The artificial sweetener neotame negatively regulates the intestinal epithelium directly through T1R3-signaling and indirectly through pathogenic changes to model gut bacteria.

Introduction: Recent studies have indicated considerable health risks associated with the consumption of artificial sweeteners. Neotame is a relatively new sweetener in the global market however there is still limited data on the impact of neotame on the intestinal epithelium or the commensal microbiota. Methods: In the present study, we use a model of the intestinal epithelium (Caco-2) and microbiota (Escherichia coli and Enterococcus faecalis) to investigate how physiologically-relevant exposure of neotame impacts intestinal epithelial cell function, gut bacterial metabolism and pathogenicity, and gut epithelium-microbiota interactions. Results: Our findings show that neotame causes intestinal epithelial cell apoptosis and death with siRNA knockdown of T1R3 expression significantly attenuating the neotame-induced loss to cell viability. Similarly, neotame exposure results in barrier disruption with enhanced monolayer leak and reduced claudin-3 cell surface expression through a T1R3-dependent pathway. Using the gut bacteria models, E. coli and E. faecalis, neotame significantly increased biofilm formation and metabolites of E. coli, but not E. faecalis, reduced Caco-2 cell viability. In co-culture studies, neotame exposure increased adhesion capacity of E. coli and E. faecalis onto Caco-2 cells and invasion capacity of E. coli. Neotame-induced biofilm formation, E.coli-specific Caco-2 cell death, adhesion and invasion was identified to be meditated through a taste-dependent pathway. Discussion: Our study identifies novel pathogenic effects of neotame on the intestinal epithelium or bacteria alone, and in co-cultures to mimic the gut microbiome. These findings demonstrate the need to better understand food additives common in the global market and the molecular mechanisms underlying potential negative health impacts.

Electronic vape fluid activates the pulmonary endothelium and disrupts vascular integrity in vitro through an ARF6-dependent pathway.

The use of e-cigarettes or vapes is increasingly popular amongst a range of different demographics however the research in this area is surprisingly sparse. Clinical reports of e-cigarette- or vaping use-associated lung injury (EVALI) and vascular disruption, in both nicotine-containing and nicotine-free e-cigarette smokers, prompts the need for further research with a focus on the pulmonary endothelium. Using a common brand of e-cigarette (eVape) and an in vitro model of the human lung microvasculature, we investigated the effect of nicotine-free eVape fluid on pulmonary endothelial barrier integrity, oxidative stress and inflammation profile. Findings demonstrate reactive oxygen species-dependent breakdown of the pulmonary endothelium and release of inflammatory cytokines. These phenotypic changes, following exposure to nicotine-free eVape fluid, were accompanied by dysregulation of a number of adheren junctions-related genes of which ARF6 was most abundantly overexpressed. Further investigation of ARF6 identified it as a key regulator in eVape-induced barrier disruption and ROS accumulation. This study demonstrates, for the first time, the barrier disruptive effect of nicotine-free e-cigarette fluid on the pulmonary microvasculature and the ARF6 and ROS-dependent molecular mechanisms underlying this damage. Whilst these studies focus on a human in vitro model of the pulmonary microvasculature, the results support clinical case studies on EVALI and demonstrate a need for further investigation of the impact of nicotine-free e-cigarettes on the lung.

Investigating the use and awareness of artificial sweeteners among diabetic patients in Bangladesh.

BACKGROUND: As with many countries around the world, the incidence of diabetes in Bangladesh is increasing significantly. Whilst there is controversy in the field regarding the health impact of artificial sweeteners in Western communities, the link between sweetener consumption and awareness in Bangladesh has not been established. METHODS: In the present study, 260 diabetic patients completed a questionnaire survey to investigate the use and awareness of sweeteners and how this links to demographics and potential co-morbidities. RESULTS: Findings show that daily artificial sweetener consumption is significantly associated with hypertension but not other co-morbidities such as kidney disease or obesity. We further demonstrate that there is limited checking of artificial sweeteners in food or drink products by participants. the rurality of diabetic participants was found to significantly correlates with lower awareness of any health impact of artificial sweeteners. CONCLUSIONS: The findings from this study demonstrate that there is a need to increase the awareness of artificial sweetener use in diabetic patients in Bangladesh. Combined with a more robust understanding of the health impact of artificial sweeteners, these findings suggest that there is potential to improve outcomes for diabetic patients by improving this awareness.

Pre-sleep protein supplementation does not improve performance, body composition, and recovery in British Army recruits (part 1).

Dietary protein is crucial for optimising physical training adaptations such as muscular strength and mass, which are key aims for athletic populations, including British Army recruits. New recruits fail to meet the recommended protein intake during basic training (BT), with negligible amounts consumed in the evening. This study assessed the influence of a daily bolus of protein prior to sleep on performance adaptations, body composition and recovery in British Army recruits. 99 men and 23 women [mean ± standard deviation (SD): age: 21.3 ± 3.5 years, height: 174.8 ± 8.4 cm, body mass 75.4 ± 12.2 kg] were randomised into a dietary control (CON), carbohydrate placebo (PLA), moderate (20 g) protein (MOD) or high (60 g) protein (HIGH) supplementation group. Supplements were isocaloric and were consumed on weekday evenings between 2000 and 2100 for 12 weeks during BT. Performance tests (mid-thigh pull, medicine ball throw, 2 km run time, maximal push-up, and maximal vertical jump) and body composition were assessed at the start and end of BT. Dietary intake, energy expenditure, salivary hormones, urinary nitrogen balance, perceived muscle soreness, rating of perceived exertion, mood, and fatigue were assessed at the start, middle and end of BT. Protein supplementation increased protein intake in HIGH (2.16 ± 0.50 g⸱kg-1⸱day-1) and MOD (1.71 ± 0.48 g⸱kg-1⸱day-1) compared to CON (1.17 ± 0.24 g⸱kg-1⸱day-1) and PLA (1.31 ± 0.29 g⸱kg-1⸱day-1; p < 0.001). Despite this, there was no impact of supplementation on mid-thigh pull performance (CON = 7 ± 19%, PLA = 7 ± 19%, MOD = 0 ± 16%, and HIGH = 4 ± 14%; p = 0.554) or any other performance measures (p > 0.05). Fat-free mass changes were also similar between groups (CON = 4 ± 3%, PLA = 4 ± 4%, MOD = 3 ± 3%, HIGH = 5 ± 4%, p = 0.959). There was no impact of protein supplementation on any other body composition or recovery measure. We conclude no benefits of pre-bed protein supplementation to improve performance, body composition and recovery during BT. It is possible the training stimulus was great enough, limiting the impact of protein supplementation. However, the high degree of inter-participant variability suggests an individualised use of protein supplementation should be explored, particularly in those who consume sub-optimal (<1.6 g⸱kg-1⸱day-1) habitual amounts of protein. Clinical trial registration: The study was registered with ClinicalTrials.gov, U.S. national institutes (identifier: NCT05998590).

Pre-sleep protein supplementation does not improve recovery from load carriage in British Army recruits (part 2).

British Army basic training (BT) is physically demanding with new recruits completing multiple bouts of physical activity each day with limited recovery. Load carriage is one of the most physically demanding BT activities and has been shown to induce acute exercise-induced muscle damage (EIMD) and impair muscle function. Protein supplementation can accelerate muscle recovery by attenuating EIMD and muscle function loss. This study investigated the impact of an additional daily bolus of protein prior to sleep throughout training on acute muscle recovery following a load carriage test in British Army recruits. Ninety nine men and 23 women (mean ± SD: age: 21.3 ± 3.5 yrs., height: 174.8 ± 8.4 cm, body mass 75.4 ± 12.2 kg) were randomized to dietary control (CON), carbohydrate placebo (PLA), moderate (20 g; MOD) or high (60 g; HIGH) protein supplementation. Muscle function (maximal jump height), perceived muscle soreness and urinary markers of muscle damage were assessed before (PRE), immediately post (POST), 24-h post (24 h-POST) and 40-h post (40 h-POST) a load carriage test. There was no impact of supplementation on muscle function at POST (p = 0.752) or 40 h-POST (p = 0.989) load carriage but jump height was greater in PLA compared to HIGH at 24 h-POST (p = 0.037). There was no impact of protein supplementation on muscle soreness POST (p = 0.605), 24 h-POST (p = 0.182) or 40 h-POST (p = 0.333). All groups had increased concentrations of urinary myoglobin and 3-methylhistidine, but there was no statistical difference between groups at any timepoint (p > 0.05). We conclude that pre-sleep protein supplementation does not accelerate acute muscle recovery following load carriage in British Army recruits during basic training. The data suggests that consuming additional energy in the form of CHO or protein was beneficial at attenuating EIMD, although it is acknowledged there were no statistical differences between groups. Although EIMD did occur as indicated by elevated urinary muscle damage markers, it is likely that the load carriage test was not arduous enough to reduce muscle function, limiting the impact of protein supplementation. Practically, protein supplementation above protein intakes of 1.2 g⸱kg-1⸱day-1 following load carriage over similar distances (4 km) and carrying similar loads (15-20 kg) does not appear to be warranted.

The Effect of a Hydroxytyrosol-Rich, Olive-Derived Phytocomplex on Aerobic Exercise and Acute Recovery.

There is current scientific interest in naturally sourced phenolic compounds and their potential benefits to health, as well as the effective role polyphenols may provide in an exercise setting. This study investigated the chronic effects of supplementation with a biodynamic and organic olive fruit water phytocomplex (OliPhenolia® [OliP]), rich in hydroxytyrosol (HT), on submaximal and exhaustive exercise performance and respiratory markers of recovery. Twenty-nine recreationally active participants (42 ± 2 yrs; 71.1 ± 2.1 kg; 1.76 ± 0.02 m) consumed 2 × 28 mL∙d−1 of OliP or a taste- and appearance-matched placebo (PL) over 16 consecutive days. Participants completed a demanding, aerobic exercise protocol at ~75% maximal oxygen uptake (V˙O2max) for 65 min 24 h before sub- and maximal performance exercise tests prior to and following the 16-day consumption period. OliP reduced the time constant (τ) (p = 0.005) at the onset of exercise, running economy (p = 0.015) at lactate threshold 1 (LT1), as well as the rating of perceived exertion (p = 0.003) at lactate turnpoint (LT2). Additionally, OliP led to modest improvements in acute recovery based upon a shorter time to achieve 50% of the end of exercise V˙O2 value (p = 0.02). Whilst OliP increased time to exhaustion (+4.1 ± 1.8%), this was not significantly different to PL (p > 0.05). Phenolic compounds present in OliP, including HT and related metabolites, may provide benefits for aerobic exercise and acute recovery in recreationally active individuals. Further research is needed to determine whether dose-response or adjunct use of OliP alongside longer-term training programs can further modulate exercise-associated adaptations in recreationally active individuals, or indeed support athletic performance.

Preparation and Characterisation of Zinc Diethyldithiocarbamate-Cyclodextrin Inclusion Complexes for Potential Lung Cancer Treatment.

Zinc diethyldithiocarbamate (Zn (DDC)2), a disulfiram metabolite (anti-alcoholism drug), has shown a strong anti-cancer activity in vitro. However, its application was limited by its low aqueous solubility and rapid metabolism. In this study, the solubility enhancement of Zn (DDC)2 is investigated by forming inclusion complexes with cyclodextrins. The inclusion complexes were prepared using two different types of beta-cyclodextrins, SBE-CD and HP-CD. Phase solubility diagrams for the resulting solutions were assessed; subsequently, the solutions were freeze-dried for further characterisation studies using DSC, TGA, XRD, and FTIR. The cytotoxic activity of the produced inclusion complexes was evaluated on human lung carcinoma cells using the MTT assay. The solubility of Zn (DDC)2 increased significantly upon adding beta-cyclodextrins, reaching approximately 4 mg/mL for 20% w/w CD solutions. The phase solubility diagram of Zn (DDC)2 was of the Ap-type according to the Higuchi and Connors model. Characterisation studies confirmed the inclusion of the amorphous drug in the CD-Zn (DDC)2 complexes. The cytotoxicity of Zn (DDC)2 was enhanced 10-fold by the inclusion complexes compared to the free drug. Overall, the resulting CD-Zn (DDC)2 inclusion complexes have a potential for treatment against lung cancer.

The Impact of a Natural Olive-Derived Phytocomplex (OliPhenolia®) on Exercise-Induced Oxidative Stress in Healthy Adults.

The role of natural polyphenols in reducing oxidative stress and/or supporting antioxidant mechanisms, particularly relating to exercise, is of high interest. The aim of this study was to investigate OliPhenolia® (OliP), a biodynamic and organic olive fruit water phytocomplex, rich in hydroxytyrosol (HT), for the first time within an exercise domain. HT bioavailability from OliP was assessed in fifteen healthy volunteers in a randomized, double-blind, placebo controlled cross-over design (age: 30 ± 2 yrs; body mass: 76.7 ± 3.9 kg; height: 1.77 ± 0.02 m), followed by a separate randomized, double-blinded, cohort trial investigating the short-term impact of OliP consumption (2 × 28 mL∙d−1 of OliP or placebo (PL) for 16-days) on markers of oxidative stress in twenty-nine recreationally active participants (42 ± 2 yrs; 71.1 ± 2.1 kg; 1.76 ± 0.02 m). In response to a single 28 mL OliP bolus, plasma HT peaked at 1 h (38.31 ± 4.76 ng∙mL−1), remaining significantly elevated (p < 0.001) until 4 h. Plasma malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT), reduced glutathione (GSH) and HT were assessed at rest and immediately following exercise (50 min at ~75% V˙O2max then 10 min intermittent efforts) and at 1 and 24 h post-exercise, before and after the 16-day supplementation protocol. Plasma HT under resting conditions was not detected pre-intervention, but increased to 6.3 ± 1.6 ng·mL−1 following OliP only (p < 0.001). OliP demonstrated modest antioxidant effects based on reduced SOD activity post-exercise (p = 0.016) and at 24 h (p ≤ 0.046), and increased GSH immediately post-exercise (p = 0.009) compared with PL. No differences were reported for MDA and CAT activity in response to the exercise protocol between conditions. The phenolic compounds within OliP, including HT, may have specific antioxidant benefits supporting acute exercise recovery. Further research is warranted to explore the impact of OliP following longer-term exercise training, and clinical domains pertinent to reduced oxidative stress.

Agonists for Bitter Taste Receptors T2R10 and T2R38 Attenuate LPS-Induced Permeability of the Pulmonary Endothelium in vitro.

One of the hallmarks of acute respiratory distress syndrome (ARDS) is an excessive increase in pulmonary vascular permeability. In settings of ARDS, the loss of barrier integrity is mediated by cell-cell contact disassembly and actin remodelling. Studies into molecular mechanisms responsible for improving microvascular barrier function are therefore vital in the development of therapeutic targets for reducing vascular permeability seen in ARDS. Bitter taste receptors (T2Rs) belong to the superfamily of G-protein-coupled receptors found in several extraoral systems, including lung epithelial and smooth muscle cells. In the present study, we show for the first time that several T2Rs are expressed in human pulmonary arterial endothelial cells (HPAECs). Our results focus on those which are highly expressed as: T2R10, T2R14 and T2R38. Agonists for T2R10 (denatonium) and T2R38 (phenylthiourea), but not T2R14 (noscapine), significantly attenuated lipopolysaccharide (LPS)-induced permeability and VE-cadherin internalisation in HPAECs. In T2R10- or T2R38-siRNA knockdown cells, these endothelial-protective effects were abolished, indicating a direct effect of agonists in regulating barrier integrity. Our further findings indicate that T2R10 and T2R38 exert their barrier-protective function through cAMP but via Rac1-dependent and independent pathways, respectively. However, using an in vivo model of ARDS, the T2R38 agonist, phenylthiourea, was not able to protect against pulmonary edema formation. Taken together, these studies identify bitter taste sensing in the pulmonary endothelium to regulate barrier integrity in vitro through cAMP-Rac1 signalling.

Investigating the Opposing Effect of Two Different Green Tea Supplements on Oxidative Stress, Mitochondrial Function and Cell Viability in HepG2 Cells.

Green tea extract (GTE) improves exercise outcomes and reduces obesity. However, case studies indicate contradictory physiology regarding liver function and toxicity. We studied the effect of two different decaffeinated GTE (dGTE) products, from a non-commercial (dGTE1) and commercial (dGTE2) supplier, on hepatocyte function using the human cell model, HepG2. dGTE1 was protective against hydrogen peroxide (H2O2)-induced apoptosis and cell death by attenuating oxidative stress pathways. Conversely, dGTE2 increased cellular and mitochondrial oxidative stress and apoptosis. A bioavailability study with dGTE showed the major catechin in GTE, EGCG, reached 0.263 µg·ml-1. In vitro, at this concentration, EGCG mimicked the protective effect of dGTE1. GC/MS analysis identified steric acid and higher levels of palmitic acid in dGTE2 versus dGTE1 supplements. We demonstrate the significant biological differences between two GTE supplements which may have potential implications for manufacturers and consumers to be aware of the biological effects of supplementation.

Saccharin and Sucralose Protect the Glomerular Microvasculature In Vitro against VEGF-Induced Permeability.

Diabetic kidney disease (DKD) has become a global health concern, with about 40% of people living with type 1 and type 2 diabetes mellitus developing DKD. Upregulation of vascular endothelial growth factor (VEGF) in the kidney is a significant pathology of DKD associated with increased glomerular vascular permeability. To date, however, current anti-VEGF therapies have demonstrated limited success in treating DKD. Recent studies have shown that artificial sweeteners exhibit anti-VEGF potential. The aim of this study was therefore to assess the effects of aspartame, saccharin, and sucralose on VEGF-induced leak using an in vitro model of the glomerular endothelium. Saccharin and sucralose but not aspartame protected against VEGF-induced permeability. Whilst the sweeteners had no effect on traditional VEGF signalling, GC-MS analysis demonstrated that the sweetener sucralose was not able to enter the glomerular endothelial cell to exert the protective effect. Chemical and molecular inhibition studies demonstrated that sweetener-mediated protection of the glomerular endothelium against VEGF is dependent on the sweet taste receptor, T1R3. These studies demonstrate the potential for sweeteners to exert a protective effect against VEGF-induced increased permeability to maintain a healthy endothelium and protect against vascular leak in the glomerulus in settings of DKD.

Artificial Sweeteners Negatively Regulate Pathogenic Characteristics of Two Model Gut Bacteria, E. coli and E. faecalis.

Artificial sweeteners (AS) are synthetic sugar substitutes that are commonly consumed in the diet. Recent studies have indicated considerable health risks which links the consumption of AS with metabolic derangements and gut microbiota perturbations. Despite these studies, there is still limited data on how AS impacts the commensal microbiota to cause pathogenicity. The present study sought to investigate the role of commonly consumed AS on gut bacterial pathogenicity and gut epithelium-microbiota interactions, using models of microbiota (Escherichia coli NCTC10418 and Enterococcus faecalis ATCC19433) and the intestinal epithelium (Caco-2 cells). Model gut bacteria were exposed to different concentrations of the AS saccharin, sucralose, and aspartame, and their pathogenicity and changes in interactions with Caco-2 cells were measured using in vitro studies. Findings show that sweeteners differentially increase the ability of bacteria to form a biofilm. Co-culture with human intestinal epithelial cells shows an increase in the ability of model gut bacteria to adhere to, invade and kill the host epithelium. The pan-sweet taste inhibitor, zinc sulphate, effectively blocked these negative impacts. Since AS consumption in the diet continues to increase, understanding how this food additive affects gut microbiota and how these damaging effects can be ameliorated is vital.

Diet-induced iron deficiency in rats impacts small intestinal calcium and phosphate absorption.

AIMS: Recent reports suggest that iron deficiency impacts both intestinal calcium and phosphate absorption, although the exact transport pathways and intestinal segment responsible have not been determined. Therefore, we aimed to systematically investigate the impact of iron deficiency on the cellular mechanisms of transcellular and paracellular calcium and phosphate transport in different regions of the rat small intestine. METHODS: Adult, male Sprague-Dawley rats were maintained on a control or iron-deficient diet for 2 weeks and changes in intestinal calcium and phosphate uptake were determined using the in situ intestinal loop technique. The circulating levels of the hormonal regulators of calcium and phosphate were determined by ELISA, while the expression of transcellular calcium and phosphate transporters, and intestinal claudins were determined using qPCR and western blotting. RESULTS: Diet-induced iron deficiency significantly increased calcium absorption in the duodenum but had no impact in the jejunum and ileum. In contrast, phosphate absorption was significantly inhibited in the duodenum and to a lesser extent the jejunum, but remained unchanged in the ileum. The changes in duodenal calcium and phosphate absorption in the iron-deficient animals were associated with increased claudin 2 and 3 mRNA and protein levels, while levels of parathyroid hormone, fibroblast growth factor-23 and 1,25-dihydroxy vitamin D3 were unchanged. CONCLUSION: We propose that iron deficiency alters calcium and phosphate transport in the duodenum. This occurs via changes to the paracellular pathway, whereby upregulation of claudin 2 increases calcium absorption and upregulation of claudin 3 inhibits phosphate absorption.

The Impact of Decaffeinated Green Tea Extract on Fat Oxidation, Body Composition and Cardio-Metabolic Health in Overweight, Recreationally Active Individuals.

This study investigated the effect of decaffeinated green tea extract (dGTE), with or without antioxidant nutrients, on fat oxidation, body composition and cardio-metabolic health measures in overweight individuals engaged in regular exercise. Twenty-seven participants (20 females, 7 males; body mass: 77.5 ± 10.5 kg; body mass index: 27.4 ± 3.0 kg·m2; peak oxygen uptake (O2peak): 30.2 ± 5.8 mL·kg-1·min-1) were randomly assigned, in a double-blinded manner, either: dGTE (400 mg·d-1 (-)-epigallocatechin-3-gallate (EGCG), n = 9); a novel dGTE+ (400 mg·d-1 EGCG, quercetin (50 mg·d-1) and α-lipoic acid (LA, 150 mg·d-1), n = 9); or placebo (PL, n = 9) for 8 weeks, whilst maintaining standardised, aerobic exercise. Fat oxidation ('FATMAX' and steady state exercise protocols), body composition, cardio-metabolic and blood measures (serum glucose, insulin, leptin, adiponectin, glycerol, free fatty acids, total cholesterol, high [HDL-c] and low-density lipoprotein cholesterol [LDL-c], triglycerides, liver enzymes and bilirubin) were assessed at baseline, week 4 and 8. Following 8 weeks of dGTE+, maximal fat oxidation (MFO) significantly improved from 154.4 ± 20.6 to 224.6 ± 23.2 mg·min-1 (p = 0.009), along with a 22.5% increase in the exercise intensity at which fat oxidation was deemed negligible (FATMIN; 67.6 ± 3.6%O2peak, p = 0.003). Steady state exercise substrate utilisation also improved for dGTE+ only, with respiratory exchange ratio reducing from 0.94 ± 0.01 at week 4, to 0.89 ± 0.01 at week 8 (p = 0.004). This corresponded with a significant increase in the contribution of fat to energy expenditure for dGTE+ from 21.0 ± 4.1% at week 4, to 34.6 ± 4.7% at week 8 (p = 0.006). LDL-c was also lower (normalised fold change of -0.09 ± 0.06) for dGTE+ by week 8 (p = 0.038). No other significant effects were found in any group. Eight weeks of dGTE+ improved MFO and substrate utilisation during exercise, and lowered LDL-c. However, body composition and cardio-metabolic markers in healthy, overweight individuals who maintained regular physical activity were largely unaffected by dGTE.

Extracellular vesicles released from p18 overexpressing pulmonary endothelial cells are barrier protective - potential implications for acute respiratory distress syndrome.

The novel endosome protein, p18, and the early endosome GTPase, Rab4, play a significant role in protecting the pulmonary vasculature against permeability associated with acute respiratory distress syndrome. Recently, endothelial-derived extracellular vesicles have been identified to play a key role in the endothelial permeability associated with acute respiratory distress syndrome. Therefore, we investigated the effect of these microparticles, released from endothelial cells overexpressing p18 and Rab4, on pulmonary endothelial barrier function. Endothelial-derived extracellular vesicles isolated from lung microvascular endothelial cells which overexpressed cDNA for wild-type p18 protected a naïve monolayer against lipopolysaccharide-induced permeability. In contrast, endothelial-derived extracellular vesicles from cells overexpressing the non-endosomal binding p18 mutant (p18N39) exerted no protective effect on the endothelial monolayer. Cells overexpressing either dominant active or inactive Rab4 released endothelial-derived extracellular vesicles which had no effect on lipopolysaccharide-induced permeability. miRNA analysis and permeability studies of endothelial-derived extracellular vesicle isolated from wild-type p18-overexpressing cells demonstrates that let-7i-5p, miR-96-5p, and miR-137-3p are endothelial-derived extracellular vesicle cargo which exert protective effects on the pulmonary endothelium. Finally, we observed down-regulation of p18 protein expression in both the lung and endothelium in an in vivo and in vitro model of acute respiratory distress syndrome. These results demonstrate that endothelial-derived extracellular vesicle released from cells overexpressing p18, but not Rab4, contain miRNA cargo which likely promote a barrier-protective effect on the pulmonary endothelium in settings of acute respiratory distress syndrome. Findings indicate the importance of p18 in the pulmonary vasculature and demonstrate that targeting this protein may provide a novel therapeutic strategy to reduce endothelial permeability associated with acute respiratory distress syndrome.

Lutein and zeaxanthin attenuates VEGF-induced neovascularisation in human retinal microvascular endothelial cells through a Nox4-dependent pathway.

Age-related macular degeneration (AMD) and proliferative diabetic retinopathy (DR) are two of the most common and severe causes of vision loss in the population. Both conditions are associated with excessive levels of vascular endothelial growth factor (VEGF) in the eye which results in an increase in the formation of new blood vessels through a process called neovascularisation. As such, anti-VEGF therapies are currently utilised as a treatment for patients with AMD however they are associated with painful administration of injections and potential degeneration of healthy endothelium. There is therefore growing interest in alternate treatment options to reduce neovascularisation in the eye. The use of carotenoids, lutein (L) and zeaxanthin (Z), has been shown to improve vision loss parameters in patients with AMD, however the underlying mechanisms are not well-understood. We studied the impact of these compounds on neovascularisation processes using an in vitro cell model of the retinal microvascular endothelium. Our findings show that L and Z reduced VEGF-induced tube formation whilst, in combination (5:1 ratio), the compounds significantly blocked VEGF-induced neovascularisation. The carotenoids, individually and in combination, reduced VEGF-induced oxidative stress concomitant with increased activity of the NADPH oxidase, Nox4. We further demonstrated that the Nox4 inhibitor, GLX7013114, attenuated the protective effect of L and Z. Taken together, these findings indicate the protective effect of the carotenoids, L and Z, in reducing VEGF-mediated neovascularisation via a Nox4-dependent pathway. These studies implicate the potential for these compounds to be used as a therapeutic approach for patients suffering from AMD and proliferative DR.

Artificial Sweeteners Disrupt Tight Junctions and Barrier Function in the Intestinal Epithelium through Activation of the Sweet Taste Receptor, T1R3.

The breakdown of the intestinal epithelial barrier and subsequent increase in intestinal permeability can lead to systemic inflammatory diseases and multiple-organ failure. Nutrition impacts the intestinal barrier, with dietary components such as gluten increasing permeability. Artificial sweeteners are increasingly consumed by the general public in a range of foods and drinks. The sweet taste receptor (T1R3) is activated by artificial sweeteners and has been identified in the intestine to play a role in incretin release and glucose transport; however, T1R3 has not been previously linked to intestinal permeability. Here, the intestinal epithelial cell line, Caco-2, was used to study the effect of commonly-consumed artificial sweeteners, sucralose, aspartame and saccharin, on permeability. At high concentrations, aspartame and saccharin were found to induce apoptosis and cell death in intestinal epithelial cells, while at low concentrations, sucralose and aspartame increased epithelial barrier permeability and down-regulated claudin 3 at the cell surface. T1R3 knockdown was found to attenuate these effects of artificial sweeteners. Aspartame induced reactive oxygen species (ROS) production to cause permeability and claudin 3 internalization, while sweetener-induced permeability and oxidative stress was rescued by the overexpression of claudin 3. Taken together, our findings demonstrate that the artificial sweeteners sucralose, aspartame, and saccharin exert a range of negative effects on the intestinal epithelium through the sweet taste receptor T1R3.

Culture of pulmonary artery endothelial cells from pulmonary artery catheter balloon tips: considerations for use in pulmonary vascular disease.

Endothelial dysfunction is a hallmark of pulmonary arterial hypertension (PAH) but there are no established methods to study pulmonary artery endothelial cells (PAECs) from living patients. We sought to culture PAECs from pulmonary artery catheter (PAC) balloons used during right-heart catheterisation (RHC) to characterise successful culture attempts and to describe PAEC behaviour.PAECs were grown in primary culture to confluence and endothelial cell phenotype was confirmed. Standard assays for apoptosis, migration and tube formation were performed between passages three to eight. We collected 49 PAC tips from 45 subjects with successful PAEC culture from 19 balloons (39%).There were no differences in subject demographic details or RHC procedural details in successful versus unsuccessful attempts. However, for subjects who met haemodynamic criteria for PAH, there was a higher but nonsignificant (p=0.10) proportion amongst successful attempts (10 out of 19, 53%) versus unsuccessful attempts (nine out of 30, 30%). A successful culture was more likely in subjects with a lower cardiac index (p=0.03) and higher pulmonary vascular resistance (p=0.04). PAECs from a subject with idiopathic PAH were apoptosis resistant compared to commercial PAECs (p=0.04) and had reduced migration compared to PAECs from a subject with portopulmonary hypertension with high cardiac output (p=0.01). PAECs from a subject with HIV-associated PAH formed fewer (p=0.01) and shorter (p=0.02) vessel networks compared to commercial PAECs.Sustained culture and characterisation of PAECs from RHC balloons is feasible, especially in PAH with high haemodynamic burden. This technique may provide insight into endothelial dysfunction during PAH pathogenesis.

Sore eyes as the most significant ocular symptom experienced by people with COVID-19: a comparison between pre-COVID-19 and during COVID-19 states.

OBJECTIVE: Conjunctivitis has been reported in people suffering from COVID-19. However, many ocular symptoms are associated with the term 'conjunctivitis' which may be misleading. It is also unknown whether ocular symptoms were different in chronic sufferers of anterior eye diseases, when they were experienced or how long they lasted for compared with other COVID-19 symptoms. METHODS: An online structured questionnaire obtained self-report data from people who had a confirmed diagnosis of COVID-19. Data for the type, frequency and duration of different COVID-19 symptoms were ascertained. Anterior eye symptoms experienced by participants in the pre-COVID-19 state were compared with during the COVID-19 state. RESULTS: Data from 83 participants showed that the most reported COVID-19 symptoms were dry cough (66%), fever (76%), fatigue (90%) and loss of smell/taste (70%). The three most common ocular symptoms experienced by participants were photophobia (18%), sore eyes (16%) and itchy eyes (17%). The frequency of sore eyes was significantly higher (p=0.002) during COVID-19 state (16%) compared with pre-COVID-19 state (5%). There were no differences between males and females (p>0.05). 81% of participants reported to have experienced ocular symptoms within 2 weeks of other COVID-19 symptoms, and 80% reported they lasted for less than 2 weeks. CONCLUSION: The most significant ocular symptom experienced by people suffering from COVID-19 was sore eyes. Other symptoms associated with other types of conjunctivitis, such as mucous discharge and gritty eyes linked to bacterial infection, did not reach significance. The term 'conjunctivitis' is too broad and should be used with caution.

Endosomes and Autophagy: Regulators of Pulmonary Endothelial Cell Homeostasis in Health and Disease.

Significance: Alterations in oxidant/antioxidant balance injure pulmonary endothelial cells and are important in the pathogenesis of lung diseases, such as Acute Respiratory Distress Syndrome (ARDS), ischemia/reperfusion injury, pulmonary arterial hypertension (PAH), and emphysema. Recent Advances: The endosomal and autophagic pathways regulate cell homeostasis. Both pathways support recycling or degradation of macromolecules or organelles, targeted to endosomes or lysosomes, respectively. Thus, both processes promote cell survival. However, with environmental stress or injury, imbalance in endosomal and autophagic pathways may enhance macromolecular or organelle degradation, diminish biosynthetic processes, and cause cell death. Critical Issues: While the role of autophagy in cellular homeostasis in pulmonary disease has been investigated, the role of the endosome in the lung vasculature is less known. Furthermore, autophagy can either decrease or exacerbate endothelial injury, depending upon inciting insult and disease process. Future Directions: Diseases affecting the pulmonary endothelium, such as emphysema, ARDS, and PAH, are linked to altered endosomal or autophagic processing, leading to enhanced degradation of macromolecules and potential cell death. Efforts to target this imbalance have yielded limited success as treatments for lung injuries, which may be due to the complexity of both processes. It is possible that endosomal trafficking proteins, such as Rab GTPases and late endosomal/lysosomal adaptor, MAPK and MTOR activator 1, may be novel therapeutic targets. While endocytosis or autophagy have been linked to improved function of the pulmonary endothelium in vitro and in vivo, further studies are needed to identify targets for modulating cellular homeostasis in the lung.