'I'm a failure again, I can't do it': Attitudes towards, and experiences of, exercise participation in adults with class III obesity.

OBJECTIVES: Living within a larger body brings unique challenges to exercise participation, which are poorly understood. This qualitative study explored the attitudes towards, and experiences of, exercise participation in adults with class III obesity. DESIGN: Individual semi-structured qualitative interviews. METHODS: We recruited 30 adults with class III obesity (body mass index: 45.8 ± 8.6 kg/m2) from a specialist multidisciplinary weight management service. Participants took part in semi-structured interviews while participating in a 6-month home-based aerobic and resistance exercise intervention. Open-ended questions were used flexibly to explore their views and experiences of exercise, encompassing barriers, motives and perceived benefits. Transcripts were analysed using reflexive thematic analysis. RESULTS: Three themes were developed: (1) a web of barriers; (2) tailored exercise facilitates positive experiences; and (3) a desire to live a normal life. People with class III obesity perceived that they were unable to do exercise; a view that was attributed to perceived judgement, low physical function, pain during everyday activities and failed weight loss attempts. These complex physical and psychosocial barriers to exercise were described as contributing to exercise avoidance. High value was placed on tailored exercise that accommodates the unique needs of moving in a larger body. A desire to carry out everyday tasks underpinned motivations for exercise. CONCLUSIONS: Our findings suggest that multi-component obesity interventions should move away from generic exercise prescriptions designed to maximize energy expenditure, and instead move towards addressing the unique physical and psychosocial needs of people who have class III obesity with tailored person-centred and weight-neutral exercise prescriptions.

Early Human Pathophysiological Responses to Exertional Hypobaric Decompression Stress.

INTRODUCTION: Consistent blood biomarkers of hypobaric (altitude) decompression stress remain elusive. Recent laboratory investigation of decompression sickness risk at 25,000 ft (7620 m) enabled evaluation of early pathophysiological responses to exertional decompression stress.METHODS: In this study, 15 healthy men, aged 20-50 yr, undertook 2 consecutive (same-day) ascents to 25,000 ft (7620 m) for 60 and 90 min, breathing 100% oxygen, each following 1 h of prior denitrogenation. Venous blood was sampled at baseline (T0), immediately after the second ascent (T8), and next morning (T24). Analyses encompassed whole blood hematology, endothelial microparticles, and soluble markers of cytokine response, endothelial function, inflammation, coagulopathy, oxidative stress, and brain insult, plus cortisol and creatine kinase.RESULTS: Acute hematological effects on neutrophils (mean 72% increase), eosinophils (40% decrease), monocytes (37% increase), and platelets (7% increase) normalized by T24. Consistent elevation (mean five-fold) of the cytokine interleukin-6 (IL-6) at T8 was proinflammatory and associated with venous gas emboli (microbubble) load. Levels of C-reactive protein and complement peptide C5a were persistently elevated at T24, the former by 100% over baseline. Additionally, glial fibrillary acidic protein, a sensitive marker of traumatic brain injury, increased by a mean 10% at T24.CONCLUSIONS: This complex composite environmental stress, comprising the triad of hyperoxia, decompression, and moderate exertion at altitude, provoked pathophysiological changes consistent with an IL-6 cytokine-mediated inflammatory response. Multiple persistent biomarker disturbances at T24 imply incomplete recovery the day after exposure. The elevation of glial fibrillary acidic protein similarly implies incomplete resolution following recent neurological insult.Connolly DM, Madden LA, Edwards VC, D'Oyly TJ, Harridge SDR, Smith TG, Lee VM. Early human pathophysiological responses to exertional hypobaric decompression stress. Aerosp Med Hum Perform. 2023; 94(10):738-749.

Immobilised-enzyme microreactors for the identification and synthesis of conjugated drug metabolites.

The study of naturally circulating drug metabolites has been a focus of interest, since these metabolites may have different therapeutic and toxicological effects compared to the parent drug. The synthesis of metabolites outside of the human body is vital in order to conduct studies into the pharmacological activities of drugs and bioactive compounds. Current synthesis methods require significant purification and separation efforts or do not provide sufficient quantities for use in pharmacology experiments. Thus, there is a need for simple methods yielding high conversions whilst bypassing the requirement for a separation. Here we have developed and optimised flow chemistry methods in glass microfluidic reactors utilising surface-immobilised enzymes for sulfonation (SULT1a1) and glucuronidation (UGT1a1). Conversion occurs in flow, the precursor and co-factor are pumped through the device, react with the immobilised enzymes and the product is then simply collected at the outlet with no separation from a complex biological matrix required. Conversion only occurred when both the correct co-factor and enzyme were present within the microfluidic system. Yields of 0.97 ± 0.26 µg were obtained from the conversion of resorufin into resorufin sulfate over 2 h with the SULT1a1 enzyme and 0.47 µg of resorufin glucuronide over 4 h for UGT1a1. This was demonstrated to be significantly more than static test tube reactions at 0.22 µg (SULT1a1) and 0.19 µg (UGT1a1) over 4 h. With scaling out and parallelising, useable quantities of hundreds of micrograms for use in pharmacology studies can be synthesised simply.

Digital Health Solutions for Weight Loss and Obesity: A Narrative Review.

Personal exercise programmes have long been used and prescribed for weight loss and the improvement of quality of life in obese patients. While individualised programmes are usually the preferred option, they can be more costly and challenging to deliver in person. A move to digital programmes with a wider reach has commenced, and demand has increased due to the SARS-CoV-2 pandemic. In this review, we evaluate the current status of digital exercise programme delivery and its evolution over the past decade, with a focus on personalisation. We used specific keywords to search for articles that met our predetermined inclusion and exclusion criteria in order to provide valuable evidence and insights for future research. We identified 55 studies in total in four key areas of focus, from the more recent development of apps and personal digital assistants to web-based programmes and text or phone call interventions. In summary, we observed that apps may be useful for a low-intensity approach and can improve adherence to programmes through self-monitoring, but they are not always developed in an evidence-based manner. Engagement and adherence are important determinants of weight loss and subsequent weight maintenance. Generally, professional support is required to achieve weight loss goals.

Vascularized Co-Culture Clusteroids of Primary Endothelial and Hep-G2 Cells Based on Aqueous Two-Phase Pickering Emulsions.

Three-dimensional cell culture has been extensively involved in biomedical applications due to its high availability and relatively mature biochemical properties. However, single 3D cell culture models based on hydrogel or various scaffolds do not meet the more in-depth requirements of in vitro models. The necrotic core formation inhibits the utilization of the 3D cell culture ex vivo as oxygen permeation is impaired in the absence of blood vessels. We report a simple method to facilitate the formation of angiogenic HUVEC (human umbilical vein endothelial cells) and Hep-G2 (hepatocyte carcinoma model) co-culture 3D clusteroids in a water-in-water (w/w) Pickering emulsions template which can overcome this limitation. This method enabled us to manipulate the cells proportion in order to achieve the optimal condition for stimulating the production of various angiogenic protein markers in the co-cultured clusteroids. The HUVEC cells respond to the presence of Hep-G2 cells and their byproducts by forming endothelial cell sprouts in Matrigel without the exogenous addition of vascular endothelial growth factor (VEGF) or other angiogenesis inducers. This culture method can be easily replicated to produce other types of cell co-culture spheroids. The w/w Pickering emulsion template can facilitate the fabrication of 3D co-culture models to a great extent and be further utilized in drug testing and tissue engineering applications.

Fabrication of Angiogenic Sprouting Coculture of Cell Clusteroids Using an Aqueous Two-Phase Pickering Emulsion System.

Tumor cell spheroids and 3D cell culture have generated a lot of interest in the past decade due to their relative ease of production and biomedical research applications. To date, the frontier in tumor 3D models has been pushed to the level of personalized cancer treatment and customized tissue engineering applications. However, without vascularization, the central parts of these artificial constructs cannot survive without an adequate oxygen and nutrient supply. The formation of a necrotic core into in vitro 3D cell models still serves as the major obstacle in their wider practical application. Here, we propose a rapid formation protocol based on using a water-in-water (w/w) Pickering emulsion template to generate phenotypically endothelial/hepatic (ECV304/Hep-G2) coculture cell clusteroids with angiogenic capability. The w/w Pickering emulsion template was based on a dextran/poly(ethylene oxide) aqueous two-phase system stabilized by whey protein particles. The initial cell proportion in the coculture clusteroids can easily be manipulated for optimal performance. The cocultured pattern of the endothelial/hepatic cells could significantly promote the production of angiogenesis-related proteins. Our study confirmed that cocultured clusteroids can stimulate cell sprouting without the addition of vascular endothelial growth factor (VEGF) or other angiogenesis inducers at a 1:2 ratio of Hep-G2/ECV304. Angiogenesis gene production in the coculture clusteroids was enhanced with VEGF, urea, and insulin-like growth factor-binding protein along with angiogenesis-related marker CD34 levels, also indicating angiogenesis progress. Our aqueous two-phase Pickering emulsion templates provided a convenient approach to vascularize a target cell type in 3D cell coculture without additional stimulating factors, which could potentially apply to either cell lines or biopsy tissues, expanding the clusteroids downstream applications.

Enhanced clearing of Candida biofilms on a 3D urothelial cell in vitro model using lysozyme-functionalized fluconazole-loaded shellac nanoparticles.

Candida urinary tract biofilms are increasingly witnessed in nosocomial infections due to reduced immunity of patients and the hospital ecosystem. The indwelling devices utilized to support patients with urethral diseases that connect the unsterilized external environment with the internal environment of the patient are another significant source of urinary tract biofilm infections. Recently, nanoparticle (NP)-associated therapeutics have gained traction in a number of areas, including fighting antibiotic-resistant bacterial biofilm infection. However, most studies on nanotherapeutic delivery have only been carried out in laboratory settings rather than in clinical trials due to the lack of precise in vitro and in vivo models for testing their efficiency. Here we develop a novel biofilm-infected 3D human urothelial cell culture model to test the efficiency of nanoparticle (NP)-based antifungal therapeutics. The NPs were designed based on shellac cores, loaded with fluconazole and coated with the cationic enzyme lysozyme. Our formulation of 0.2 wt% lysozyme-coated 0.02 wt% fluconazole-loaded 0.2 wt% shellac NPs, sterically stabilised by 0.25 wt% poloxamer 407, showed an enhanced efficiency in removing Candida albicans biofilms formed on 3D layer of urothelial cell clusteroids. The NP formulation exhibited low toxicity to urothelial cells. This study provides a reliable in vitro model for Candida urinary tract biofilm infections, which could potentially replace animal models in the testing of such antifungal nanotechnologies. The reproducibility and availability of a well-defined biofilm-infected 3D urothelial cell culture model give valuable insights into the formation and clearing of fungal biofilms and could accelerate the clinical use of antifungal nanotherapeutics.

Biofilm-Infected Human Clusteroid Three-Dimensional Coculture Platform to Replace Animal Models in Testing Antimicrobial Nanotechnologies.

Microbial biofilms are a major concern in wound care, implant devices, and organ infections. Biofilms allow higher tolerance to antimicrobial drugs, can impair wound healing, and potentially lead to sepsis. There has been a recent focus on developing novel nanocarrier-based delivery vehicles to enhance the biofilm penetration of traditional antibacterial drugs. However, a feasible in vitro human skin model to mimic the biofilm formation and its treatment for clearance have not yet been reported. This study describes the benefits of using an innovative bacterial biofilm-infected keratinocyte clusteroid model for the first time. It paves a new way for testing innovative nanomedicine delivery systems in a rapid and reproducible way on a realistic human cell-based platform, free of any animal testing. Herein, we have developed a novel composite 3D biofilm/human keratinocyte clusteroid coculture platform, which was used to measure biofilm clearance efficiency of nanoparticle (NP)-based therapeutics. We tested this model by treating the biofilm-infected 3D coculture layers by a ciprofloxacin-loaded Carbopol nanogel particles, surface-functionalized by the cationic protease Alcalase. We measured the antibacterial efficiency of the NP treatment on clearing Staphylococcus aureus and Pseudomonas aeruginosa biofilms on the 3D keratinocyte clusteroid/biofilm coculture model. Our experiments showed that these bacteria can infect the 3D layer of keratinocyte clusteroids and produce a stable biofilm. The biofilms were efficiently cleared by treatment with a formulation of 0.0032 wt % ciprofloxacin-loaded in 0.2 wt % Carbopol NPs surface-functionalized with 0.2 wt % Alcalase. Taken together, these promising results demonstrate that our coculture model can be exploited as a novel platform for testing the biofilm-eliminating efficiency of various NP formulations emulating skin and wound infections and could have wider applicability to replace animal models in similar experiments. This 3D cell culture-based platform could help in developing and testing of more effective antibacterial agents for clinical applications of antiplaque dental treatments, implants, infection control, and wound dressings.

Advanced biomedical applications based on emerging 3D cell culturing platforms.

It is of great value to develop reliable in vitro models for cell biology and toxicology. However, ethical issues and the decreasing number of donors restrict the further use of traditional animal models in various fields, including the emerging fields of tissue engineering and regenerative medicine. The huge gap created by the restrictions in animal models has pushed the development of the increasingly recognized three-dimensional (3D) cell culture, which enables cells to closely simulate authentic cellular behaviour such as close cell-to-cell interactions and can achieve higher functionality. Furthermore, 3D cell culturing is superior to the traditional 2D cell culture, which has obvious limitations and cannot closely mimic the structure and architecture of tissues. In this study, we review several methods used to form 3D multicellular spheroids. The extracellular microenvironment of 3D spheroids plays a role in many aspects of biological sciences, including cell signalling, cell growth, cancer cell generation, and anti-cancer drugs. More recently, they have been explored as basic construction units for tissue and organ engineering. We review this field with a focus on the previous research in different areas using spheroid models, emphasizing aqueous two-phase system (ATPS)-based techniques. Multi-cellular spheroids have great potential in the study of biological systems and can closely mimic the in vivo environment. New technologies to form and analyse spheroids such as the aqueous two-phase system and magnetic levitation are rapidly overcoming the technical limitations of spheroids and expanding their applications in tissue engineering and regenerative medicine.

Bioimprint Mediated Label-Free Isolation of Pancreatic Tumor Cells from a Healthy Peripheral Blood Cell Population.

New techniques are required for earlier diagnosis and response to treatment of pancreatic cancer. Here, a label-free approach is reported in which circulating pancreatic tumor cells are isolated from healthy peripheral blood cells via cell bioimprinting technology. The method involves pre-fabrication of pancreatic cell layers and sequential casting of cell surfaces with a series of custom-made resins to produce negative cell imprints. The imprint is functionalized with a combination of polymers to engineer weak attraction to the cells which is further amplified by the increased area of contact with the matching cells. A flow-through bioimprint chip is designed and tested for selectivity toward two pancreatic tumor cell lines, ASPC-1 and Mia-PaCa-2. Healthy human peripheral blood mononuclear cells (PBMCs) are spiked with pancreatic tumor cells at various concentrations. Bioimprints are designed for preferential retention of the matching pancreatic tumor cells and with respect to PBMCs. Tumor bioimprints are capable of capturing and concentrating pancreatic tumor cells from a mixed cell population with increased retention observed with the number of seedings. ASPC-1 bioimprints preferentially retain both types of pancreatic tumor cells. This technology could be relevant for the collection and interrogation of liquid biopsies, early detection, and relapse monitoring of pancreatic cancer patients.

Resistance training leads to large improvements in strength and moderate improvements in physical function in adults who are overweight or obese: a systematic review.

QUESTIONS: What are the effects of resistance training on muscle strength, physical function and muscle power in adults who are overweight or obese? Which factors moderate the effects? DESIGN: Systematic review of randomised controlled trials, with random effects meta-analyses and meta-regressions. PARTICIPANTS: Adults who are overweight or obese. INTERVENTION: Resistance training lasting ≥ 4 weeks. OUTCOME MEASURES: Muscle strength, muscle power and physical function. RESULTS: Thirty trials with 1,416 participants met the eligibility criteria. Pooled analyses indicated that resistance training has a large beneficial effect on muscle strength (SMD 1.39, 95% CI 1.05 to 1.73, I2 = 85%) and a moderate effect on physical function (SMD 0.67, 95% CI 0.25 to 1.08, I2 = 71%) in adults who are overweight or obese. However, the effect of resistance training on muscle power was unclear (SMD 0.42, 95% CI -3.3 to 4.2, I2 = 46%). The effect of resistance training on strength was greatest for the upper body (versus lower/whole body: ß = 0.35, 95% CI 0.05 to 0.66) and in dynamic strength tests (versus isometric/isokinetic: ß = 1.20, 95% CI 0.60 to 1.81), although trials judged to have good methodological quality reported statistically smaller effects (versus poor/fair quality: ß = -1.21, 95% CI -2.35 to -0.07). Concomitant calorie restriction did not modify strength gains but reduced the effect of resistance training on physical function (ß = -0.79, 95% CI -1.41 to -0.17). Small study effects were evident for strength outcomes (ß = 5.9, p < 0.001). CONCLUSIONS: Resistance training has a large positive effect on muscle strength and a moderate effect on physical function in adults who are overweight or obese. However, the effect of resistance training on muscle power is uncertain. In addition, concomitant calorie restriction may compromise the functional adaptations to resistance training. REGISTRATION: PROSPERO CRD42019146394.

The effects of empagliflozin vs metformin on endothelial microparticles in overweight/obese women with polycystic ovary syndrome.

CONTEXT: Endothelial microparticles (EMPs) are novel, surrogate biomarkers of endothelial function and have been shown to be elevated in women with polycystic ovary syndrome (PCOS). It remains poorly understood how pharmacological options for managing PCOS affect EMP levels. OBJECTIVE: To characterise and compare the effects of empagliflozin vs metformin on the circulating levels of EMPs in overweight/obese women with PCOS. METHODS: This was a randomised, comparative, 12-week single-centre trial conducted at the Academic Diabetes, Endocrinology and Metabolism Research Centre, Hull, UK. This analysis includes data from 39 overweight/obese women with PCOS who completed the study and were randomised to empagliflozin (15 mg/day) (n = 19) or metformin (1500 mg/day) (n = 20). Blood samples were collected at baseline and 12 weeks after treatment and analysed for specific surface proteins (ICAM-1, VCAM-1, PECAM-1, E-selectin and endoglin) expressed by circulating EMPs using flow cytometry. RESULTS: In the empagliflozin group, ICAM-1 (P = 0.006), E-selectin (P = 0.016) and VCAM-1 (P = 0.001) EMPs increased significantly following 12 weeks of treatment, but no changes were seen in PECAM-1 (P = 0.93) or endoglin (P = 0.13) EMPs. In the metformin group, VCAM-1 EMPs (P < 0.001) increased significantly after 12 weeks of treatment, whereas all other EMPs remained unchanged. When data were expressed as percentage change from baseline in each group, no significant differences were seen between groups for any biomarker (P-values from 0.22 to 0.80). CONCLUSIONS: Short-term administration of empagliflozin and metformin in overweight/obese women with PCOS appear to increase EMPs expressed by endothelial cells during their activation.

Removal of Human Leukemic Cells from Peripheral Blood Mononuclear Cells by Cell Recognition Chromatography with Size Matched Particle Imprints.

We report a cell recognition chromatography approach for blood cancer cell separation from healthy peripheral blood mononuclear cells (PBMCs) based on size-matched functionalized particle imprints. Negative imprints were prepared from layers of 15 µm polymeric microbeads closely matching the size of cultured human leukemic cells (HL60). We replicated these imprints on a large scale with UV curable polyurethane resin using nanoimprinting lithography. The imprints were functionalized with branched polyethylene imine (bPEI) and passivated by Poloxamer 407 to promote a weak attraction toward cells. When a matching cell fits into an imprint cavity, its contact area with the imprint is maximized, which amplifies the attraction and the binding selectivity. We tested these imprints specificity for depleting myeloblasts from a mixture with healthy human PBMCs in a cell recognition chromatography setup hosting the imprint. The mixture of fixed HL60/PBMCs ratio was circulated over the imprint and at each step the selectivity toward HL60 was assessed by flow cytometry. The role of the imprint length, flow rate, channel depth, and the bPEI coating concentration were examined. The results show that HL60 cells, closely matching the imprint cavities, get trapped on the imprint, while the smaller PBMCs are carried away by the drag force of the flow. Lower flow rates, longer imprints, and interim channel depth favor HL60 specific retention. The bPEI concentration higher than 1 wt % on the imprint made it less selective toward the HL60 because of indiscriminate attraction with all cells. Particle imprint based cell recognition chromatography was able to achieve selective myeloblast depletion from initial 11.7% HL60 (88.3% PBMC) to less than 1.3% HL60 for 3 h of circulation. The cell recognition chromatography with size-matched microbead imprints can be employed as an efficient cell separation technique and potentially lead to alternative therapies for myeloblasts removal from peripheral blood of patients with acute myeloid leukemia.

Test-Retest Reliability of a Commercial Linear Position Transducer (GymAware PowerTool) to Measure Velocity and Power in the Back Squat and Bench Press.

Orange, ST, Metcalfe, JW, Marshall, P, Vince, RV, Madden, LA, and Liefeith, A. Test-retest reliability of a commercial linear position transducer (GymAware PowerTool) to measure velocity and power in the back squat and bench press. J Strength Cond Res 34(3): 728-737, 2020-This study examined the test-retest reliability of the GymAware PowerTool (GYM) to measure velocity and power in the free-weight back squat and bench press. Twenty-nine academy rugby league players (age: 17.6 ± 1.0 years; body mass: 87.3 ± 20.8 kg) completed 2 test-retest sessions for the back squat followed by 2 test-retest sessions for the bench press. GYM measured mean velocity (MV), peak velocity (PV), mean power (MP), and peak power at 20, 40, 60, 80, and 90% of 1 repetition maximum (1RM). GYM showed good reliability (intraclass correlation coefficient [ICC] and standard error of measurement percentage, respectively) for the measurement of MV at loads of 40 (0.77, 3.9%), 60 (0.83, 4.8%), 80 (0.83, 5.8%), and 90% (0.79, 7.9%) of 1RM in the back squat. In the bench press, good reliability was evident for PV at 40 (0.82, 3.9%), 60 (0.81, 5.1%), and 80% (0.77, 8.4%) of 1RM, and for MV at 80 (0.78, 7.9%) and 90% (0.87, 9.9%) of 1RM. The measurement of MP showed good to excellent levels of reliability across all relative loads (ICC ≥0.75). In conclusion, GYM provides practitioners with reliable kinematic information in the back squat and bench press, at least with loads of 40-90% of 1RM. This suggests that strength and conditioning coaches can use the velocity data to regulate training load according to daily readiness and target specific components of the force-velocity curve. However, caution should be taken when measuring movement velocity at loads <40% of 1RM.

Feasibility of a novel exercise prehabilitation programme in patients scheduled for elective colorectal surgery: a feasibility randomised controlled trial.

BACKGROUND AND OBJECTIVES: To investigate the feasibility of delivering a functional exercise-based prehabilitation intervention and its effects on postoperative length of hospital stay, preoperative physical functioning and health-related quality of life in elective colorectal surgery. MATERIALS AND METHODS: In this randomised controlled feasibility trial, 22 elective colorectal surgery patients were randomly assigned to exercise prehabilitation (n = 11) or standard care (n = 11). Feasibility of delivering the intervention was assessed based on recruitment and compliance to the intervention. Impact on postoperative length of hospital stay and complications, preoperative physical functioning (timed up and go test, five times sit to stand, stair climb test, handgrip dynamometry and 6-min walk test) and health-related quality of life were also assessed. RESULTS: Over 42% of patients (84/198) screened were deemed ineligible for prehabilitation due to insufficient time existing prior to scheduled surgery. Of those who were eligible, approximately 18% consented to the trial. Median length of hospital stay was 8 [range 6-27] and 10 [range 5-12] days respectively for the standard care and prehabilitation groups. Patterns towards preoperative improvements for the timed up and go test, stair climb test and 6-min walk test were observed for all participants receiving prehabilitation but not standard care. CONCLUSIONS: Despite prehabilitation appearing to convey positive benefits on physical functioning, short surgical wait times and patient engagement represent major obstacles to implementing exercise prehabilitation programmes in colorectal cancer patients.

Isolated tumour microparticles induce endothelial microparticle release in vitro.

: Cancer induces a hypercoagulable state, resulting in an increased risk of venous thromboembolism. One of the mechanisms driving this is tissue factor (TF) production by the tumour, released in small lipid bound microparticles. We have previously demonstrated that tumour cell line media-induced procoagulant changes in HUVEC. The aim of this study was to investigate the effect of tumour microparticles and recombinant human TF (rhTF) on the endothelium. Procoagulant microparticles from the PANC-1 cell line were harvested by ultrafiltration. HUVEC were then incubated with these procoagulant microparticles or rhTF. Flow cytometry was used to investigate the effect of endothelial cell surface protein expression and microparticle release. Microparticles but not soluble TF was responsible for the procoagulant activity of cell-free tumour media. We also demonstrated an increase in endothelial microparticle release with exposure to tumour microparticles, with a positive linear relationship observed (R = 0.6630 P ≤ 0.0001). rhTF did not induce any of the changes observed with microparticles. Here we demonstrate that procoagulant activity of tumour cell line media is dependent on microparticles, and that exposure of endothelial cells to these microparticles results in an increase in microparticle release from HUVEC. This suggests a mechanism of transfer of procoagulant potential from the cancer to the remote endothelium.

Impaired heat shock protein 72 expression in women with polycystic ovary syndrome following a supervised exercise programme.

Induction of heat shock protein expression and the heat shock (stress) response are seen in exercise. This exercise-induced response is thought protective against cellular stress through the expression of heat shock proteins. The highly inducible heat shock protein 72 (HSP72) has been shown to be expressed in a number of stress-related conditions, but not investigated in women with polycystic ovary syndrome (PCOS). Twenty-one women (10 controls, 11 with PCOS) concluded an 8-week supervised, moderate-intensity exercise programme. Monocytes and lymphocytes were analysed by flow cytometry for HSP72 expression from blood samples prior to, mid-way and at the completion of the programme. The monocyte HSP72 expression showed an increase from baseline values through mid-way (p = 0.025), and at the completion of the programme (p = 0.011) only in the control group, the PCOS group showed no significant change. This pattern was similar for lymphocyte HSP72 expression where a significant increase was found at the completion of the programme (p = 0.01) only in the control group. The magnitude of increased HSP72 expression following completion of the programme was linked to baseline values only in the control group. In conclusion, increased HSP72 expression to exercise over an 8-week period was seen in control but not in PCOS women, suggesting that there is an impairment of HSP72 expression in response to exercise in these women.

Procoagulant tumor microvesicles attach to endothelial cells on biochips under microfluidic flow.

Tumor patients are at a high risk of venous thromboembolism (VTE), and the mechanism by which this occurs may involve tumor-derived microvesicles (MVs). Previously, it has been shown that tumor MVs become attached to endothelial cells in static conditions. To investigate whether this process occurs under physiologically relevant flow rates, tumor MVs were perfused across a microfluidic device coated with growing human umbilical vein endothelial cells (HUVECs). Cell lines were screened for their ability to form tumor spheroids, and two cell lines, ES-2 and U87, were selected; spheroids formed were transferred to a microfluidic chip, and a second endothelial cell biochip was coated with HUVECs and the two chips were linked. Media flowed through the spheroid chip to the endothelial chip, and procoagulant activity (PCA) of the tumor media was determined by a one-stage prothrombin time assay. Tumor MVs were also quantified by flow cytometry before and after interaction with HUVECs. Confocal images showed that HUVECs acquired fluorescence from MV attachment. Labeled MVs were proportionally lost from MV rich media with time when flowed over HUVECs and were not observed on a control chip. The loss of MV was accompanied by a proportional reduction in PCA. Flow cytometry, confocal microscopy, and live flow imagery captured under pulsatile flow confirmed an association between tumor MVs and HUVECs. Tumor MVs attached to endothelial cells under physiological flow rates, which may be relevant to the VTE pathways in cancer patients.

The CD105:CD106 microparticle ratio is CD106 dominant in polycystic ovary syndrome compared to type 2 diabetes and healthy subjects.

BACKGROUND: A retrospective analysis was carried out from patients and controls during the past 5 years from a series of studies investigating endothelial microparticles (MP). METHODS: In total, 319 samples from 207 individuals were included in this analysis, from patients with type 2 diabetes (T2D, n = 105), women with polycystic ovary syndrome (PCOS, n = 145) and healthy volunteers (n = 69). All data were generated via the same flow cytometry protocol with the same antibody clones. Endothelial markers CD105 (Endoglin) and CD106 (Vascular cell adhesion molecule-1) were used to enumerate MP in venous blood. RESULTS: The ratio of CD105MP:CD106MP was significantly different between groups (F = 63.43, p < 0.0001). Women with PCOS were found to have a median CD105MP:CD106MP ratio of 0.40 (IQR 0.24-0.57), suggesting approximately two CD106MP were found per CD105MP. The T2D group showed a median ratio of 2.32 (1.51-3.69) whereas in healthy volunteers the ratio was 2.21 (1.63-3.55). Serum intercellular adhesion molecule-1 was also shown to be significantly increased in PCOS when compared with control or T2D groups (F = 14.5, p < 0.001). CONCLUSION: These data suggest that women with PCOS have an altered endothelial MP release in favour of CD106. Thus a potential activated endothelial state exists in women with PCOS with a shift towards a predominantly CD106MP profile.

Fabrication of Human Keratinocyte Cell Clusters for Skin Graft Applications by Templating Water-in-Water Pickering Emulsions.

Most current methods for the preparation of tissue spheroids require complex materials, involve tedious physical steps and are generally not scalable. We report a novel alternative, which is both inexpensive and up-scalable, to produce large quantities of viable human keratinocyte cell clusters (clusteroids). The method is based on a two-phase aqueous system of incompatible polymers forming a stable water-in-water (w/w) emulsion, which enabled us to rapidly fabricate cell clusteroids from HaCaT cells. We used w/w Pickering emulsion from aqueous solutions of the polymers dextran (DEX) and polyethylene oxide (PEO) and a particle stabilizer based on whey protein (WP). The HaCaT cells clearly preferred to distribute into the DEX-rich phase and this property was utilized to encapsulate them in the water-in-water (DEX-in-PEO) emulsion drops then osmotically shrank to compress them into clusters. Prepared formulations of HaCaT keratinocyte clusteroids in alginate hydrogel were grown where the cells percolated to mimic 3D tissue. The HaCaT cell clusteroids grew faster in the alginate film compared to the individual cells formulated in the same matrix. This methodology could potentially be utilised in biomedical applications.