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Implementation of the NASA AG-Bed Rest Pilot

To examine the efficacy of artificial gravity (AG) as a countermeasure to spaceflight deconditioning, intermittent AG produced by a horizontal short-radius centrifuge (SRC) was utilized on human test subjects deconditioned by bed rest. This poster will present the subject screening, study design, logistics, and implementation of the 41 day pilot study conducted at the University of Texas Medical Branch, Galveston, TX bed rest facility. An extensive screening process was employed to exclude subjects that were dissimilar to the U.S. astronaut population. Candidates underwent a modified U.S. Air Force Class III physical and tests of bone density, cardiovascular fitness, vestibular system function, psychological fitness and centrifuge tolerance. 15 subjects completed the study; 7 control and 8 AG treatment. All provided written consent to volunteer after the nature of the study and its hazards were clearly explained to them. Standard conditions were strictly regulated; Ta = 72 +/- 2 F, humidity = 70 +/- 5%, light/dark cycle 16h:8h. All fluid intake (minimum 28.5 ml/kg body weight/day) and urine output was monitored. Caloric intake was adjusted as necessary to maintain body weight. Carbohydrate, fat and protein were provided in a ratio of 55:30:15. Phosphorus intake was 1400 mg/d, sodium intake was 2 mmol/kg/d, potassium intake was 1.3 mmol/kg/d, and dietary calcium intake was 1000 mg/d. A physician examined each subject daily. During the first 11 days of the study protocol, subjects were ambulatory, but confined to the facility. Subjects participated in multiple baseline tests of bone, muscle, cardiovascular, sensory-motor, immunological, and psychological function. On the 12th day, subjects entered the bed rest phase of the study, during which they were confined to strict 6? head down tilt bed rest for 21 days. Beginning 24 hrs into this period, treatment subjects received 1 hour daily exposures to artificial gravity which was produced by spinning the subjects on a 3.0 m radius SRC. They were oriented radially in the supine position so that the centrifugal force was aligned with their long body axis, and while spinning, they #stood# on a force plate, supporting the centrifugal loading (2.5 g at the feet, 1.0 g at the heart). The subject station allowed free translation over approximately 10 cm to ensure full loading of the lower extremities and to allow for anti-orthostatic muscle contractions. Control subjects were positioned on the centrifuge but did not spin. Following the bed rest phase, subjects were allowed to ambulate again, but remained within the facility for an additional 9 days and participated in multiple follow-up tests of physiological function.

Warren, L. E.↗

Thermoregulatory effects of caffeine ingestion during rest and exercise in men

Body temperatures and thermoregulatory responses were measured at rest and during submaximal exercise under normal ambient conditions in 11 aerobically-conditioned men (age = 29.2 +/- 6.2 yr, VO2(max) = 3.73 +/- 0.46 min(sup -1), relative body fat = 12.3 +/- 3.7 percent, mean +/- SD) with (CT) and without (NCT) the ingestion of 10 mg of caffeine per kg of body weight. Oxygen uptake (VO2), heart rate (HR), and rectal (T(sub re)) and mean skin (T-bar(sub sk)) temperatures were recorded for 100 minutes starting one minute after ingestion of caffeine or a placebo. Data were collected throughout 30 minutes of rest (sitting) and the following 70 minutes of sitting leg ergometer exercise using the same constant load (1,088 +/- 153 kgm/min) in both NCT and CT. The load resulted in a mean relative exercise intensity equal to approximately 68 percent of VO2(sub max). Skin heat conductance (H(sub sk)) and sweat rate were calculated. Two-way analysis of covariance revealed no significant (P greater than 0.05) differences between NCT and CT in VO2, HR, T(sub re), T-bar(sub sk), or H(sub sk). A dependent t-test indicated no significant difference between NCT and CT in sweat rate. Thus, a high level of caffeine ingestion has no detrimental effects on body temperatures and thermoregulatory responses during moderately heavy exercise in normal ambient conditions.

Dunagan, Nancy↗

Exercise thermoregulation in men after 1 and 24-hours of 6 degrees head-down tilt

BACKGROUND: Exercise thermoregulation is dependent on heat loss by increased skin blood flow (convective and conductive heat loss) and through enhanced sweating (evaporative heat loss). Reduction of plasma volume (PV), increased plasma osmolality, physical deconditioning, and duration of exposure to simulated and actual microgravity reduces the ability to thermoregulate during exercise. HYPOTHESIS: We hypothesized that 24 h of head down tilt (HDT24) would alter thermoregulatory responses to a submaximal exercise test and result in a higher exercise rectal temperature (Tre) when compared with exercise Tre after 1 h of head down tilt (HDT1). METHODS: Seven men (31+/-SD 6 yr, peak oxygen uptake (VpO2peak) of 44+/-6 ml x kg(-1) x min(-1)) were studied during 70 min of supine cycling at 58+/-SE 1.5% VO2peak at 22.0 degrees C Tdb and 47% rh. RESULTS: Relative to pre-tilt sitting chair rest data, HDT1 resulted in a 6.1+/-0.9% increase and HDT24 in a 4.3+/-2.3% decrease in PV (delta = 10.4% between experiments, p<0.05) while plasma osmolality remained unchanged (NS). Pre-exercise Tre was elevated after HDT24 (36.71 degrees C +/-0.06 HDT1 vs. 36.93 degrees C+/-0.11 HDT24, p<0.05). The 70 min of exercise did not alter this relationship (p<0.05) with respective end exercise increases in Tre to 38.01 degrees C and 38.26 degrees C (degrees = 1.30 degrees C (HDT1) and 1.33 degrees C (HDT24)). While there were no pre-exercise differences in mean skin temperature (Tsk), a significant (p<0.05) time x treatment interaction occurred during exercise: after min 30 in HDT24 the Tsk leveled off at 31.1 degrees C, while it continued to increase reaching 31.5 degrees C at min 70 in HDT1. A similar response (NS) occurred in skin blood velocity. Neither local sweating rates nor changes in body weight during exercise of -1.63+/-0.24 kg (HDT1) or - 1.33+/-0.09 kg (HDT24) were different (NS) between experiments. CONCLUSION: While HDT24 resulted in elevated pre-exercise Tre, reduced PV, attenuation of Tsk and skin blood velocity during exercise, the absolute increase in exercise Tre was not altered. But if sweat rate and cutaneous vascular responses were similar at different core temperatures (unchanged thermoregulation), the Tre offset could have been caused by the HDT-induced hypovolemia.

NASA Program Biomedical Research and Countermeasur↗

Pilot Field Test: Performance of a Sit-to-Stand Test After Long-Duration Space Flight

BACKGROUND: Astronauts returning from the International Space Station are met by a team of recovery personnel typically providing physical assistance and medical support immediately upon landing. That is because long-duration spaceflight impacts astronauts' functional abilities. Future expeditions to planets or asteroids beyond the low Earth orbit, however, may require crewmembers to egress the vehicle and perform other types of physical tasks unassisted. It is therefore important to characterize the extent and longevity of functional deficits experienced by astronauts in order to design safe exploration class missions. Pilot Field Test (PFT) experiment conducted with participation of ISS crewmembers traveling on Soyuz expeditions 34S - 41S comprised several tasks designed to study the recovery of sensorimotor abilities of astronauts during the first 24 hours after landing and beyond. METHODS: The first test in the PFT battery sequence, and also the least demanding one from the sensorimotor perspective, was a Sit-to-Stand test. Test subjects were seated in the chair and had to stand up on command and remain standing for ten seconds. The subjects were instructed to stand up unassisted as quickly as they were able to, while maintaining postural control. Synchronized wireless inertial sensors mounted on the head, chest, lower back, wrists, and ankles were used to continuously log body kinematics. Crewmembers' blood pressure and heart rate were monitored and recorded with the Portapres and Polar systems. Each session was recorded with a digital video camera. During data collections occurring within the 24-hour postflight period, crewmembers were also asked to (1) evaluate their perceived motion sickness symptoms on a 20-point scale before and after completion of the test and (2) estimate how heavy they felt compared to their normal (preflight) body weight. Consent to participate in PFT was obtained from 18 crewmembers (11 US Orbital Segment [USOS] astronauts and 7 Russian cosmonauts). For 10 subjects, the first set of data was collected in the medical tent in Soyuz landing zone (1-2 hours after landing); the other 8 subjects were tested at the Kazakhstan deployment airport (4-5 hours after landing). 8 of the 11 astronauts were tested twice more within the first 24 hours postflight, at a refueling stop on the way to Houston (approximately 13 hours after landing) and at the Johnson Space Center (approximately 24 hours after landing). Later postflight data were collected in the first two weeks on some crewmembers. Finally, 6 astronauts were tested 60+ days after landing to establish a delayed baseline. RESULTS/DISCUSSION: Two of the 18 PFT participants felt too ill to attempt any tests in Kazakhstan (at either the landing zone or deployment airport). The remaining test subjects completed the Sit-to-Stand test and their reported motion sickness scores were unaffected by this task. The task completion times and body kinematics data analysis are currently underway. Preliminary analysis of astronaut data shows a steep improvement in the time to complete the task on the second data take, and in some cases, the trend continues through day six postflight. Head and trunk pitch angles and pitch rates were also examined and increases in all measures are evident throughout the observed recovery period (60+ days postflight). Interesting patterns of head and trunk pitch coordination have also emerged. One of the data analysis objectives is comparison of initial postflight performance and recovery of experienced crewmembers and first-time fliers. Another one - possible differences in performance between USOS and Russian crewmembers.

Kofman, I. S.↗

Comparison of ground-based and space flight energy expenditure and water turnover in middle-aged healthy male US astronauts

Energy requirements during space flight are poorly defined because they depend on metabolic-balance studies, food disappearance, and dietary records. Water turnover has been estimated by balance methods only. The purpose of this study was to determine energy requirements and water turnover for short-term space flights (8-14 d). Subjects were 13 male astronauts aged 36-51 y with normal body mass indexes (BMIs). Total energy expenditure (TEE) was determined during both a ground-based period and space flight and compared with the World Health Organization (WHO) calculations of energy requirements and dietary intake. TEE was not different for the ground-based and the space-flight periods (12.40 +/- 2.83 and 11.70 +/- 1.89 MJ/d, respectively), and the WHO calculation using the moderate activity correction was a good predictor of TEE during space flight. During the ground-based period, energy intake and TEE did not differ, but during space flight energy intake was significantly lower than TEE; body weight was also less at landing than before flight. Water turnover was lower during space flight than during the ground-based period (2.7 +/- 0.6 compared with 3.8 +/- 0.5 L/d), probably because of lower fluid intakes and perspiration loss during flight. This study confirmed that the WHO calculation can be used for male crew members' energy requirements during short space flights.

STS Shuttle Project↗

Automated Fluidics Device for Extraction and Quantification of miRNA Biomarkers From Blood

Radiation Assessment DuRing Exposure And long-Duration Spaceflight (RADREADS) demonstrates space-compatible point-of-care technology for quantitative biological monitoring of blood miRNA biomarkers in response to long-term low dose radiation exposure. This individualized monitoring approach will inform targeted treatment strategies to maximize medical resource utilization by accounting for individual susceptibility to radiation-related illnesses. As human spaceflight progresses beyond Earth’s magnetic shielding, radiation exposure poses a significant risk to astronaut health and safety. Extended operation in this environment comes with an increased risk of radiation exposure, leading to higher risks of radiation sickness, cancer, central nervous system effects, and degenerative diseases. While conventional physical dosimetry techniques capture radiation dose, individualistic susceptibility to radiation damage is varied. Multiple characteristics, including age, body weight, sex, genetics, and immune status, have been found to influence radiosensitivity (Liu et al. 2011, and Bouffler 2016). This differential response necessitates individualized monitoring and targeted treatment strategies to maximize medical resource utilization; however, a practical diagnostic platform for quantifying long-term, low dose radiation-induced tissue damage does not currently exist. MicroRNAs (miRNAs) are a class of small, non-coding RNAs that regulate gene expression by mediating the degradation of messenger RNA. The levels of particular miRNAs are influenced by biological processes such as inflammation and serve as biomarkers for a variety of conditions including cancer (Singh et al. 2017). MicroRNAs are found in various bodily fluids and are amenable to collection via liquid biopsies, providing a minimally invasive and easily quantifiable readout for a variety of radiosensitive reporters. A preliminary signature of 15 spaceflight sensitive miRNA has been identified in rodent and human studies, including miR-21-5p, miR-24-3p, miR-92a-3p, miR-17-5p, miR-16a-3p, miR-34a-3p, and miR-223-3p. These targets generally increased expression with radiation dose and linear energy transfer, though variation between individuals is not yet described. Current gaps in the field include a lack of understanding of longitudinal biological responses to long-term, low dose radiation exposure and the absence of space-compatible point-of-care technology for quantitative biological monitoring. In this body of work, we aim to develop an automated bleed-to-read system to process whole blood for the detection of miRNA biomarkers in order to monitor individualistic responses to radiation exposure. This will be achieved via separating serum (or plasma) from whole blood, followed by extraction, amplification, and quantification of the miRNA using a RT-qPCR reaction. Previously, the WetLab-2 hardware enabled execution of a RT-qPCR reaction aboard ISS; however, it is a manual system that requires crew manipulation and bulky components (Parra et al. 2017). To address these issues, automated fluid handling hardware was developed for each stage of sample preparation. Extraction of total RNA is achieved by sequentially pumping reagents through an off-the-shelf nucleic acid binding column (miRNeasy Serum/Plasma Advanced Kit, Qiagen). This approach eliminates several manual pipetting and centrifuging steps and limits the use of toxic chemicals commonly found in other sample processing techniques. The resulting elution will then be automatically dispensed for RT-qPCR analysis using a compact rotary qPCR (Mic qPCR Cycler, Bio Molecular Systems) that will improve spaceflight compatibility by removing bubbles from the detection region, another challenge highlighted by WetLab-2 (Parra et al. 2017). Efforts are also being made to simplify the RT-qPCR reaction to a 1-step air-dryable mix to improve long-term reagent stability at room temperature and reduce system complexity. By automating the RT-qPCR processes via microfluidic manipulation, RADREADS will reduce crewmember hands-on time and enable the personalized detection of radiation-induced tissue damage during long duration missions. Minimally invasive, longitudinal monitoring of individual’s response to radiation exposure will inform how the physiological system responds to long-term low dose space radiation and enables development of targeted countermeasures by the medical team. Ultimately, this portable technology will require minimal technical expertise and can also be used to monitor miRNA biomarkers associated with other diseases.

Tristen Head↗

The nutritional status of astronauts is altered after long-term space flight aboard the International Space Station

Defining optimal nutrient requirements is critical for ensuring crew health during long-duration space exploration missions. Data pertaining to such nutrient requirements are extremely limited. The primary goal of this study was to better understand nutritional changes that occur during long-duration space flight. We examined body composition, bone metabolism, hematology, general blood chemistry, and blood levels of selected vitamins and minerals in 11 astronauts before and after long-duration (128-195 d) space flight aboard the International Space Station. Dietary intake and limited biochemical measures were assessed during flight. Crew members consumed a mean of 80% of their recommended energy intake, and on landing day their body weight was less (P = 0.051) than before flight. Hematocrit, serum iron, ferritin saturation, and transferrin were decreased and serum ferritin was increased after flight (P < 0.05). The finding that other acute-phase proteins were unchanged after flight suggests that the changes in iron metabolism are not likely to be solely a result of an inflammatory response. Urinary 8-hydroxy-2'-deoxyguanosine concentration was greater and RBC superoxide dismutase was less after flight (P < 0.05), indicating increased oxidative damage. Despite vitamin D supplement use during flight, serum 25-hydroxycholecalciferol was decreased after flight (P < 0.01). Bone resorption was increased after flight, as indicated by several markers. Bone formation, assessed by several markers, did not consistently rise 1 d after landing. These data provide evidence that bone loss, compromised vitamin D status, and oxidative damage are among critical nutritional concerns for long-duration space travelers.

Space Flight↗

Myosin heavy chain expression in rodent skeletal muscle: effects of exposure to zero gravity

This study ascertained the effects of 9 days of zero gravity on the relative (percentage of total) and calculated absolute (mg/muscle) content of isomyosin expressed in both antigravity and locomotor skeletal muscle of ground control (CON) and flight-exposed (FL) rats. Results showed that although there were no differences in body weight between FL and CON animals, a significant reduction in muscle mass occurred in the vastus intermedius (VI) (P < 0.05) but not in the vastus lateralis (VL) or the tibialis anterior. Both total muscle protein and myofibril protein content were not different between the muscle regions examined in the FL and CON groups. In the VI, there were trends for reductions in the relative content of type I and IIa myosin heavy chains (MHCs) that were offset by increases in the relative content of both type IIb and possibly type IIx MHC protein (P > 0.05). mRNA levels were consistent with this pattern (P < 0.05). The same pattern held true for the red region of the VL as examined at both the protein and mRNA level (P < 0.05). When the atrophy process was examined, there were net reductions in the absolute content of both type I and IIa MHCs that were offset by calculated increases in type IIb MHC in both VI and red VL. Collectively, these findings suggest that there are both absolute and relative changes occurring in MHC expression in the "red" regions of antigravity skeletal muscle during exposure to zero gravity that could affect muscle function.

short duration↗

Implicit Formulation of Muscle Dynamics in OpenSim

Astronauts lose bone and muscle mass during spaceflight. Exercise countermeasure is the primary method for counteracting bone and muscle mass loss in space. New spacecraft exercise device concepts are currently being developed for the NASAs new crew exploration vehicle. The NASA Digital Astronaut Project (DAP) uses computational modeling to help determine if the new exercise devices will be effective as countermeasures. The NASA Digital Astronaut Project is developing the ability to utilize predictive simulation to provide insight into the change in kinematics and kinetics with a change in device and gravitational environment (1-g versus 0-g). For example, in space exercise the subject's body weight is applied in addition to the loads prescribed for musculoskeletal maintenance. How and where these loads are applied obviously directly impacts bone and tissue loads. Additionally, due to space vehicle structural requirements, exercise devices are often placed on vibration isolation systems. This changes the apparent impedance or stiffness of the device as seen by the user. Data collection under these conditions is often impractical and limited. Predictive modeling provides a means to have a virtual subject to test hypotheses. Predictive simulation provides a virtual subject for which we are able to perform studies such as sensitivity to device loading and vibration isolation without the need for laboratory kinematic or kinetic test data.Direct Collocation optimization provides an efficient means to perform task based optimization and predictive modeling. It is relatively straight forward to structure a physical exercise task in a Direct Collocation mathematical formulation: perform a motion such that you start at an initial pose, achieve a given amount of deflection i.e a squat, return to the initial pose, and minimize muscle activation cost. Direct Collocation is advantageous in that it does not require numerical integration to evaluate the objective function. Instead, the system dynamics are transformed to discrete time and the optimizer is constrained such that the solution is not considered to be a valid unless the dynamic equations are satisfied at all time points. The simulation and optimization are effectively done simultaneously. Due to the implicit integration, time steps can be more coarse than in a differential equation solver. In a gait scenario this means that that the model constraints and cost function are evaluated at 100 nodes in the gait cycle versus 10,000 integration steps in a variable-step forward dynamic simulation. Furthermore, no time is wasted on accurate simulations of movements that are far from the optimum. Constrained optimization algorithms require a Jacobian matrix that contains the partial derivatives of each of the dynamic constraints with respect to of each of the state and control variables at all time points. This is a large but sparse matrix. An implicit dynamics formulation requires computation of the dynamic residuals f as a function of the states x and their derivatives, and controls u:f(x, dxdt, u) 0If the dynamics of musculoskeletal system are formulated implicitly, the Jacobian elements are often available analytically, eliminating the need for numerical differentiation; this is obviously computationally advantageous. Additionally, implicit formulation of musculoskeletal dynamics do not suffer from singularities from low mass bodies, zero muscle activation, or other stiff system or

physical exercise↗

Broccoli seed extract rich in polysaccharides and glucoraphanin ameliorates DSS ‐induced colitis via intestinal barrier protection and gut microbiota modulation in mice

Abstract BACKGROUND Broccoli has received widespread attention because of its anti‐inflammatory and antioxidant effects. The present study aimed to explore the composition of broccoli seed extract (BSE) and its effect on colitis induced by dextran sulfate sodium (DSS). RESULTS BSE mainly comprises glucoraphanin and polysaccharides composed of arabinose, galactose, glucose and mannose. Animal experiments suggested that BSE intervention effectively reversed body weight loss, suppressed the levels of proinflammatory interleukin‐6, tumor necrosis factor‐α and interleukin‐1β, and elevated the levels of anti‐inflammatory interleukin‐10 and the activities of superoxide dismutase and glutathione in DSS‐induced colitis mice. According to histopathologic and immunohistochemical analysis of colon tissue, BSE intervention may repair the intestinal barrier by upregulating mRNA levels and the expression of tight junction proteins (claudin‐1, occludin and zonula occludens‐1). Gas chromatography–mass spectrometry (MS) analysis demonstrated that cecal short‐chain fatty acids in mice with BSE administration were significantly increased compared with the model group. Sulforaphane and sulforaphane‐ N ‐acetylcysteine were only detected in BSE group mice by ultra‐performance liquid chromatography‐MS analysis. In addition, BSE intervention evidently increased the abundance of Alistipeds , Coriobacteriaceae UCG‐002 and Bifidobacterium and decreased the abundance of Escheichia‐Shinella , Lachnospiraceae others , Parabacteroides , Ruminococcaceae others and Turicibacter , which possibly promoted carbohydrate metabolism and short‐chain fatty acid production. CONCLUSION The present study aimed to elucidate the effect of BSE on colitis and found that BSE, as a novel food ingredient, has great potential for the improvement of colitis. © 2022 Society of Chemical Industry.

Wu, Jiaying↗

Excessive Aurantiochytrium acetophilum docosahexaenoic acid supplementation decreases growth performance and breast muscle mass of broiler chickens

We report Docosahexaenoic acid (DHA) is an n-3 polyunsaturated fatty acid with health-promoting potential. This study was to investigate effects of supplemental DHA from Aurantiochytrium acetophilum on growth performance, health status, meat quality, and protein synthesis signaling of broiler chickens. Day-old male chicks were housed in an environmental control room (6 cages/treatment, 8 chicks/cage), and fed a corn-soybean meal basal diet supplemented with the DHA-rich A. acetophilum biomass (Heliae, Gilbert, AZ) at 0, 1, 2, and 4% (0, 1.7, 3.4 and 6.8 g DHA/kg diet) for 6 weeks. Growth performance was measured weekly. Blood samples were collected at weeks 3 and 6 (2 chicks/cage). Four tissues were sampled (2 chicks/cage) for biochemical and meat quality analyses. Data were analyzed by one-way ANOVA and regression. Compared with the control, the 4% A. acetophilum diet decreased (p < 0.05) body weight gain (19%) and gain to feed ratio (19%) during weeks 4–6. The A. acetophilum supplementation dose-dependently decreased (p < 0.05, R 2 = 0.21–0.54) plasma alanine amino transferase activity and glucose concentrations, but had little effect on plasma activity of alkaline phosphatase or concentrations of inorganic phosphorus and uric acid at weeks 3 and 6. Compared with the control, the 4% A. acetophilum diet decreased (p < 0.05) breast muscle weight by 21%, and down-regulated (p < 0.05) mRNA levels of mammalian target of rapamycin and ribosomal s6 protein (S6), and protein levels of phosphorylated S6 to S6 and phosphorylated S6 kinase beta 1 to S6 kinase beta 1. The A. acetophilum supplementation linearly increased (p < 0.01) lipid peroxidation (R 2 = 0.62–0.90) and hardness and chewiness (R 2 = 0.34–0.44) of breast and thigh muscles. In conclusion, supplemental 4% (6.8 g DHA/kg), but not 1 or 2%, of A. acetophilum impaired growth performance, breast muscle mass accumulation, and(or) protein synthesis signaling of broilers.

59 BASIC BIOLOGICAL SCIENCES↗

The effects of protease, xylanase, and xylo-oligosaccharides on growth performance, nutrient utilization, short-chain fatty acids, and microbiota in Eimeria -challenged broiler chickens fed low-protein diet

A total of 392 Cobb 500 off-sex male broiler chicks were used in a 21-day experiment to study the effect of protease, xylanase, and xylo-oligosaccharides (XOS) on improving growth performance, nutrient utilization (ileal digestibility and total tract retention), gene expression of nutrient transporters, cecal short-chain fatty acids (SCFAs), and microbiota profile of broilers challenged with Eimeria spp. Chicks at 0-day old were allocated to 8 treatments in a 4 × 2 factorial arrangement: 1) corn-soybean meal diet with no enzyme (Con); 2) Con plus 0.2 g/kg protease alone (PRO); 3) Con plus 0.2 g/kg protease combined with 0.1 g/kg xylanase (PRO + XYL); or 4) Con plus 0.5 g/kg xylo-oligosaccharides (XOS); with or without Eimeria challenge. The 4 diets were formulated to be marginally low in crude protein (183 g/kg). Challenged groups were inoculated with a solution containing E. maxima, E. acervulina, and E. tenella oocysts on d 15. Eimeria depressed (P < 0.01) growth performance and nutrient utilization. Supplemental protease improved (P < 0.05) body weight gain and feed intake in the prechallenge phase (d 0-15) but had no effect during the infection period (d 15-21). There was no interaction between infection and feed supplementation for nutrient utilization. The supplementations of either PRO or XOS alone increased (P < 0.01) total tract retention of Ca and tended (P < 0.1) to improve total tract retention of N, P, AME, and AMEn. Eimeria decreased (P < 0.05) expressions of GLUT2, GLUT5, PepT1, ATP2B1, CaSR, Calbidin D28K, NPT2, and ZnT1 but increased (P < 0.01) expression of GLUT1. XOS supplementation increased (P < 0.05) ATP2B1 expression. Protease decreased (P < 0.05) isobutyrate concentration in unchallenged treatments but not in challenged treatments. Eimeria decreased (P < 0.01) cecal saccharolytic SCFAs acetate and propionate but increased (P < 0.01) branched-chain fatty acid isovalerate. The supplementation of PRO + XYL or XOS increased (P < 0.05) cecal butyrate or decreased cecal isobutyrate concentrations, respectively. PRO + XYL and XOS decreased cecal protein levels in unchallenged birds but not challenged ones. Eimeria challenge significantly (P < 0.05) decreased the microbial richness (Observed features) and diversity (Shannon index and phylogenetic diversity) and changed the microbial composition by reducing the abundance of certain bacteria, such as Ruminococcus torques, and increasing the abundance of others, such as Anaerostipes. In contrast, none of the additives had any significant effect on the cecal microbial composition. In conclusion, PRO or XOS supplementation individually improved nutrient utilization. All the additives decreased the cecal content of branched-chain fatty acids, consistent with decreased cecal N concentration, although the effects were more pronounced in unchallenged birds. In addition, none of the feed additives impacted the Eimeria-induced microbial perturbation.

60 APPLIED LIFE SCIENCES↗

Indole-3-carbinol ameliorates necroptosis and inflammation of intestinal epithelial cells in mice with ulcerative colitis by activating aryl hydrocarbon receptor

Highlights: • AHR activated by I3C plays an important role in protecting IECs from necroptosis and inflammation, in vivo and in vitro. • AHR could ubiquitinate RIPK1, promote IAPs expression and inhibit NF-κB activation in NCM460 cells. • I3C is a good candidate as a natural product to prevent and treat UC. Ulcerative colitis (UC) is a disease characterized by inflammation and disruption of the intestinal epithelial barrier. Necroptosis plays a critical role in disease progression. Indole-3-carbinol (I3C), a natural dietary agonist of aryl hydrocarbon receptor (AHR), has shown alleviating effects on UC. However, its mechanisms of action have not been comprehensively elucidated. Therefore, we aimed at investigating the protective role of I3C in DSS-induced colitis mice models. I3C significantly ameliorated body weight loss, colon length shortening and colonic pathological damage in colitis mice, reduced disease activity index (DAI) and histological (HI) scores, as well as alleviated colonic necroptosis and inflammation. In vitro, I3C attenuated necroptosis and inflammation of colonoids and NCM460 cells. AHR, activated by I3C, inhibits activation of receptor-interacting protein kinase 1 (RIPK1) and the subsequent assembly of necrosome in a time-dependent manner, as well as suppressing NF-κB activation and decreasing TNF-α, IL-1β, IL-6 and IL-8 expression. Silencing of AHR aggravated necroptosis and inflammation of NCM460 cells, and did not be ameliorated by I3C. Furthermore, AHR activation induces the expression of inhibitor of apoptosis proteins (IAPs) and the ubiquitination of RIPK1. In conclusion, I3C exerts a protective effect in DSS-induced colitis mice models by alleviating the necroptosis and inflammation of IECs through activating AHR.

60 APPLIED LIFE SCIENCES↗

Safety and efficacy of C. muridarum vaccines adjuvanted with CpG-1826 and four concentrations of Montanide-ISA-720-VG

It is recommended that the adjuvant Montanide ISA 720 VG be used at a concentration of 70% v/v. At this concentration, Montanide causes at the site of immunization a local granuloma that can last for several weeks. To determine the safety and protective efficacy of a Chlamydia muridarum MOMP vaccine, formulated with CpG-1826 and four different concentrations of Montanide (70%, 50%, 30% and 10%), BALB/c (H-2 d ) female mice were immunized twice intramuscularly. Local reactogenicity was significant for vaccines formulated with 70% or 50% Montanide but not for those inoculated with 30% or 10% Montanide. Robust humoral and cell mediated memory immune responses were elicited by the 70%, 50% and 30% Montanide formulations. Mice were challenged intranasally with 10 4 C. muridarum inclusion forming units (IFU). Based on changes in body weight, lungs’s weight and number of IFU recovered, mice vaccinated with the 70%, 50% and 30% Montanide formulations were significantly protected, but not mice receiving 10% Montanide. To conclude, we recommend the 30% Montanide concentration to be tested in humans and animal models to determine its safety and efficacy, in comparison to the 70% Montanide concentration currently used. The 30% Montanide formulation could significantly facilitate licensing of this adjuvant for human use.

60 APPLIED LIFE SCIENCES↗

Relationship of estrogen synthesis capacity in the brain with obesity and self-control in men and women

Significance Obesity is a major public health problem in a growing proportion of children and adults in the developed world. Estrogen influences body weight and behavioral responses to appetitive stimuli. Estrogen biosynthesis is catalyzed by the enzyme aromatase in all organs measured, including the brain. Using the aromatase radiotracer [ 11 C]vorozole, we measured aromatase availability in the amygdala in healthy-weight to obese adults. Obesity was associated with lower aromatase availability and less constraint independent of sex and age. Variability in brain estrogen synthesis may contribute to obesity by directly regulating feeding and broadly by affecting self-control. These findings suggest that brain aromatase imaging offers a method for characterizing the role of brain estrogen in obesity and other impairments in self-regulation.

Biegon, Anat↗

Protein intake is more stable than carbohydrate or fat intake across various US demographic groups and international populations

The optimal macronutrient composition of the diet is controversial and many adults attempt to regulate the intake of specific macronutrients for various health-related reasons. The objective was to compare stability and ranges of intakes of different macronutrients across diverse adult populations in the USA and globally. US dietary intake data from NHANES 2009–2014 were used to determine macronutrient intake as a percentage of total energy intake. Variability in macronutrient intake was estimated by calculating the difference between 75th and 25th percentile (Q3–Q1) IQRs of macronutrient intake distributions. In addition, intake data from 13 other countries with per capita gross domestic product (GDP) over $10,000 US dollars (USD) were used to assess variability of intake internationally since there are large differences in types of foods consumed in different countries. Protein, carbohydrate, and fat intake (NHANES 2009–2014) was 15.7 ± 0.1, 48.1 ± 0.1, and 32.9 ± 0.1% kcal, respectively, in US adults. The IQR of protein intake distribution (3.73 ± 0.11% kcal) was 41% of carbohydrate intake distribution (9.18 ± 0.20% kcal) and 58% of fat intake distribution (6.40 ± 0.14% kcal). The IQRs of carbohydrate and fat intake distributions were significantly (P <0.01) influenced by age and race; however, the IQR of protein intake was not associated with demographic and lifestyle factors including sex, race, income, physical activity, and body weight. International mean protein intake was 16.3 ± 0.2% kcal, similar to US intake, and there was less variation in protein than carbohydrate or fat intake. Protein intake of the US population and multiple international populations, regardless of demographic and lifestyle factors, was consistently ~16% of total energy, suggesting biological control mechanism(s) tightly regulate protein intake and, consequently, influence intake of other macronutrients and food constituents. Substantial differences in intake of the other macronutrients observed in US and international populations had little influence on protein intake. This trial was registered at the ISRCTN registry as ISRCTN46157745

dietary preferences↗

The Efficacy and Safety of an Intra-articular Dual-Acting Antibacterial Agent (TNP-2092) for Implant Infection–Associated Methicillin-Resistant Staphylococcus aureus

Abstract Owing to the presence of microbial biofilm on the implant, the eradication of biofilm-associated infections poses a challenge for antibiotic therapies. The study aimed to investigate the efficacy and safety of the novel antibiotic agent TNP-2092 in the context of implant infections. In vivo, rats with periprosthetic joint infection (PJI) treated with antibiotics showed an increase in body weight and decrease in swelling, temperature, and width of knee, compared with the control group. Meanwhile, inflammatory markers in synovium and serum were decreased in the TNP-2092 group, consistent with the pathological results. Moreover, TNP-2092 was effective in eliminating bacteria and disruption biofilm formation, and further alleviated the abnormal bone absorption and reactive bone changes around the prosthesis. In conclusion, intra-articular injection of TNP-2092 is safe and effective in treating knee PJI in a rat model. The study provides a foundation for the future utilization of TNP-2092 in the management of implant-related infections.

Dai, Tianyu↗

Evaluation of digestively resistant or soluble fibers, short- and medium-chain fatty acids, trace minerals, and antibiotics in nonchallenged nursery pigs on performance, digestibility, and intestinal integrity

Abstract Three experiments (EXP) were conducted to determine the effect of feed additives on performance, intestinal integrity, gastrointestinal volatile fatty acids (VFA), and energy and nutrient digestion in nonchallenged nursery pigs. In EXP 1, 480 pigs (6.36-kg body weight, BW) were placed into 96 pens with 5 pigs/pen, and allotted to 1 of 10 dietary treatments: 1) negative control containing no feed additive (NC), 2) NC + 44 mg chlortetracycline and 38.5 mg tiamulin/kg diet (CTsb), 3) NC + 5% resistant potato starch (RSpo), 4) NC + 5% soluble corn fiber (SCF), 5) NC + 5% sugar beet pulp (SBP), 6) NC + 0.30% fatty acid mix (FAM), 7) NC + 0.10% phytogenic blend of essential oils and flavoring compounds (PHY), 8) NC + 50 mg Cu and 1,600 mg zinc oxide/kg diet (CuZn), 9) NC + 5% resistant corn starch (RScn), and 10) NC + 0.05% β-glucan (BG) for 28 d. There was no impact of dietary treatment on BW gain or feed intake (P ≥ 0.22). Pigs fed diets containing SCF, CTsb, and RSpo resulted in microbial community differences compared to pigs fed the NC (P < 0.05). In EXP 2, 48 barrows (12.8 kg BW) were selected at the end of EXP 1 and fed the same dietary treatments they had previously received: 1) NC, 2) NC + 5% RScn, 3) NC + 5% SCF, and 4) NC + FAM for 8 d. There was no effect of feeding diets containing RScn, SCF, or FAM on in vivo intestinal permeability (P ≤ 0.21). Ileal or colon pH, concentrations of VFA did not differ due to dietary treatment (P ≥ 0.36), but pigs fed diets containing FAM resulted in a greater butyric acid concentration in the cecum compared to pigs fed the NC (P ≤ 0.05). In EXP 3, 156 pigs (6.11 kg BW) were placed into 52 pens with 3 pigs/pen and allotted to 1 of 4 dietary treatments arranged in a factorial manner: 1) NC, 2) NC + 5% RSpo, 3) NC + 0.30% FAM, and 4) NC + 5% RSpo + 0.30% FAM for 24 d. Feeding pigs diets containing RSpo did not affect BW gain (P = 0.91) while pigs fed diets containing FAM grew improved BW gain (P = 0.09). Colonic butyric acid concentrations were greater in pigs fed diets containing RSpo (P = 0.03), while pigs fed diets containing FAM exhibited reduced total VFA concentrations (P = 0.11). The results indicate that supplementing diets with digestively resistant but fermentable fibers, short- and medium-chain fatty acids, or antibiotics do not have a consistent effect, positive or negative, on markers of intestinal integrity or barrier function, intestinal VFA patterns, ATTD of energy and nutrients, or on pig performance.

Agriculture↗