The effect of diet on Plasmodium falciparum development revealed by NMR metabolomics and image analysis

The development of axenic in vitro growth models of the human malaria parasite Plasmodium falciparum, has been pivotal in accelerating knowledge of this very important human pathogen. Despite the importance of this pathogen, there have been very few studies relating to the metabolism of the parasite. Furthermore, much of the preceding studies have been undertaken using culture conditions that do not accurately represent the physiological environment of the human host. There is a need to address whether different nutrient environments would trigger a parasite response at the systems level promoting a metabolic rewiring that would have an effect in progeny generation or life cycle progression. Because of its robustness, reproducibility and suitability for footprinting studies, NMR spectroscopy was chosen as the analytic technique for the study. One of the disadvantages of NMR is limited availability of software for identification and quantification of metabolites. This was taken as an opportunity to develop a pipeline using free, open-source programming framework. This tool was used to find unique and discriminatory metabolic profiles for both uninfected and P. falciparum infected red blood cells at various life-cycle stages using cell extracts and extracellular material. With the aim of studying parasite development in physiological conditions a culture medium mimicking human blood conditions was developed and tested on P. falciparum infected RBCs finding both phenotypic and metabolic differences. Further studies consisted of the development of tightly synchronised parasite cultures that were followed during 54 h using NMR-based metabolomics to assess consumption and excretion of metabolites in media, and high content imaging and bright field microscopy to assess parasite size and progeny. The measurements were taken under three different nutritional conditions: usual in vitro, physiological-like and hypoglycaemic. In usual culturing conditions P. falciparum 3D7 life cycle lasted around 45 h. During the early stages there was moderate consumption of glucose and glutamine and excretion of lactate, alanine and glycerol. During the mature trophozoite stages and schizonts, glucose uptake dramatically increased with a consequent augmentation of the lactate, alanine and glycerol production. These were excreted but their function was not clear. It was observed that these “wasteful" products were proportionally lower in the early developmental stages than in the later ones, suggesting a higher demand of raw materials (glucose) for biomass production during the early stages. During the late trophozoite stage the most abundant amino acids in the haemoglobin chain (leucine, valine and glycine) were excreted, a likely consequence of the need for space to nish maturation. Myoinositol, which is essential for creation of membranes was also avidly consumed. When comparing these findings with parasites growing in more physiological conditions there was a noticeable delay in the life cycle of at least 9 h. Consequently haemoglobin digestive products were excreted later in the time course. A decrease in the progeny resulting from schizonts containing significantly fewer merozoites was also observed. Parasites growing in physiological conditions but challenged with lower glucose availability also presented a further delay of the life-cycle and a decreased number of merozoites with respect to usual laboratory conditions. Haemoglobin degradation products were also excreted later in the life cycle and at lower rates compared to the parasites grown in complete media. These results suggest that there are significant differences between in vivo and in vitro life-cycles of P. falciparum. Such effects as the reduction in growth rates and elongation of the life cycle, if not accounted for, could severely compromise the in vivo results of in vitro drug killing rates assay

The proliferating cell hypothesis: a metabolic framework for Plasmodium growth and development.

We hypothesise that intraerythrocytic malaria parasite metabolism is not merely fulfilling the need for ATP generation, but is evolved to support rapid proliferation, similar to that seen in other rapidly proliferating cells such as cancer cells. Deregulated glycolytic activity coupled with impaired mitochondrial metabolism is a metabolic strategy to generate glycolytic intermediates essential for rapid biomass generation for schizogony. Further, we discuss the possibility that Plasmodium metabolism is not only a functional consequence of the 'hard-wired' genome and argue that metabolism may also have a causal role in triggering the cascade of events that leads to developmental stage transitions. This hypothesis offers a framework to rationalise the observations of aerobic glycolysis, atypical mitochondrial metabolism, and metabolic switching in nonproliferating stages

MMP13 and TIMP1 are functional markers for two different potential modes of action by mesenchymal stem/stromal cells when treating osteoarthritis

Mesenchymal stem cells (MSCs) have been investigated as a potential injectable therapy for the treatment of knee osteoarthritis, with some evidence of success in preliminary human trials. However, optimization and scale‐up of this therapeutic approach depends on the identification of functional markers that are linked to their mechanism of action. One possible mechanism is through their chondrogenic differentiation and direct role in neo‐cartilage synthesis. Alternatively, they could remain undifferentiated and act through the release of trophic factors that stimulate endogenous repair processes within the joint. Here, we show that extensive in vitro aging of bone marrow‐derived human MSCs leads to loss of chondrogenesis but no reduction in trophic repair, thereby separating out the two modes of action. By integrating transcriptomic and proteomic data using Ingenuity Pathway Analysis, we found that reduced chondrogenesis with passage is linked to downregulation of the FOXM1 signaling pathway while maintenance of trophic repair is linked to CXCL12. In an attempt at developing functional markers of MSC potency, we identified loss of mRNA expression for MMP13 as correlating with loss of chondrogenic potential of MSCs and continued secretion of high levels of TIMP1 protein as correlating with the maintenance of trophic repair capacity. Since an allogeneic injectable osteoar therapy would require extensive cell expansion in vitro, we conclude that early passage MMP13+, TIMP1‐secretinghigh MSCs should be used for autologous OA therapies designed to act through engraftment and chondrogenesis, while later passage MMP13−, TIMP1‐secretinghigh MSCs could be exploited for allogeneic OA therapies designed to act through trophic repair

Exploring the potential of rapid evaporative ionization mass spectrometry (Intelligent Knife) for point-of-care testing in aortic surgery

Abstract OBJECTIVES Many intraoperative decisions regarding the extent of thoracic aortic surgery are subjective and are based on the appearance of the aorta, perceived surgical risks and likelihood of early recurrent disease. Our objective in this work was to carry out a cross-sectional study to demonstrate that rapid evaporative ionization mass spectrometry (REIMS) of electrosurgical aerosol is able to empirically discriminate ex vivo aneurysmal human thoracic aorta from normal aorta, thus providing supportive evidence for the development of the technique as a point-of-care test guiding intraoperative surgical decision-making. METHODS Human aortic tissue was obtained from patients undergoing surgery for thoracic aortic aneurysms (n = 44). Normal aorta was obtained from a mixture of post-mortem and punch biopsies from patients undergoing coronary surgery (n = 13). Monopolar electrocautery was applied to samples and surgical aerosol aspirated and analysed by REIMS to produce mass spectral data. RESULTS Models generated from REIMS data can discriminate aneurysmal from normal aorta with accuracy and precision of 88.7% and 85.1%, respectively. In addition, further analysis investigating aneurysmal tissue from patients with bicuspid and tricuspid aortic valves was discriminated from normal tissue and each other with accuracies and precision of 93.5% and 91.4% for control, 83.8% and 76.7% for bicuspid aortic valve and 89.3% and 86.0% for tricuspid aortic valve, respectively. CONCLUSIONS Analysis of electrosurgical aerosol from ex vivo aortic tissue using REIMS allowed us to discriminate aneurysmal from normal aorta, supporting its development as a point-of-care test (Intelligent Knife) for guiding surgical intraoperative decision-making. </jats:sec

Rapid evaporative ionization mass spectrometry (intelligent knife) for point-of-care testing in acute aortic dissection surgery

Abstract OBJECTIVES Rapid evaporative ionization mass spectrometry (REIMS) can discriminate aneurysmal from normal aortic tissue. Our objective in this work was to probe the integrity of acute dissection tissue using biomechanical, biochemical and histological techniques and demonstrate that REIMS can be used to discriminate identified differences. METHODS Human aortic tissue was obtained from patients undergoing surgery for acute aortic dissection. Biomechanical, biochemical and histological assessment was carried out to probe mechanical properties and elastin, collagen and glycosaminoglycan composition of the tissue. Monopolar electrocautery was applied to samples and surgical aerosol aspirated and analysed by REIMS to produce mass spectral data. RESULTS Tissue was obtained from 10 patients giving rise to 26 tissue pieces: 10 false lumen (FL), 10 dissection flap and 6 true lumen samples. Models generated from biomechanical and biochemical data showed that FL tissue was distinct from true lumen and dissection flap tissue. REIMS identified the same pattern being able to classify tissue types with 72.4% accuracy and 69.3% precision. Further analysis of REIMS data for FL tissue suggested patients formed 3 distinct clusters. Histological and biochemical assessment revealed patterns of extracellular matrix degradation within the clusters that are associated with altered tissue integrity identified using biomechanical testing. CONCLUSIONS Structural integrity of the FL in acute Type A dissection could dictate future clinical distal disease progression. REIMS can detect differences in tissue integrity, supporting its development as a point-of-care test to guide surgical intraoperative decision-making. </jats:sec

Outcomes in Newly Diagnosed Atrial Fibrillation and History of Acute Coronary Syndromes: Insights from GARFIELD-AF

BACKGROUND: Many patients with atrial fibrillation have concomitant coronary artery disease with or without acute coronary syndromes and are in need of additional antithrombotic therapy. There are few data on the long-term clinical outcome of atrial fibrillation patients with a history of acute coronary syndrome. This is a 2-year study of atrial fibrillation patients with or without a history of acute coronary syndromes

Analysis of Outcomes in Ischemic vs Nonischemic Cardiomyopathy in Patients With Atrial Fibrillation A Report From the GARFIELD-AF Registry

IMPORTANCE Congestive heart failure (CHF) is commonly associated with nonvalvular atrial fibrillation (AF), and their combination may affect treatment strategies and outcomes