Randomized Trial to Assess the Safety and Tolerability of Daily Intake of an Allulose Amino Acid-Based Hydration Beverage in Men and Women.

BACKGROUND: Maintaining adequate hydration is critical to optimal health, well-being, and performance. Those who are physically active in stressful environments, such as warm and/or humid scenarios, may be at particular risk for dehydration with ensuing loss of electrolytes, leading to sluggishness and impaired physical performance. METHODS: We evaluated an electrolyte and amino acid product containing L-alanine and L-glutamine, as well as select vitamins [B3 (niacin), B5 (pantothenic acid), B6 (pyridoxine), B12 (cobalamin), and vitamin C (ascorbic acid)]. Subjects (n = 40; four groups, n = 10) were randomized to consume either a placebo packet or one, two, or three packets daily of the test product for 4 weeks with site visits at 0, 2, and 4 weeks. We tested safety and tolerability by analyzing hematological parameters (complete blood counts), metabolic parameters (hepatic, renal, acid-base balance), urinalysis end products, thyroid status [T3 (triiodothyronine), T4 (thyroxine), TSH (thyroid-stimulating hormone)], tolerability (via questionnaire), vital signs, and dietary intake. RESULTS: Statistical analyses displayed ten significant main effects (p < 0.05) with white blood cells, lymphocytes, neutrophils, urinary pH, thyroxine, urination frequency, calcium, calories, fat, and cholesterol. Interactions for time and group (p < 0.05) were observed for MCV, eGFR, potassium, overall tolerability, bloating, and cramping-demonstrating mild GA disturbances. Little to no change of physiological relevance was noted for any outcome variable, regardless of dosing level. CONCLUSIONS: Our results indicate the product was well-tolerated at all dosing levels and no significant adverse changes occurred in any of the test parameters compared to the placebo group, indicating relative safety of ingestion over a 4-week treatment period, at the volumes used, and outside the context of physical stress.

The dietary supplement Cyplexinol® alleviates joint pain in men and women.

Background and Aim: Joint pain afflicts millions of adults worldwide. The effect of a bone morphogenetic protein complex on joint pain is assessed in this study. Methods: We compared the impact of a dietary supplement protein complex (Cyplexinol®) and placebo in 18 men and women (aged 43 ± 10 years) with self-reported joint pain. Subjects were randomly assigned to each condition, consumed twice daily for 14 days (900 mg/day). Subjects completed questionnaires (e.g., Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) and subjective pain using a visual analog scale [VAS]) at the start and end of each treatment phase. Blood samples were analyzed for bone morphogenic protein (BMP), alkaline phosphatase, and cytokines (tumor necrosis factor [TNF]-α, interleukin [IL]-6, IL-10, IL-1ß, and TGF-ß). Blood was also collected on days 1 and 15 to determine the acute impact of treatment on these measures. Results: Pain and discomfort scores improved (P ≤ 0.05) for subjects following use of Cyplexinol® but not placebo. Improvements were noted for WOMAC pain (P = 0.05), stiffness (P = 0.039), and total pain (P = 0.026), as well as VAS pain (P = 0.015), recreational activity interference (P = 0.023), mood interference (P = 0.012), and total pain (P = 0.024). A trend was noted for WOMAC physical function (P = 0.052). An approximate 50% increase in BMP5 was noted following Cyplexinol® (P = 0.01), with a similar increase noted for placebo (P = 0.022). A near doubling in TGF-ß (P = 0.001) was noted for Cyplexinol®. No other changes of significance were noted across time, nor were any differences noted in cytokines following acute intake of the conditions (P > 0.05). Conclusions: Cyplexinol® can alleviate joint pain in middle-aged men and women, while elevating BMP5 and TGF-ß. Cyplexinol® does not influence cytokines, at least within a short 2-week supplementation period or within the 2-h post-ingestion period. Relevance for Patients: Individuals suffering with joint pain in the knee and/or hip may benefit from daily use of Cyplexinol®, as we observed decreased pain and stiffness following treatment.

Risk Factors for Cardiometabolic Disease in Professional Firefighters.

OBJECTIVE: Firefighters are plagued with cardiometabolic disease (CMD). Obesity, poor cardiorespiratory and muscular fitness, and blood lipids (low-density lipoprotein cholesterol, triglycerides, low high-density lipoprotein cholesterol) are risk factors for CMD. However, markers of oxidative stress, inflammation, and insulin resistance can provide further insight regarding CMD risk. METHODS: This study investigated the relationships between fitness metrics (cardiorespiratory and muscular fitness, percent body fat, waist circumference), blood lipids, blood pressure, and years of experience as a firefighter to blood markers of insulin resistance: Homeostatic Model Assessment for Insulin Resistance (HOMA-IR), oxidative stress: advanced oxidation protein products (AOPPs), and inflammation: C-reactive protein. RESULTS: Waist circumference and blood concentrations of triglycerides were significantly related to AOPPs and HOMA-IR. Cardiorespiratory fitness was inversely related to AOPPs, HOMA-IR and C-reactive protein. CONCLUSION: These findings demonstrate the importance of high cardiorespiratory fitness and low waist circumference to reduce markers of CMD.

A Topical Botanical Ointment for Self-Reported Hip and/or Knee Pain: A Randomized Placebo-Controlled Clinical Trial.

Joint pain is a common ailment among adults worldwide. Six men and 14 women (aged 51 ± 10 years) with self-reported joint pain were randomly assigned, using a cross-over design, to a botanical ointment (Yeahhh Baby!®) or placebo, twice daily for 14 days. Subjects completed questionnaires regarding their joint pain and discomfort (eg, WOMAC and subjective pain using a visual analog scale [VAS]) each evening and underwent a washout period of two weeks before crossing into the other condition. Pain and discomfort scores improved for subjects when using Yeahhh Baby!® ointment from day 1 to the average of days 2-15. For certain measures, similar, albeit insignificant, improvements were noted when subjects used the placebo-demonstrating the powerful placebo effect. Specifically, with Yeahhh Baby!® ointment, effects were noted for WOMAC pain (P = .008), WOMAC physical function (P = .024), WOMAC total (P = .019), and VAS mood interference (P = .042). The most pronounced improvement was noted for WOMAC pain (P = .048), with a 25% reduction observed with Yeahhh Baby!®, with a 10% reduction noted for placebo. These findings indicate that, as compared to a placebo, Yeahhh Baby!® ointment may provide relief to individuals suffering from joint pain in their knees and/or hips.

Firefighters With Higher Cardiorespiratory Fitness Demonstrate Lower Markers of Cardiovascular Disease Risk.

OBJECTIVE: High cardiorespiratory fitness (CRF) is associated with reduced markers of oxidative stress and cardiovascular disease (CVD) risk factors; however, this relationship has not been elucidated in firefighters. The purpose of this study was to examine differences in markers of CVD risk between firefighters who have either high or low levels of CRF. METHODS: Forty-six firefighters participated in a maximal graded exercise test and a dual-energy x-ray absorptiometry scan and provided a fasted blood sample. V˙O 2max values were categorized based on American College of Sports Medicine guidelines to establish high- and low-fitness groups. RESULTS: High fitness firefighters demonstrated significantly higher high-density lipoprotein cholesterol and lower markers of CVD risk: cholesterol, triglycerides, low-density lipoprotein cholesterol, insulin, homeostatic model assessment for insulin resistance, C-reactive protein, and advanced oxidation protein products concentrations. CONCLUSION: Firefighters are encouraged to maintain high CRF to reduce risk of CVD.

Impact of AmaTea® Max on physiological measures and gaming performance in active gamers: A placebo-controlled, double-blind, randomized study.

Background and Aim: The activity of "gaming" has increased greatly in popularity in recent years, with many gamers using nutritional supplements to aid mood and gaming performance. We evaluated the impact of AmaTea® Max (referred to as AmaTea® throughout; a patented dietary supplement consisting of a blend of caffeine and polyphenol antioxidants), compared to both caffeine and a placebo, on gaming and cognitive performance in active gamers. Methods: Subjects reported to the lab on three occasions, separated by approximately 1 week. On each day, they had baseline measurements taken and then played the game Fortnite for four 1-h periods. Measures of cognitive performance, gaming performance, heart rate and blood pressure (BP), and blood cortisol were measured before and at selected times following gameplay. Results: Neither caffeine nor AmaTea® impacted gaming or cognitive performance in a statistically significant manner. However, a trend (P=0.075) was noted for the condition effect for kills/match, with values 21% higher for AmaTea® (1.84) compared to placebo (1.51), and 12% higher for AmaTea® compared to caffeine (1.63). Subjective mood was relatively unaffected, although a condition effect was noted for jittery (P=0.05), with values lower for placebo than for caffeine (P=0.02). BP was minimally elevated with both AmaTea® and caffeine, while cortisol followed the normal diurnal variation and was lower for placebo than AmaTea® and caffeine. Conclusion: AmaTea® modestly increased kills/match during gameplay. It is possible that a different gaming stimulus, varied time of gameplay, or different dosage of the supplement may have yielded different results. Relevance for Subjects: Active gamers who seek to use a dietary supplement for purposes of gaming performance may benefit slightly from ingestion of AmaTea® before gameplay while experiencing greater vigor and lower fatigue as compared to placebo.

GATA4 regulates mesenchymal stem cells via direct transcriptional regulation of the WNT signalosome.

GATA4 is a transcription factor that regulates osteoblast differentiation. However, GATA4 is expressed at a higher level in mesenchymal stem cells (MSCs) than in osteoblasts. Therefore, the role of GATA4 in limb bud mesenchyme differentiation was investigated in mice by knocking out Gata4 using Cre-recombinase controlled by the Prx1 promoter (herein called Gata4 Prx-cKO mice). µCT analysis of the Gata4 Prx-cKO mice showed a decrease in trabecular bone properties compared with wildtype (Gata4fl/fl) littermates. Gata4 Prx-cKO mice have fewer MSCs as measured by CFU-F assays, mesenchymal progenitor cells (MPC2) (flow cytometry of Sca1+/CD45-/CD34-/CD44hi) and nestin immunofluorescence. Gata4 Prx-cKO bone marrow-derived MSCs have a significant reduction in WNT ligands, including WNT10B, and WNT signalosome components compared to control cells. Chromatin immunoprecipitation demonstrates that GATA4 is recruited to enhancers near Wnt3a, Wnt10b, Fzd6 and Dkk1. GATA4 also directly represses YAP in wildtype cells, and the absence of Gata4 leads to increased YAP expression. Together, we show that the decrease in MSCs is due to loss of Gata4 and a WNT10B-dependent positive autoregulatory loop. This leads to a concurrent increase of YAP and less activated ß-catenin. These results explain the decreased trabecular bone in Gata4 Prx-cKO mice. We suggest that WNT signalosome activity in MSCs requires Gata4 and Wnt10b expression for lineage specification.

Impact of Nuun Electrolyte Tablets on Fluid Balance in Active Men and Women.

BACKGROUND: Maintaining adequate hydration is important for overall health and has major implications for athletes involved in physically demanding tasks. While water is viewed as an effective means to rehydrate, and is inexpensive and readily available, electrolyte beverages appear to be more beneficial, in particular for athletes who routinely lose electrolytes through sweating. Nuun tablets contain a mix of electrolytes and are quickly dissolved in water to create an electrolyte-rich beverage. We determined the impact of Nuun tablets on the fluid balance of healthy, exercise-trained men and women at rest. METHODS: Eight men (25.9 ± 4.5 yrs) and 10 women (28.2 ± 9.4 yrs) ingested either water only or water with Nuun electrolyte tablets, at both a single and double strength concentration, in random order, on three separate occasions separated by approximately one week, in a fasted and euhydrated state. A total of 1 liter of fluid was ingested at each visit over a 30 minute period. Urine was collected from each subject at 0, 1, 2, 3, and 4 hours post-ingestion. Urine mass values were used to calculate fluid balance and the beverage hydration index (BHI; i.e., the volume of urine produced after drinking the Nuun beverages, relative to that of water only-control condition). Heart rate and blood pressure were measured throughout the four-hour period, while body weight was measured at the start and end of the experiment. RESULTS: Neither heart rate nor blood pressure were impacted by beverage consumption. Nuun tablets resulted in a lower urine output compared to water, with fluid balances for both concentrations more favorable compared to water (p < 0.05), beginning at 2 h post-ingestion and continuing at the 3 h and 4 h times. Body weight loss was less with Nuun at the single dose (0.38 kg; p = 0.02) and double dose (0.43 kg; p = 0.08), compared to water (0.57 kg). The BHI was higher for Nuun (single dose in particular) compared to water at both 2 h (p = 0.05) and 4 h (p = 0.02). CONCLUSION: The addition of Nuun electrolyte tablets to water improves the fluid balance and BHI in healthy men and women. Results were similar for both concentrations, suggesting that additional electrolytes are not necessary when in a rested state. Future studies should determine the impact of various concentrations of the Nuun beverage during physical exercise-in particular, exercise in the heat, when sweat loss may be highest.

Proangiogenic Activity of Endometrial Epithelial and Stromal Cells in Response to Estradiol in Gelatin Hydrogels.

Biomaterial vascularization remains a major focus in the field of tissue engineering. Biomaterial culture of endometrial cells is described as a platform to inform the design of proangiogenic biomaterials. The endometrium undergoes rapid growth and shedding of dense vascular networks during each menstrual cycle mediated via estradiol and progesterone in vivo. Cocultures of endometrial epithelial and stromal cells encapsulated within a methacrylamide-functionalized gelatin hydrogel are employed. It is reported that proangiogenic gene expression profiles and vascular endothelial growth factor production are hormone dependent in endometrial epithelial cells, but that hormone signals have no effect on human telomerase reverse transcriptase (hTERT)-immortalized endometrial stromal cells. This study subsequently examines whether the magnitude of epithelial cell response is sufficient to induce changes in human umbilical vein endothelial cell network formation. Incorporation of endometrial stromal cells improves vessel formation, but co-culture with endometrial epithelial cells leads to a decrease in vascular formation, suggesting the need for stratified cocultures of endometrial epithelial and stromal cells with endothelial cells. Given the transience of hormonal signals within 3D biomaterials, the inclusion of sex hormone binding globulin (SHBG) to alter the bioavailability of estradiol within the hydrogel is reported, demonstrating a strategy to reduce diffusive losses via SHBG-mediated estradiol sequestration.

The induction of pro-angiogenic processes within a collagen scaffold via exogenous estradiol and endometrial epithelial cells.

Nutrient transport remains a major limitation in the design of biomaterials. One approach to overcome this constraint is to incorporate features to induce angiogenesis-mediated microvasculature formation. Angiogenesis requires a temporal presentation of both pro- and anti-angiogenic factors to achieve stable vasculature, leading to increasingly complex biomaterial design scheme. The endometrium, the lining of the uterus and site of embryo implantation, exemplifies a non-pathological model of rapid growth, shedding, and re-growth of dense vascular networks regulated by the dynamic actions of estradiol and progesterone. In this study, we examined the individual and combined response of endometrial epithelial cells and human umbilical vein endothelial cells to exogenous estradiol within a three-dimensional collagen scaffold. While endothelial cells did not respond to exogenous estradiol, estradiol directly stimulated endometrial epithelial cell transduction pathways and resulted in dose-dependent increases in endogenous VEGF production. Co-culture experiments using conditioned media demonstrated estradiol stimulation of endometrial epithelial cells can induce functional changes in endothelial cells within the collagen biomaterial. We also report the effect of direct endometrial epithelial and endothelial co-culture as well as covalent immobilization of estradiol within the collagen biomaterial. These efforts establish the suitability of an endometrial-inspired model for promoting pro-angiogenic events within regenerative medicine applications. These results also suggest the potential for developing biomaterial-based models of the endometrium.

Strategies to balance covalent and non-covalent biomolecule attachment within collagen-GAG biomaterials.

Strategies to integrate instructive biomolecular signals into a biomaterial are becoming increasingly complex and bioinspired. While a large majority of reports still use repeated treatments with soluble factors, this approach can be prohibitively costly and difficult to translate in vivo for applications where spatial control over signal presentation is necessary. Recent efforts have explored the use of covalent immobilization of biomolecules to the biomaterial, via both bulk (ubiquitous) as well as spatially-selective light-based crosslinking, as a means to both enhance stability and bioactivity. However, little is known about how processing conditions during immobilization impact the degree of unintended non-covalent interactions, or fouling, that takes place between the biomaterial and the biomolecule of interest. Here we demonstrate the impact of processing conditions for bulk carbodiimide (EDC) and photolithography-based benzophenone (BP) crosslinking on specific attachment vs. fouling of a model protein (Concanavalin A, ConA) within collagen-glycosaminoglycan (CG) scaffolds. Collagen source significantly impacts the selectivity of biomolecule immobilization. EDC crosslinking intensity and ligand concentration significantly impacted selective immobilization. For benzophenone photoimmobilization we observed that increased UV exposure time leads to increased ConA immobilization. Immobilization efficiency for both EDC and BP strategies was maximal at physiological pH. Increasing ligand concentration during immobilization process led to enhanced immobilization for EDC chemistry, no impact on BP immobilization, but significant increases in non-specific fouling. Given recent efforts to covalently immobilize biomolecules to a biomaterial surface to enhance bioactivity, improved understanding of the impact of crosslinking conditions on selective attachment versus non-specific fouling will inform the design of instructive biomaterials for applications across tissue engineering.

Photopatterning of vascular endothelial growth factor within collagen-glycosaminoglycan scaffolds can induce a spatially confined response in human umbilical vein endothelial cells.

Biomolecular signals within the native extracellular matrix are complex, with bioactive factors found in both soluble and sequestered states. In the design of biomaterials for tissue engineering applications it is increasingly clear that new approaches are required to locally tailor the biomolecular environment surrounding cells within the matrix. One area of particular focus is strategies to improve the speed or quality of vascular ingrowth and remodeling. While the addition of soluble vascular endothelial growth factor (VEGF) has been shown to improve vascular response, strategies to immobilize such signals within a biomaterial offer the opportunity to optimize efficiency and to explore spatially defined patterning of such signals. Here we describe the use of benzophenone (BP) photolithography to decorate three-dimensional collagen-glycosaminoglycan (CG) scaffolds with VEGF in a spatially defined manner. In this effort we demonstrate functional patterning of a known agonist of vascular remodeling and directly observe phenotypic effects induced by this immobilized cue. VEGF was successfully patterned in both stripes and square motifs across the scaffold with high specificity (on:off pattern signal). The depth of patterning was determined to extend up to 500 µm into the scaffold microstructure. Notably, photopatterned VEGF retained native functionality as it was shown to induce morphological changes in human umbilical vein cells indicative of early vasculogenesis. Immobilized VEGF led to greater cell infiltration into the scaffold and the formation of immature vascular network structures. Ultimately, these results suggest that BP-mediated photolithography is a facile method to spatially control the presentation of instructive biological cues to cells within CG scaffolds.