the protein components of the PHA system in P.putida highlighting the complex nature of regulatory interactions responsive to nutrient stress.

An Automated Imaging-Based Screen for Genetic Modulators of ER Organisation in Cultured Human Cells.

Hereditary spastic paraplegias (HSPs) are a heterogeneous group of mono-genetic inherited neurological disorders, whose primary manifestation is the disruption of the pyramidal system, observed as a progressive impaired gait and leg spasticity in patients. Despite the large list of genes linked to this group, which exceeds 80 loci, the number of cellular functions which the gene products engage is relatively limited, among which endoplasmic reticulum (ER) morphogenesis appears central. Mutations in genes encoding ER-shaping proteins are the most common cause of HSP, highlighting the importance of correct ER organisation for long motor neuron survival. However, a major bottleneck in the study of ER morphology is the current lack of quantitative methods, with most studies to date reporting, instead, on qualitative changes. Here, we describe and apply a quantitative image-based screen to identify genetic modifiers of ER organisation using a mammalian cell culture system. An analysis reveals significant quantitative changes in tubular ER and dense sheet ER organisation caused by the siRNA-mediated knockdown of HSP-causing genes ATL1 and RTN2. This screen constitutes the first attempt to examine ER distribution in cells in an automated and high-content manner and to detect genes which impact ER organisation.

Novel roles of cardiac-derived erythropoietin in cardiac development and function.

The role of erythropoietin (EPO) has extended beyond hematopoiesis to include cytoprotection, inotropy, and neurogenesis. Extra-renal EPO has been reported for multiple tissue/cell types, but the physiological relevance remains unknown. Although the EPO receptor is expressed by multiple cardiac cell types and human recombinant EPO increases contractility and confers cytoprotection against injury, whether the heart produces physiologically meaningful amounts of EPO in vivo is unclear. We show a distinct circadian rhythm of cardiac EPO mRNA expression in adult mice and increased mRNA expression during embryogenesis, suggesting physiological relevance to cardiac EPO production throughout life. We then generated constitutive, cardiomyocyte-specific EPO knockout mice driven by the Mlc2v promoter (EPOfl/fl:Mlc2v-cre+/-; EPOΔ/Δ-CM). During cardiogenesis, cardiac EPO mRNA expression and cellular proliferation were reduced in EPOΔ/Δ-CM hearts. However, in adult EPOΔ/Δ- CM mice, total heart weight was preserved through increased cardiomyocyte cross-sectional area, indicating the reduced cellular proliferation was compensated for by cellular hypertrophy. Echocardiography revealed no changes in cardiac dimensions, with modest reductions in ejection fraction, stroke volume, and tachycardia, whereas invasive hemodynamics showed increased cardiac contractility and lusitropy. Paradoxically, EPO mRNA expression in the heart was elevated in adult EPOΔ/Δ-CM, along with increased serum EPO protein content and hematocrit. Using RNA fluorescent in situ hybridization, we found that Epo RNA colocalized with endothelial cells in the hearts of adult EPOΔ/Δ-CM mice, identifying the endothelial cells as a cell responsible for the EPO hyper-expression. Collectively, these data identify the first physiological roles for cardiomyocyte-derived EPO. We have established cardiac EPO mRNA expression is a complex interplay of multiple cell types, where loss of embryonic cardiomyocyte EPO production results in hyper-expression from other cells within the adult heart.

Bilateral nephrectomy impairs cardiovascular function and cerebral perfusion in a rat model of acute hemodilutional anemia.

Anemia and renal failure are independent risk factors for perioperative stroke, prompting us to assess the combined impact of acute hemodilutional anemia and bilateral nephrectomy (2Nx) on microvascular brain Po2 (PBro2) in a rat model. Changes in PBro2 (phosphorescence quenching) and cardiac output (CO, echocardiography) were measured in different groups of anesthetized Sprague-Dawley rats (1.5% isoflurane, n = 5-8/group) randomized to Sham 2Nx or 2Nx and subsequently exposed to acute hemodilutional anemia (50% estimated blood volume exchange with 6% hydroxyethyl starch) or time-based controls (no hemodilution). Outcomes were assessed by ANOVA with significance assigned at P < 0.05. At baseline, 2Nx rats demonstrated reduced CO (49.9 ± 9.4 vs. 66.3 ± 19.3 mL/min; P = 0.014) and PBro2 (21.1 ± 2.9 vs. 32.4 ± 3.1 mmHg; P < 0.001) relative to Sham 2Nx rats. Following hemodilution, 2Nx rats demonstrated a further decrease in PBro2 (15.0 ± 6.3 mmHg, P = 0.022). Hemodiluted 2Nx rats did not demonstrate a comparable increase in CO after hemodilution compared with Sham 2Nx (74.8 ± 22.4 vs. 108.9 ± 18.8 mL/min, P = 0.003) that likely contributed to the observed reduction in PBro2. This impaired CO response was associated with reduced fractional shortening (33 ± 9 vs. 51 ± 5%) and increased left ventricular end-systolic volume (156 ± 51 vs. 72 ± 15 µL, P < 0.001) suggestive of systolic dysfunction. By contrast, hemodiluted Sham 2Nx animals demonstrated a robust increase in CO and preserved PBro2. These data support the hypothesis that the kidney plays a central role in maintaining cerebral perfusion and initiating the adaptive increase in CO required to optimize PBro2 during acute anemia.NEW & NOTEWORTHY This study has demonstrated that bilateral nephrectomy acutely impaired cardiac output (CO) and microvascular brain Po2 (PBro2), at baseline. Following acute hemodilution, nephrectomy prevented the adaptive increase in CO associated with acute hemodilution leading to a further reduction in PBro2, accentuating the degree of cerebral tissue hypoxia. These data support a role for the kidney in maintaining PBro2 and initiating the increase in CO that optimized brain perfusion during acute anemia.

Intron retention is a mechanism of erythropoietin regulation in brain cell models.

Intron retention is a mechanism of post-transcriptional gene regulation, including genes involved in erythropoiesis. Erythropoietin (EPO) is a hormone without evidence of intracellular vesicle storage that regulates erythropoiesis. We hypothesize that EPO uses intron retention as a mechanism of post-transcriptional regulation in response to hypoxia and ischemia. Cell models of hypoxia and ischemia for kidney, liver, and brain cells were examined for intron retention by real time quantitative PCR. EPO expression increased in most cells except for blood brain barrier and liver cells. The intron retained transcript ratio decreased in brain cells, except for Astrocytes, but showed no change in kidney or liver after 24 h of ischemia. The shift in intron ratio was maintained when using poly (A) enriched cDNA, suggesting that intron retention is not due to immature transcripts. The expression of EPO was elevated at variable time points amongst cell models with the intron ratio also changing over a time course of 2 to 16 h after ischemia. We conclude that intron retention is a mechanism regulating EPO expression in response to ischemia in a tissue specific manner.

Involving patients in healthcare research is well documented but can it work in lab-based research?

Public and Patient Involvement in research is becoming a requirement on most research funding applications; this includes both healthcare and lab-based research. Whilst case studies and practical guides have been developed and are well documented for PPI in healthcare research, there is very little guidance available for PPI in lab-based research. In this piece we discuss our experience of how we have successfully involved patients in our translational cancer research, which is focused on developing personalised treatment for high-grade serous ovarian cancer. We discuss the benefits it has made to both our research and to us as researchers. The patients involved write about their experience, what they enjoyed, and the benefits they felt. Although PPI is quite topical and is being widely discussed, there is hesitancy among researchers, especially those in lab-based research about getting started because of a lack of practical guidance about how to implement it. Here, we have shared our experience, hopefully providing a practical example of how PPI can be incorporated into a lab-based research project.

This piece is co-authored by researchers and ovarian cancer patients and presents their experience of patient involvement in a laboratory-based cancer research project focused on the personalised treatment of high-grade serous ovarian cancer. Discussions with five ovarian cancer patients about their treatment experience highlighted the fact that drugs showing equivalent clinical efficacy are not necessarily tolerated equally by individual patients. This led researchers to alter their original experimental design, by including a number of the same drug type instead of focusing on only one. The researchers also discuss the benefits it has made to both the research and to them as researchers. The patients involved write about their experience, what they enjoyed, and the benefits they felt.

Cell-Penetrating Milk-Derived Peptides with a Non-Inflammatory Profile.

Milk-derived peptides are known to confer anti-inflammatory effects. We hypothesised that milk-derived cell-penetrating peptides might modulate inflammation in useful ways. Using computational techniques, we identified and synthesised peptides from the milk protein Alpha-S1-casein that were predicted to be cell-penetrating using a machine learning predictor. We modified the interpretation of the prediction results to consider the effects of histidine. Peptides were then selected for testing to determine their cell penetrability and anti-inflammatory effects using HeLa cells and J774.2 mouse macrophage cell lines. The selected peptides all showed cell penetrating behaviour, as judged using confocal microscopy of fluorescently labelled peptides. None of the peptides had an effect on either the NF-κB transcription factor or TNFα and IL-1ß secretion. Thus, the identified milk-derived sequences have the ability to be internalised into the cell without affecting cell homeostatic mechanisms such as NF-κB activation. These peptides are worthy of further investigation for other potential bioactivities or as a naturally derived carrier to promote the cellular internalisation of other active peptides.

Cannabidiol Inhibits the Proliferation and Invasiveness of Prostate Cancer Cells.

Prostate cancer is the fifth leading cause of cancer death in men, responsible for over 375,000 deaths in 2020. Novel therapeutic strategies are needed to improve outcomes. Cannabinoids, chemical components of the cannabis plant, are a possible solution. Preclinical evidence demonstrates that cannabinoids can modulate several cancer hallmarks of many tumor types. However, the therapeutic potential of cannabinoids in prostate cancer has not yet been fully explored. The aim of this study was to investigate the antiproliferative and anti-invasive properties of cannabidiol (CBD) in prostate cancer cells in vitro. CBD inhibited cell viability and proliferation, accompanied by reduced expression of key cell cycle proteins, specifically cyclin D3 and cyclin-dependent kinases CDK2, CDK4, and CDK1, and inhibition of AKT phosphorylation. The effects of CBD on cell viability were not blocked by cannabinoid receptor antagonists, a transient receptor potential vanilloid 1 (TRPV1) channel blocker, or an agonist of the G-protein-coupled receptor GPR55, suggesting that CBD acts independently of these targets in prostate cancer cells. Furthermore, CBD reduced the invasiveness of highly metastatic PC-3 cells and increased protein expression of E-cadherin. The ability of CBD to inhibit prostate cancer cell proliferation and invasiveness suggests that CBD may have potential as a future chemotherapeutic agent.

Pseudomonas umsongensis GO16 as a platform for the in vivo synthesis of short and medium chain length polyhydroxyalkanoate blends.

Polyhydroxyalkanoates (PHAs) are biological polyesters, viewed as a replacement for petrochemical plastic. However, they suffer from suboptimal physical and mechanical properties. Here, it was shown that a metabolically versatile Pseudomonas umsongensis GO16 can synthesise a blend of short chain length (scl) and medium chain length (mcl)-PHA. A defined mix of butyric (BA) and octanoic acid (OA) in different ratios was used. The PHA monomer composition varied depending on the feeding strategy. When OA and BA were fed at 80:20 ratio it showed 14, 8, 77 and 1 mol% of (R)-3-hydroxybutyrate, (R)-3-hydroxyhexanoate, (R)-3-hydroxyoctanoate and (R)-3-hydroxydecanoate respectively. The polymer characterisation clearly shows that polyhydroxybutyrate (PHB) and mcl-PHA are produced individually. The two polymers are blended on the PHA granule level, as demonstrated by fluorescence microscopy and yeast two-hybrid assay. The resulting blend has a specific viscoelasticity compared to PHB and PHO. Mcl-PHA acts as a plasticiser and reduces PHB brittleness.

Rab6-mediated retrograde trafficking from the Golgi: the trouble with tubules.

Next year marks one-quarter of a century since the discovery of the so-called COPI-independent pathway, which operates between the Golgi apparatus and the endoplasmic reticulum (ER) in eukaryotic cells. Unlike almost all other intracellular trafficking pathways, this pathway is not regulated by the physical accumulation of multisubunit proteinaceous coat molecules, but instead by the small GTPase Rab6. What also sets it apart from other pathways is that the transport carriers themselves often take the form of tubules, rather than conventional vesicles. In this review, we assess the relevant literature that has accumulated to date, in an attempt to provide a concerted description of how this pathway is regulated. We discuss the possible cargo molecules that are carried in this pathway, and the likely mechanism of Rab6 tubule biogenesis, including how the cargo itself may play a critical role. We also provide perspective surrounding the various molecular motors of the kinesin, myosin and dynein families that have been implicated in driving Rab6-coated tubular membranes long distances through the cell prior to delivering their cargo to the ER. Finally, we also raise several important questions that require resolution, if we are to ultimately provide a comprehensive molecular description of how the COPI-independent pathway is controlled.

Importance of assessing biomarkers and physiological parameters of anemia-induced tissue hypoxia in the perioperative period

Abstract Anemia is associated with increased risk of Acute Kidney Injury (AKI), stroke and mortality in perioperative patients. We sought to understand the mechanism(s) by assessing the integrative physiological responses to anemia (kidney, brain), the degrees of anemia-induced tissue hypoxia, and associated biomarkers and physiological parameters. Experimental measurements demonstrate a linear relationship between blood Oxygen Content (CaO2) and renal microvascular PO2 (y = 0.30x + 6.9, r2= 0.75), demonstrating that renal hypoxia is proportional to the degree of anemia. This defines the kidney as a potential oxygen sensor during anemia. Further evidence of renal oxygen sensing is demonstrated by proportional increase in serum Erythropoietin (EPO) during anemia (y = 93.806*10−0.02, r2= 0.82). This data implicates systemic EPO levels as a biomarker of anemia-induced renal tissue hypoxia. By contrast, cerebral Oxygen Delivery (DO2) is defended by a profound proportional increase in Cerebral Blood Flow (CBF), minimizing tissue hypoxia in the brain, until more severe levels of anemia occur. We hypothesize that the kidney experiences profound early anemia-induced tissue hypoxia which contributes to adaptive mechanisms to preserve cerebral perfusion. At severe levels of anemia, renal hypoxia intensifies, and cerebral hypoxia occurs, possibly contributing to the mechanism(s) of AKI and stroke when adaptive mechanisms to preserve organ perfusion are overwhelmed. Clinical methods to detect renal tissue hypoxia (an early warning signal) and cerebral hypoxia (a later consequence of severe anemia) may inform clinical practice and support the assessment of clinical biomarkers (i.e., EPO) and physiological parameters (i.e., urinary PO2) of anemia-induced tissue hypoxia. This information may direct targeted treatment strategies to prevent adverse outcomes associated with anemia.

A universal stress protein upregulated by hypoxia has a role in Burkholderia cenocepacia intramacrophage survival: Implications for chronic infection in cystic fibrosis.

Universal stress proteins (USPs) are ubiquitously expressed in bacteria, archaea, and eukaryotes and play a lead role in adaptation to environmental conditions. They enable adaptation of bacterial pathogens to the conditions encountered in the human niche, including hypoxia, oxidative stress, osmotic stress, nutrient deficiency, or acid stress, thereby facilitating colonization. We previously reported that all six USP proteins encoded within a low-oxygen activated (lxa) locus in Burkholderia cenocepacia showed increased abundance during chronic colonization of the cystic fibrosis (CF) lung. However, the role of USPs in chronic cystic fibrosis infection is not well understood. Structural modeling identified surface arginines on one lxa-encoded USP, USP76, which suggested it mediated interactions with heparan sulfate. Using mutants derived from the B. cenocepacia strain, K56-2, we show that USP76 is involved in host cell attachment. Pretreatment of lung epithelial cells with heparanase reduced the binding of the wild-type and complement strains but not the Δusp76 mutant strain, indicating that USP76 is directly or indirectly involved in receptor recognition on the surface of epithelial cells. We also show that USP76 is required for growth and survival in many conditions associated with the CF lung, including acidic conditions and oxidative stress. Moreover, USP76 also has a role in survival in macrophages isolated from people with CF. Overall, while further elucidation of the exact mechanism(s) is required, we can conclude that USP76, which is upregulated during chronic infection, is involved in bacterial survival within CF macrophages, a hallmark of Burkholderia infection.

Dietary Nitrate and Corresponding Gut Microbiota Prevent Cardiac Dysfunction in Obese Mice.

Impaired heart function can develop in individuals with diabetes in the absence of coronary artery disease or hypertension, suggesting mechanisms beyond hypertension/increased afterload contribute to diabetic cardiomyopathy. Identifying therapeutic approaches that improve glycemia and prevent cardiovascular disease are clearly required for clinical management of diabetes-related comorbidities. Since intestinal bacteria are important for metabolism of nitrate, we examined whether dietary nitrate and fecal microbial transplantation (FMT) from nitrate-fed mice could prevent high-fat diet (HFD)-induced cardiac abnormalities. Male C57Bl/6N mice were fed a low-fat diet (LFD), HFD, or HFD+Nitrate (4 mmol/L sodium nitrate) for 8 weeks. HFD-fed mice presented with pathological left ventricle (LV) hypertrophy, reduced stroke volume, and increased end-diastolic pressure, in association with increased myocardial fibrosis, glucose intolerance, adipose inflammation, serum lipids, LV mitochondrial reactive oxygen species (ROS), and gut dysbiosis. In contrast, dietary nitrate attenuated these detriments. In HFD-fed mice, FMT from HFD+Nitrate donors did not influence serum nitrate, blood pressure, adipose inflammation, or myocardial fibrosis. However, microbiota from HFD+Nitrate mice decreased serum lipids, LV ROS, and similar to FMT from LFD donors, prevented glucose intolerance and cardiac morphology changes. Therefore, the cardioprotective effects of nitrate are not dependent on reducing blood pressure, but rather mitigating gut dysbiosis, highlighting a nitrate-gut-heart axis. ARTICLE HIGHLIGHTS: Identifying therapeutic approaches that prevent cardiometabolic diseases are clearly important, and nitrate represents one such potential compound given its multifactorial metabolic effects. We aimed to determine whether nitrate could prevent high-fat diet (HFD)-induced cardiac abnormalities and whether this was dependent on the gut microbiome. Dietary nitrate attenuated HFD-induced pathological changes in cardiac remodelling, left ventricle reactive oxygen species, adipose inflammation, lipid homeostasis, glucose intolerance, and gut dysbiosis. Fecal microbial transplantation from nitrate-fed mice also prevented serum dyslipidemia, left ventricle reactive oxygen species, glucose intolerance, and cardiac dysfunction. Therefore, the cardioprotective effects of nitrate are related to mitigating gut dysbiosis, highlighting a nitrate-gut-heart axis.

Importance of assessing biomarkers and physiological parameters of anemia-induced tissue hypoxia in the perioperative period.

Anemia is associated with increased risk of Acute Kidney Injury (AKI), stroke and mortality in perioperative patients. We sought to understand the mechanism(s) by assessing the integrative physiological responses to anemia (kidney, brain), the degrees of anemia-induced tissue hypoxia, and associated biomarkers and physiological parameters. Experimental measurements demonstrate a linear relationship between blood Oxygen Content (CaO2) and renal microvascular PO2 (y = 0.30x + 6.9, r2 = 0.75), demonstrating that renal hypoxia is proportional to the degree of anemia. This defines the kidney as a potential oxygen sensor during anemia. Further evidence of renal oxygen sensing is demonstrated by proportional increase in serum Erythropoietin (EPO) during anemia (y = 93.806*10-0.02, r2 = 0.82). This data implicates systemic EPO levels as a biomarker of anemia-induced renal tissue hypoxia. By contrast, cerebral Oxygen Delivery (DO2) is defended by a profound proportional increase in Cerebral Blood Flow (CBF), minimizing tissue hypoxia in the brain, until more severe levels of anemia occur. We hypothesize that the kidney experiences profound early anemia-induced tissue hypoxia which contributes to adaptive mechanisms to preserve cerebral perfusion. At severe levels of anemia, renal hypoxia intensifies, and cerebral hypoxia occurs, possibly contributing to the mechanism(s) of AKI and stroke when adaptive mechanisms to preserve organ perfusion are overwhelmed. Clinical methods to detect renal tissue hypoxia (an early warning signal) and cerebral hypoxia (a later consequence of severe anemia) may inform clinical practice and support the assessment of clinical biomarkers (i.e., EPO) and physiological parameters (i.e., urinary PO2) of anemia-induced tissue hypoxia. This information may direct targeted treatment strategies to prevent adverse outcomes associated with anemia.

An imaging-based RNA interference screen for modulators of the Rab6-mediated Golgi-to-ER pathway in mammalian cells.

In mammalian cells, membrane traffic pathways play a critical role in connecting the various compartments of the endomembrane system. Each of these pathways is highly regulated, requiring specific machinery to ensure their fidelity. In the early secretory pathway, transport between the endoplasmic reticulum (ER) and Golgi apparatus is largely regulated via cytoplasmic coat protein complexes that play a role in identifying cargo and forming the transport carriers. The secretory pathway is counterbalanced by the retrograde pathway, which is essential for the recycling of molecules from the Golgi back to the ER. It is believed that there are at least two mechanisms to achieve this - one using the cytoplasmic COPI coat complex, and another, poorly characterised pathway, regulated by the small GTPase Rab6. In this work, we describe a systematic RNA interference screen targeting proteins associated with membrane fusion, in order to identify the machinery responsible for the fusion of Golgi-derived Rab6 carriers at the ER. We not only assess the delivery of Rab6 to the ER, but also one of its cargo molecules, the Shiga-like toxin B-chain. These screens reveal that three proteins, VAMP4, STX5, and SCFD1/SLY1, are all important for the fusion of Rab6 carriers at the ER. Live cell imaging experiments also show that the depletion of SCFD1/SLY1 prevents the membrane fusion event, suggesting that this molecule is an essential regulator of this pathway.

Photophysical Properties and DNA Binding of Two Intercalating Osmium Polypyridyl Complexes Showing Light-Switch Effects.

The synthesis and photophysical characterization of two osmium(II) polypyridyl complexes, [Os(TAP)2dppz]2+ (1) and [Os(TAP)2dppp2]2+ (2) containing dppz (dipyrido[3,2-a:2',3'-c]phenazine) and dppp2 (pyrido[2',3':5,6]pyrazino[2,3-f][1,10]phenanthroline) intercalating ligands and TAP (1,4,5,8-tetraazaphenanthrene) ancillary ligands, are reported. The complexes exhibit complex electrochemistry with five distinct reductive redox couples, the first of which is assigned to a TAP-based process. The complexes emit in the near-IR (1 at 761 nm and 2 at 740 nm) with lifetimes of >35 ns with a low quantum yield of luminescence in aqueous solution (∼0.25%). The Δ and Λ enantiomers of 1 and 2 are found to bind to natural DNA and with AT and GC oligodeoxynucleotides with high affinities. In the presence of natural DNA, the visible absorption spectra are found to display significant hypochromic shifts, which is strongly evident for the ligand-centered π-π* dppp2 transition at 355 nm, which undergoes 46% hypochromism. The emission of both complexes increases upon DNA binding, which is observed to be sensitive to the Δ or Λ enantiomer and the DNA composition. A striking result is the sensitivity of Λ-2 to the presence of AT DNA, where a 6-fold enhancement of luminescence is observed and reflects the nature of the binding for the enantiomer and the protection from solution. Thermal denaturation studies show that both complexes are found to stabilize natural DNA. Finally, cellular studies show that the complexes are internalized by cultured mammalian cells and localize in the nucleus.