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The quality of a dietary protein refers to its ability to provide the EAAs necessary to meet dietary requirements. There are 9 dietary amino acids that cannot be metabolically produced in the body and therefore must be consumed as part of the diet to avoid adverse metabolic consequences. These essential amino acids (EAAs) serve a variety of roles in the body. The amount and profile of the dietary EAAs relative to the individual EAA requirements and the digestibility of the dietary protein are the key factors that determine its quality. Currently the Digestible Indispensable Amino Acid Score (DIAAS) is the best available approach to quantifying protein quality. The most prominent metabolic role of dietary EAAs is to stimulate protein synthesis by serving as signals to activate molecular mechanisms responsible for the initiation of protein synthesis and, most importantly, to provide the necessary precursors for the synthesis of complete proteins. Current dietary recommendations generally do not consider protein quality. Accounting for protein quality in dietary patterns can be accomplished while staying within established ranges for dietary protein consumption. Poor protein quality can be compensated for to some extent by eating more low-quality protein, but to be effective (“complementary”) the limiting EAA must differ between the low-quality protein and the base diet to which it is being supplemented. Adding a high-quality protein to a dietary pattern based on low-quality protein is more effective in meeting EAA goals than increasing the amount of low-quality protein, even if the low-quality proteins are complementary. Further, reliance entirely on low-quality protein food sources, particularly in circumstances that may benefit from a level of dietary EAAs greater than minimal requirements, is likely to include excessive caloric consumption. While protein consumption in high-income nations is generally perceived to be adequate or even excessive, assessment of dietary patterns indicates that a significant percentage of individuals may fall short of meeting optimal levels of EAA consumption, especially in circumstances such as aging in which the optimal EAA consumption is greater than basal values for healthy young individuals. The case is made that protein quality is an important consideration in meeting EAA requirements.

Essential amino acid (EAA)-based compositions have been shown to be effective stimulators of muscle protein synthesis, but the lower limit of effective dosage is not clear. We have used stable isotope tracer methodology to quantify the response of muscle protein fractional synthetic rate (FSR) to a dose of 3.6 g of a high-leucine composition of EAAs plus arginine in older subjects. Muscle protein FSR increased 0.058%/hour over 3 h following consumption. When account was taken of the total muscle mass, this increase in muscle protein FSR represented approximately 80% of ingested EAAs. We conclude that a low dose of an EAA-based composition can effectively stimulate muscle protein synthesis.

OBJECTIVES/GOALS: To construct an assessment scale capable of evaluating a trial’s gender literacy or the extent to which biologically assigned “sex” is understood as separate from culturally defined and personally embodied “gender”. This scale in tandem with a policy brief will outline recommendations for inclusive medical nomenclature in the clinical space. METHODS/STUDY POPULATION: Using clinicaltrials.gov, inclusion/exclusion criteria was recorded for PrEP interventional trials (i.e., Truvada, Descovy). To evaluate these trials, an assessment scale for “gender literacy” is necessary. This scale relies on the fact that sex and gender are distinct elements to one’s identity and ought to be reported as such. As a form of content analysis, where literary information (eligibility criteria) is evaluated based on set rubric, this scale will require validation through inter-coder agreement. Evaluated in a group of 5 college-age students, this scale was used on selected PrEP clinical trials to verify if there was high agreement in the scores given. After validation, the dataset from clinicaltrials.gov underwent evaluation using the proposed assessment scale for gender literacy. RESULTS/ANTICIPATED RESULTS: The student coders had a Kalpha of 0.4 in the first round of grading. After retraining, their Kalpha increased to 0.68. The grading involved a subjective language rating (LIR), evaluating the usage of inclusive language, and a numerical score (GR) for the demographics of inclusion in a trial. After this inter-coder agreement validation, 216 active PrEP clinical trials (as of March 2023) were downloaded from clinicaltrials.gov. Grading of these trials showed that cisgender males represented 40% of participants, while 28% represented both transgender men and women, and less than 1% represented non-binary individuals. Moreover, more than half of the trials (52%) exhibited cisgender-oriented language or made no reference to gender identity. DISCUSSION/SIGNIFICANCE: It is a scientific imperative for clinical trials to have representative participant bases in order to derive data that is generalizable to afflicted populations. Especially for PrEP clinical trials, where gender-diverse individuals need visibility, trial design must be carefully crafted so as not to exclude through dated or exclusionary language.

OBJECTIVES/GOALS: We endeavor to investigated the hypothesis that muscle protein synthesis (MPS) is stimulated more after consumption of a 4-ounce beef patty as compared to 4- and 8-ounces of a soy protein based meat alternative (SPBMA) and if a greater stimulation is related to differences in the responses of plasma essential amino acid (EAA) concentrations. METHODS/STUDY POPULATION: Participants were aged 18 to 40 years of age with a BMI between 20 and 32 kg/m2. Written informed consent was obtained from all participants, and approved by UAMS IRB. Participants were assigned to one of three intervention groups via a single-blinded permuted block randomization, stratified for sex: 4 oz beef patty; 4 oz SPBMA; 2 x 4 oz (8oz) SPBMA. The impossible burgerTM was selected as it is primarily soy protein, a high-quality plant protein, and specifically designed to mimic a beef burger. Stable isotope were infused to assess protein metabolism. Appropriate muscle and blood samples were obtained. Enrichment and plasma EAA concentrations were measured with mass spectrometry. ANOVA’s on the change from basal to postprandial were used to identify group difference, significance was accepted at p < 0.05. RESULTS/ANTICIPATED RESULTS: The MPS increase from basal to postprandial indicated a significant main effect of group (p = 0.026), with the beef group (0.020 ± 0.016%/hour) being significantly greater than the 4oz SPBMA (0.003 ± 0.010%/hour; p = 0.021) but not the 8oz PBMA group (0.013 ± 0.016%/hour; p = 0.454). Similar results were observed for whole-body protein synthesis, where the beef group (p = 0.042) and 8oz SPBMA (p = 0.033) were significantly greater than the 4oz SPBMA (p = 0.021). Whole-body protein balance was significantly greater in the 8oz SPBMA as compared to 4oz of beef and SPBMA. Lastly, we observed a significantly relationship (p = 0.046; r = 0.411) between the maximal plasma EAA concentration and change in MPS, indicating the greater rate of MPS following 4oz of beef is mediated by an higher increase in plasma EAA concentrations. DISCUSSION/SIGNIFICANCE: In conclusion, 4oz of beef stimulates muscle protein FSR more than 4oz of a SPBMA. A common SPBMA can stimulate increase in protein metabolism, however, greater amounts are required as compared to beef protein. Further, the change in the muscle protein FSR response was significantly correlated with the maximal EAA concentration.

OBJECTIVES/GOALS: To compare the herbal medicine (HM) programs of the U.S. to those of different countries–including the European Union, South Korea, China, and India–and to examine each regulatory body’s process for obtaining market approval for HM drugs. METHODS/STUDY POPULATION: The European Union, South Korea, China, and India’s respective HM regulatory programs were examined and compared to the U.S. FDA’s HM process. These specific regulatory bodies were chosen based on the country’s long history with HM and/or the robustness of their existing HM review processes. International HM programs were researched using official government websites and journals published by independent, external research institutions that were accessed via USC’s library services. Data regarding the efficacy of HM policies such as HM IND approval rates, number of marketed HM drugs, and establishment of unique HM sectors will be collected. RESULTS/ANTICIPATED RESULTS: Investigational New Drug (INDs) applications regarding HM from each country will be categorized and displayed according to their approval status in order to provide insight on a HM program’s efficiency. Results also included a table displaying common challenges for approval for HM drugs across federal regulatory bodies. If applicable, effective solutions implemented to address some of these obstacles that proved to be effective will also be displayed in the form of a table. DISCUSSION/SIGNIFICANCE: Tables displaying the collective flaws of international HM programs and the resulting regulatory solutions can provide clearer guidance for companies seeking to submit HM INDs and for the U.S. FDA seeking to develop improved HM regulations.

Understanding and augmenting cancer-specific immunity is impeded by the fact that most tumors are driven by patient-specific mutations that encode unique antigenic epitopes. The shared antigens in virus-driven tumors can help overcome this limitation. Merkel cell carcinoma (MCC) is a particularly interesting tumor immunity model because (1) 80% of cases are driven by Merkel cell polyomavirus (MCPyV) oncoproteins that must be continually expressed for tumor survival; (2) MCPyV oncoproteins are only ~400 amino acids in length and are essentially invariant between tumors; (3) MCPyV-specific T cell responses are robust and strongly linked to patient outcomes; (4) anti-MCPyV antibodies reliably increase with MCC recurrence, forming the basis of a standard clinical surveillance test; and (5) MCC has one of the highest response rates to PD-1 pathway blockade among all solid cancers. Leveraging these well-defined viral oncoproteins, a set of tools that includes over 20 peptide-MHC class I tetramers has been developed to facilitate the study of anti-tumor immunity across MCC patients. Additionally, the highly immunogenic nature of MCPyV oncoproteins forces MCC tumors to develop robust immune evasion mechanisms to survive. Indeed, several immune evasion mechanisms are active in MCC, including transcriptional downregulation of MHC expression by tumor cells and upregulation of inhibitory molecules including PD-L1 and immunosuppressive cytokines. About half of patients with advanced MCC do not persistently benefit from PD-1 pathway blockade. Herein, we (1) summarize the lessons learned from studying the anti-tumor T cell response to virus-positive MCC; (2) review immune evasion mechanisms in MCC; (3) review mechanisms of resistance to immune-based therapies in MCC and other cancers; and (4) discuss how recently developed tools can be used to address open questions in cancer immunotherapy. We believe detailed investigation of this model cancer will provide insight into tumor immunity that will likely also be applicable to more common cancers without shared tumor antigens.

Abstract Sarcopenia, or the age‐related loss of skeletal muscle mass and function, is an increasingly prevalent condition that contributes to reduced quality of life, morbidity, and mortality in older adults. Older adults display blunted anabolic responses to otherwise anabolic stimuli—a phenomenon that has been termed anabolic resistance (AR)—which is likely a casual factor in sarcopenia development. AR is multifaceted, but historically much of the mechanistic focus has been on signalling impairments, and less focus has been placed on the role of the vasculature in postprandial protein kinetics. The vascular endothelium plays an indispensable role in regulating vascular tone and blood flow, and age‐related impairments in vascular health may impede nutrient‐stimulated vasodilation and subsequently the ability to deliver nutrients (e.g. amino acids) to skeletal muscle. Although the majority of data has been obtained studying younger adults, the relatively limited data on the effect of blood flow on protein kinetics in older adults suggest that vasodilatory function, especially of the microvasculature, strongly influences the muscle protein synthetic response to amino acid feedings. In this narrative review, we examine evidence of AR in older adults following amino acid and mixed meal consumption, examine the evidence linking vascular dysfunction and insulin resistance to age‐related AR, review the influence of nitric oxide and endothelin‐1 on age‐related vascular dysfunction as it relates to AR, briefly review the potential causal role of arterial stiffness in promoting skeletal muscle microvascular dysfunction and AR, and provide a brief overview and future considerations for research examining age‐related AR.

Human nutrition, and what can be considered “ideal” nutrition, is a complex, multi-faceted topic which many researchers and practitioners deliberate. While some attest that basic human nutrition is relatively understood, it is undeniable that a global nutritional problem persists. Many countries struggle with malnutrition or caloric deficits, while others encounter difficulties with caloric overconsumption and micronutrient deficiencies. A multitude of factors contribute to this global problem. Limitations to the current scope of the recommended daily allowances (RDAs) and dietary reference intakes (DRIs), changes in soil quality, and reductions in nutrient density are just a few of these factors. In this article, we propose a new, working approach towards human nutrition designated “Foundational Nutrition”. This nutritional lens combines a whole food approach in conjunction with micronutrients and other nutrients critical for optimal human health with special consideration given to the human gut microbiome and overall gut health. Together, this a synergistic approach which addresses vital components in nutrition that enhances the bioavailability of nutrients and to potentiate a bioactive effect.

Summary: Determination of whole body rates of protein synthesis, breakdown and net balance in human subjects still has an important role in nutrition research. Quantifying the anabolic response to dietary protein intake is a particularly important application. There are different models with which to accomplish this goal, each with advantages and limitations.The nitrogen (N)-flux method in which tracer is given orally has distinct advantages in terms of lack of invasiveness. In addition, the calculated results include all aspects of whole-body protein synthesis and breakdown. However, the prolonged timeframe of the method eliminates the possibility of the “pre-post” experimental design whereby each subject serves as their own control in the evaluation of the response to a meal.Models based on the primed-constant infusion of an essential amino acid (EAA) tracer enable the determination of baseline whole-body protein kinetics within 2 h, and can quantify a dynamic change from the basal state. The greatest challenge when using an EAA model is distinguishing exogenous and endogenous sources of the tracee in the blood. One approach is to use an intrinsically-labeled protein. This method has the advantage that the exogenous tracee is clearly distinguished from endogenous tracee. On the other hand, the intrinsically-labeled protein method suffers from unmeasured dilution that is likely to cause the systematic underestimation of the rate of appearance of exogenous tracee and thus overestimate the rate of whole-body protein breakdown. Alternatively, the “bioavailability” approach estimates the contribution of exogenous tracee to the peripheral circulation from the amount of tracee ingested, the true ileal digestibility of the tracee, and the irreversible loss of tracee prior to entry into the peripheral circulation. Errors in assumed values with the bioavailability method can potentially be significant, but are not likely to result in the systematic over- or under-estimations of rates of whole-body protein synthesis and breakdown.The optimal method depends on the degree of uncertainty regarding required assumptions in a particular circumstance. With all methods, it is advisable to calculate upper and lower bounds of whole body protein kinetics, in accord with reasonable maximal and minimal assumed values. Simultaneous use of two methods requiring different assumptions can also serve to confirm the validity of single approach.

Background: Previously, young males administered 200 mg/week of testosterone enanthate during 28 days of energy deficit (EDef) gained lean mass and lost less total mass than controls (Optimizing Performance for Soldiers I study, OPS I). Despite that benefit, physical performance deteriorated similarly in both groups. However, some experimental limitations may have precluded detection of performance benefits, as performance measures employed lacked military relevance, and the EDef employed did not elicit the magnitude of stress typically experienced by Soldiers conducting operations. Additionally, the testosterone administered required weekly injections, elicited supra-physiological concentrations, and marked suppression of endogenous testosterone upon cessation. Therefore, this follow-on study will address those limitations and examine testosterone's efficacy for preserving Solder performance during strenuous operations. Methods: In OPS II, 32 males will participate in a randomized, placebo-controlled, double-blind trial. After baseline testing, participants will be administered either testosterone undecanoate (750 mg) or placebo before completing four consecutive, 5-day cycles simulating a multi-stressor, sustained military operation (SUSOPS). SUSOPS will consist of two low-stress days (1000 kcal/day exercise-induced EDef; 8 h/night sleep), followed by three high-stress days (3000 kcal/day and 4 h/night). A 23-day recovery period will follow SUSOPS. Military relevant physical performance is the primary outcome. Secondary outcomes include 4-comparment body composition, muscle and whole-body protein turnover, intramuscular mechanisms, biochemistries, and cognitive function/mood. Conclusions: OPS II will determine if testosterone undecanoate safely enhances performance, while attenuating muscle and total mass loss, without impairing cognitive function, during and in recovery from SUSOPS. Trial Registration: ClinicalTrials.gov Identifier: NCT04120363.

Abstract Surgery and anesthesia induce a catabolic response that leads to skeletal muscle protein loss. Previous investigations have observed positive effects of perioperative nutrition. Furthermore, the benefits of exogenous amino acids on muscle protein kinetics are well established. However, no investigation has focused on muscle protein kinetics with and without perioperative amino acid infusion. Thus, we aimed to assess the effect of perioperative amino acid (AA) infusion on muscle protein balance in individuals undergoing elective total hip arthroplasty (THA). Elective THA patients were randomized to undergo a metabolic study prior to surgery (n = 5; control [CON]), intraoperative AA infusion (n = 9), or no AA (n = 13; standard of care [SC]). The CON group was studied prior to surgery to provide nonoperative/non‐anesthesia muscle protein kinetic reference values. The bolus infusion method with 13C6‐phenylalanine injected at time 0, and [15N]‐phenylalanine 30 min later was used to calculate muscle protein synthesis (MPS), protein breakdown (MPB), and net balance (MPS−MPB). Perioperative AA significantly improved muscle net balance as compared to SC (−0.005 ± 0.018%/h vs. −0.052 ± 0.011%/h) but not CON (0.003 ± 0.013%/h). The AA infusion significantly increased muscle net balance via a significant increase in MPS (AA = 0.062 ± 0.007%/h; SC = 0.037 ± 0.004%/h; CON = 0.072% ± 0.005%/h), and a nonsignificant attenuation of MPB (AA = 0.067 ± 0.012%/h; SC = 0.089 ± 0.014%/h; CON = 0.075 ± 0.011%/h). Our data support the use of perioperative AA infusion during elective THA as pragmatic strategy to offset the loss of surgically induced skeletal muscle protein.

Abstract We previously demonstrated that improved net muscle protein balance, via enhanced protein synthetic efficiency, occurs 5 days after testosterone (T) administration. Whether the effects of T on muscle protein kinetics occur immediately upon exposure is not known. We investigated the effects of acute T exposure on leg muscle protein kinetics and selected amino acid (AA) transport using the arteriovenous balance model, and direct calculations of mixed-muscle protein fractional synthesis (FSR) and breakdown (FBR) rates. Four healthy men were studied over a 5 h period with and without T (infusion rate, 0.25 mg·min− 1). Muscle protein FSR, FBR, and net protein balance (direct measures and model derived) were not affected by T, despite a significant increases in arterial (p = 0.009) and venous (p = 0.064) free T area under the curve during T infusion. T infusion had minimal effects on AA transport kinetics, affecting only the outward transport and total intracellular rate of appearance of leucine. These data indicate that exposing skeletal muscle to T does not confer immediate effects on AA kinetics or muscle anabolism. There remains an uncertainty as to the earliest discernable effects of T on skeletal muscle protein kinetics after initial administration.

Abstract Backgrounds Folate Hydrolase‐1 (FOLH1; PSMA) is a type II transmembrane protein, luminally expressed by solid tumour neo‐vasculature. Monoclonal antibody (mAb), J591, is a vehicle for mAb‐based brachytherapy in FOLH1+ cancers. Brachytherapy is a form of radiotherapy that involves placing a radioactive material a short distance from the target tissue (e.g., on the skin or internally); brachytherapy is commonly accomplished with the use of catheters, needles, metal seeds and antibody or small peptide conjugates. Herein, FOLH1 expression in primary (p) and metastatic (m) Merkel cell carcinoma (MCC) is characterized to determine its targeting potential for J591‐brachytherapy. Materials & Methods Paraffin sections from pMCC and mMCC were evaluated by immunohistochemistry for FOLH1. Monte Carlo simulation was performed using the physical properties of conjugated radioisotope lutetium‐177. Kaplan–Meier survival curves were calculated based on patient outcome data and FOLH1 expression. Results Eighty‐one MCC tumours were evaluated. 67% (54/81) of all cases, 77% (24/31) pMCC and 60% (30/50) mMCC tumours were FOLH1+. Monte Carlo simulation showed highly localized ionizing tracks of electrons emitted from the targeted neo‐vessel. 42% (34/81) of patients with FOLH1+/− MCC had available survival data for analysis. No significant differences in our limited data set were detected based on FOLH1 status (p = 0.4718; p = 0.6470), staining intensity score (p = 0.6966; p = 0.9841) or by grouping staining intensity scores (− and + vs. ++, +++, +++) (p = 0.8022; p = 0.8496) for MCC‐specific survival or recurrence free survival, respectively. Conclusions We report the first evidence of prevalent FOLH1 expression within MCC‐associated neo‐vessels, in 60‐77% of patients in a large MCC cohort. Given this data, and the need for alternatives to immune therapies it is appropriate to explore the safety and efficacy of FOLH1‐targeted brachytherapy for MCC. What's already known about this topic? We report the first evidence of prevalent folate hydrolase‐1 (FOLH1; also known as prostate‐specific membrane antigen) expression within MCC‐associated neovessels. What does this study add? Herein, FOLH1 expression in Merkel cell carcinoma neovasculature is validated, and the therapeutic mechanism of specific, systemic targeting of disseminated disease with antibody‐based brachytherapy, is defined.

Background The effects of ingesting varying essential amino acid (EAA)/protein-containing food formats on protein kinetics during energy deficit are undetermined. Therefore, recommendations for EAA/protein food formats necessary to optimize both whole-body protein balance and muscle protein synthesis (MPS) during energy deficit are unknown. We measured protein kinetics after consuming iso-nitrogenous amounts of free-form essential amino acid-enriched whey (EAA + W; 34.7 g protein, 24 g EAA sourced from whey and free-form EAA), whey (WHEY; 34.7 g protein, 18.7 g EAA), or a mixed-macronutrient meal (MEAL; 34.7 g protein, 11.4 g EAA) after exercise during short-term energy deficit. Methods Ten adults (mean ± SD; 21 ± 4 y; 25.7 ± 1.7 kg/m2) completed a randomized, double-blind crossover study consisting of three, 5 d energy-deficit periods (− 30 ± 3% of total energy requirements), separated by 14 d. Whole-body protein synthesis (PS), breakdown (PB), and net balance (NET) were determined at rest and in response to combination exercise consisting of load carriage treadmill walking, deadlifts, and box step-ups at the end of each energy deficit using L-[2H5]-phenylalanine and L-[2H2]-tyrosine infusions. Treatments were ingested immediately post-exercise. Mixed-muscle protein synthesis (mixed-MPS) was measured during exercise through recovery. Results Change (Δ postabsorptive + exercise to postprandial + recovery [mean treatment difference (95%CI)]) in whole-body (g/180 min) PS was 15.8 (9.8, 21.9; P = 0.001) and 19.4 (14.8, 24.0; P = 0.001) greater for EAA + W than WHEY and MEAL, respectively, with no difference between WHEY and MEAL. ΔPB was − 6.3 (− 11.5, − 1.18; P = 0.02) greater for EAA + W than WHEY and − 7.7 (− 11.9, − 3.6; P = 0.002) greater for MEAL than WHEY, with no difference between EAA + W and MEAL. ΔNET was 22.1 (20.5, 23.8; P = 0.001) and 18.0 (16.5, 19.5; P = 0.00) greater for EAA + W than WHEY and MEAL, respectively, while ΔNET was 4.2 (2.7, 5.6; P = 0.001) greater for MEAL than WHEY. Mixed-MPS did not differ between treatments. Conclusions While mixed-MPS was similar across treatments, combining free-form EAA with whey promotes greater whole-body net protein balance during energy deficit compared to iso-nitrogenous amounts of whey or a mixed-macronutrient meal. Trial registration ClinicalTrials.gov, Identifier no. NCT04004715. Retrospectively registered 28 June 2019, first enrollment 6 June 2019

Background Clinical benefit from programmed cell death 1 receptor (PD-1) inhibitors relies on reinvigoration of endogenous antitumor immunity. Nonetheless, robust immunological markers, based on circulating immune cell subsets associated with therapeutic efficacy are yet to be validated.Methods We isolated peripheral blood mononuclear cell from three independent cohorts of melanoma and Merkel cell carcinoma patients treated with PD-1 inhibitor, at baseline and longitudinally after therapy. Using multiparameter flow cytometry and cell sorting, we isolated four subsets of CD8+ T cells, based on PD-1 and TIGIT expression profiles. We performed phenotypic characterization, T cell receptor sequencing, targeted transcriptomic analysis and antitumor reactivity assays to thoroughly characterize each of these subsets.Results We documented that the frequency of circulating PD-1+TIGIT+ (DPOS) CD8+ T-cells after 1 month of anti-PD-1 therapy was associated with clinical response and overall survival. This DPOS T-cell population was enriched in highly activated T-cells, tumor-specific and emerging T-cell clonotypes and T lymphocytes overexpressing CXCR5, a key marker of the CD8 cytotoxic follicular T cell population. Additionally, transcriptomic profiling defined a specific gene signature for this population as well as the overexpression of specific pathways associated with the therapeutic response.Conclusions Our results provide a convincing rationale for monitoring this PD-1+TIGIT+ circulating population as an early cellular-based marker of therapeutic response to anti-PD-1 therapy.

Abstract The purpose of this study was to assess the maturity‐related differences in the aerobic and anaerobic adaptations to sprint interval training (SIT) among youth male athletes. Twenty‐seven youth male athletes were assessed for years from peak height velocity (PHV) and classified into prepubescent (PRE, n = 7, years from PHV = −2.21 ± 0.47 years), peripubescent (PERI, n = 10, years from PHV = 0.25 ± 0.88 years), and postpubescent (POST, n = 10, years from PHV = 2.81 ± 0.50 years) groups based on their years from estimated peak height velocity. Participants completed a ramp exercise protocol on a cycle ergometer to determine maximal aerobic power, maximal oxygen consumption (VO2peak), and fatigue thresholds. Following baseline, all participants completed a 4‐week SIT program that consisted of eight total training sessions. During each session, participants completed repeated 20‐s sprints on a cycle ergometer against a resistance of 7.5% of body mass. The number of sprints per sessions increased from four in session 1 to seven in session 7, with four sprints in session 8. Peak and mean power from sessions 1 and 8 were recorded. All participants completed a post‐testing ramp exercise protocol that mirrored baseline. Maximal aerobic power increased (p

Background We have determined the acute response of protein kinetics to one or two servings (6.3 g and 12.6 g) of a proprietary composition containing free-form essential amino acids (EAA) (3.2 g EAA per serving) and whey protein (2.4 g per serving), as well as the response to consumption of a popular whey-based protein supplement (Gatorade Recover) (17 g; 12.6 g protein). Methods Whole-body rates of protein synthesis, breakdown and net balance (taken to be the anabolic response) were determined using primed-constant infusions of 2H5-phenylalnine and 2H2-tyrosine. Muscle protein fractional synthetic rate (FSR) was also determined with the 2H5-phenylalanine tracer. Results Plasma EAA levels increased following consumption of all beverages, with the greatest response in the high-dose EAA/protein composition. Similarly, the increase in net balance between whole-body protein synthesis and breakdown was greatest following consumption of the high-dose EAA/protein composition, while the low-dose EAA/protein composition and Gatorade Recover induced similar increases in net balance. When the net balance response was normalized for the total amount of product given, the high- and low-dose EAA/protein beverages were approximately 6- and 3-fold more anabolic than the Gatorade Recover, respectively. The greater anabolic response to the EAA/protein composition was due to greater increases in whole-body protein synthesis with both doses, and a markedly greater suppression of whole-body protein breakdown in the high-dose group. Muscle protein FSR after beverage consumption reflected changes in whole-body protein synthesis, with the larger EAA/protein dose significantly increasing FSR. Conclusion We conclude that a composition of a balanced EAA formulation combined with whey protein is highly anabolic as compared to a whey protein-based recovery product, and that the response is dose-dependent. Trial registration ClinicalTrials.gov Identifier: NCT03502941. This trial was registered on April 19, 2018.

Abstract Background Merkel cell carcinoma (MCC) is an aggressive skin cancer that frequently responds to anti-PD-1 therapy. MCC is associated with sun exposure and, in 80% of cases, Merkel cell polyomavirus (MCPyV). MCPyV-specific T and B cell responses provide a unique opportunity to study cancer-specific immunity throughout PD-1 blockade therapy. Methods Immune responses were assessed in patients (n = 26) with advanced MCC receiving pembrolizumab. Peripheral blood mononuclear cells (PBMC) were collected at baseline and throughout treatment. MCPyV-oncoprotein antibodies were quantified and T cells were assessed for MCPyV-specificity via tetramer staining and/or cytokine secretion. Pre-treatment tumor biopsies were analyzed for T cell receptor clonality. Results MCPyV oncoprotein antibodies were detectable in 15 of 17 (88%) of virus-positive MCC (VP-MCC) patients. Antibodies decreased in 10 of 11 (91%) patients with responding tumors. Virus-specific T cells decreased over time in patients who had a complete response, and increased in patients who had persistent disease. Tumors that were MCPyV(+) had a strikingly more clonal (less diverse) intratumoral TCR repertoire than virus-negative tumors (p = 0.0001). Conclusions Cancer-specific T and B cell responses generally track with disease burden during PD-1 blockade, in proportion to presence of antigen. Intratumoral TCR clonality was significantly greater in VP-MCC than VN-MCC tumors, suggesting expansion of a limited number of dominant clones in response to fewer immunogenic MCPyV antigens. In contrast, VN-MCC tumors had lower clonality, suggesting a diverse T cell response to numerous neoantigens. These findings reveal differences in tumor-specific immunity for VP-MCC and VN-MCC, both of which often respond to anti-PD-1 therapy.

Abstract Background We recently reported a 56% objective response rate in patients with advanced Merkel cell carcinoma (MCC) receiving pembrolizumab. However, a biomarker predicting clinical response was not identified. Methods Pretreatment FFPE tumor specimens (n = 26) were stained for CD8, PD-L1, and PD-1 by immunohistochemistry/immunofluorescence (IHC/IF), and the density and distribution of positive cells was quantified to determine the associations with anti-PD-1 response. Multiplex IF was used to test a separate cohort of MCC archival specimens (n = 16), to identify cell types expressing PD-1. Results Tumors from patients who responded to anti-PD-1 showed higher densities of PD-1+ and PD-L1+ cells when compared to non-responders (median cells/mm2, 70.7 vs. 6.7, p = 0.03; and 855.4 vs. 245.0, p = 0.02, respectively). There was no significant association of CD8+ cell density with clinical response. Quantification of PD-1+ cells located within 20 μm of a PD-L1+ cell showed that PD-1/PD-L1 proximity was associated with clinical response (p = 0.03), but CD8/PD-L1 proximity was not. CD4+ and CD8+ cells in the TME expressed similar amounts of PD-1. Conclusions While the binomial presence or absence of PD-L1 expression in the TME was not sufficient to predict response to anti-PD-1 in patients with MCC, we show that quantitative assessments of PD-1+ and PD-L1+ cell densities as well as the geographic interactions between these two cell populations correlate with clinical response. Cell types expressing PD-1 in the TME include CD8+ T-cells, CD4+ T-cells, Tregs, and CD20+ B-cells, supporting the notion that multiple cell types may potentiate tumor regression following PD-1 blockade.

Acquired resistance is a major problem in cancer immunotherapy. Here the authors report a study of two patients with Merkel cell carcinoma under immunotherapy treatment who develop resistance after deep responses for >1 year and identified a novel mechanism of acquired, gene-specific transcriptional suppression of HLAs.

Ingesting protein-containing supplements and foods provides essential amino acids (EAA) necessary to increase muscle and whole-body protein synthesis (WBPS). Large variations exist in the EAA composition of supplements and foods, ranging from free-form amino acids to whole protein foods. We sought to investigate how changes in peripheral EAA after ingesting various protein and free amino acid formats altered muscle and whole-body protein synthesis. Data were compiled from four previous studies that used primed, constant infusions of L-(ring-2H5)-phenylalanine and L-(3,3-2H2)-tyrosine to determine fractional synthetic rate of muscle protein (FSR), WBPS, and circulating EAA concentrations. Stepwise regression indicated that max EAA concentration (EAACmax; R2 = 0.524, p < 0.001), EAACmax (R2 = 0.341, p < 0.001), and change in EAA concentration (ΔEAA; R = 0.345, p < 0.001) were the strongest predictors for postprandial FSR, Δ (change from post absorptive to postprandial) FSR, and ΔWBPS, respectively. Within our dataset, the stepwise regression equation indicated that a 100% increase in peripheral EAA concentrations increases FSR by ~34%. Further, we observed significant (p < 0.05) positive (R = 0.420–0.724) correlations between the plasma EAA area under the curve above baseline, EAACmax, ΔEAA, and rate to EAACmax to postprandial FSR, ΔFSR, and ΔWBPS. Taken together our results indicate that across a large variety of EAA/protein-containing formats and food, large increases in peripheral EAA concentrations are required to drive a robust increase in muscle and whole-body protein synthesis.

Protein intake recommendations to optimally stimulate muscle protein synthesis (MPS) are derived from dose-response studies examining the stimulatory effects of isolated intact proteins (e.g., whey, egg) on MPS in healthy individuals during energy balance. Those recommendations may not be adequate during periods of physiological stress, specifically the catabolic stress induced by energy deficit. Providing supplemental intact protein (20–25 g whey protein, 0.25–0.3 g protein/kg per meal) during strenuous military operations that elicit severe energy deficit does not stimulate MPS-associated anabolic signaling or attenuate lean mass loss. This occurs likely because a greater proportion of the dietary amino acids consumed are targeted for energy-yielding pathways, whole-body protein synthesis, and other whole-body essential amino acid (EAA)-requiring processes than the proportion targeted for MPS. Protein feeding formats that provide sufficient energy to offset whole-body energy and protein-requiring demands during energy deficit and leverage EAA content, digestion, and absorption kinetics may optimize MPS under these conditions. Understanding the effects of protein feeding format-driven alterations in EAA availability and subsequent changes in MPS and whole-body protein turnover is required to design feeding strategies that mitigate the catabolic effects of energy deficit. In this manuscript, we review the effects, advantages, disadvantages, and knowledge gaps pertaining to supplemental free-form EAA, intact protein, and protein-containing mixed meal ingestion on MPS. We discuss the fundamental role of whole-body protein balance and highlight the importance of comprehensively assessing whole-body and muscle protein kinetics when evaluating the anabolic potential of varying protein feeding formats during energy deficit.

Military personnel may be exposed to circumstances (e.g., large energy deficits, sleep deprivation, cognitive demands, and environmental extremes) of external stressors during training and combat operations (i.e., operational stressors) that combine to degrade muscle protein. The loss of muscle protein is further exacerbated by frequent periods of severe energy deficit. Exposure to these factors results in a hypogonadal state that may contribute to observed decrements in muscle mass. In this review, lessons learned from studying severe clinical stressed states and the interventions designed to mitigate the loss of muscle protein are discussed in the context of military operational stress. For example, restoration of the anabolic hormonal status (e.g., testosterone, insulin, and growth hormone) in stressed physiological states may be necessary to restore the anabolic influence derived from dietary protein on muscle. Based on our clinical experiences, restoration of the normal testosterone status during sustained periods of operational stress may be advantageous. We demonstrated that in severe burn patients, pharmacologic normalization of the anabolic hormonal status restores the anabolic stimulatory effect of nutrition on muscle by improving the protein synthetic efficiency and limiting amino acid loss from skeletal muscle. Furthermore, an optimal protein intake, and in particular essential amino acid delivery, may be an integral ingredient in a restored anabolic response during the stress state. Interventions which improve the muscle net protein balance may positively impact soldier performance in trying conditions.