Amino Acid Flow Research Articles

Evaluating endogenous loss and standard ileal digestibility of amino acids in response to the graded severity levels of E. maxima infection

The study was conducted to evaluate the effects of graded levels of Eimeria maxima challenge on endogenous loss, apparent ileal digestibility (AID), and standard ileal digestibility (SID) of amino acids. A total of 768 fourteen-day-old male Cobb 500 broiler chickens were randomly allocated into 64 battery cages. Apart from the regular corn-soybean based diet, the nitrogen-free diet (NFD) was formulated to determine the endogenous loss of amino acids. One-half of the birds (32 cages) were fed the NFD, and another half fed the regular diet from d 14 to 20. Both groups were further assigned to 4 treatments (nonchallenged control or three levels of challenge doses) with 8 replicate cages. The challenge doses were: the low challenge dose (Low) with E. maxima 12,500 oocysts, the medium challenge dose (Medium) with 25,000 E. maxima oocysts, and the high challenge dose (High) with 50,000 E. maxima oocysts. At 6 d postinfection, ileal digesta samples were collected and the intestinal lesion score were recorded. The results indicated a significant linear increase of endogenous amino acid flow in response to the graded E. maxima challenge. Moreover, the AID and SID of amino acids were linearly reduced due to the increasing challenge dose. The study demonstrated that NFD significantly reduced lesion scores, underestimating the true endogenous losses of birds fed regular diets. Even though the endogenous loss of amino acids was underestimated, they were linearly increased in response to the graded E. maxima challenge. In conclusion, the higher Eimeria dose birds were challenged with, the more endogenous amino acids were released into the intestine and the lower dietary nutrients were digested and absorbed by broiler chickens.

Apparent and standardised ileal digestibility coefficients of amino acids in wheat, soybean meal and rapeseed meal for broilers

The apparent (AID) and(SID) ileal amino acid digestibilities in wheat, soybean meal, and rapeseed meal were determined with Ross 308 broiler chicken (n = 64) using the slaughter technique with chromium mordanted straw as an indigestible marker. The recovery of endogenous amino acids at the distal ileum was determined with a protein-free diet and it was used to calculate the SID digestibilities of the studied feed ingredients. The mean amino acid AID and SID were higher in soybean meal and wheat than in rapeseed meal (p<0.05). The mean amino acid flow measured from the ileum was the highest in rapeseed meal, intermediate in soybean meal, and the lowest in wheat (p<0.05). The main amino acids in the basal endogenous secretion were aspartic and glutamic acid and the lowest ones present were methionine and histidine (p<0.05). The difference between amino acid AID and SID values varied among ingredients and was higher in wheat than in soybean meal and rapeseed meal (p<0.05).

Basal ileal endogenous amino acid flow in broiler chickens as influenced by age

The current study was carried out to measure the basal ileal endogenous amino acid (EAA) flow in male broilers (Ross 308) at different ages (d 7, 14, 21, 28, 35, and 42), following the feeding of a nitrogen-free diet. Titanium dioxide (5 g/kg) was included as an indigestible marker. The nitrogen-free diet was offered for four days prior to ileal digesta collection to 6 replicate cages housing 14 (d 3–7), 12 (d 10–14), 10 (d 17–21), 8 (d 24–28), 8 (d 31–35), and 6 (d 38–42) birds per cage. The basal EAA flow was calculated as g/kg DM intake. The amino acid (AA) profile of endogenous protein, expressed as g/100 g protein, was also calculated. The basal endogenous flow of nitrogen and all individual and total AA decreased quadratically (P < 0.05 to 0.001), with flows being higher on d 7, then decreasing on d 14, plateauing until d 35 and decreasing further on d 42. The concentrations of Trp, Cys, and Gly in the endogenous protein increased linearly (P < 0.01 to 0.001) with advancing age, whereas a linear decrease (P < 0.001) was noted for Lys. A quadratic influence (P < 0.05 to 0.001) was observed for the concentrations of Ile, Leu, Met, Val, and Asp. These changes in the endogenous protein profile may be attributed to variations in the contribution of endogenous sources with age but delineating the exact contribution of different sources is complicated. Overall, the current findings suggest that the basal ileal EAA flow is influenced by broiler age and age-specific EAA flows may need to be considered to standardize the AA digestibility.

The amino acid permease (AAP) genes CsAAP2A and SlAAP5A/B are required for oomycete susceptibility in cucumber and tomato.

Cucurbit downy mildew (DM), caused by the obligate biotroph Pseudoperonospora cubensis, is a destructive disease in cucumber. A valuable source of DM resistance is the Indian cucumber accession PI 197088, which harbours several quantitative trait loci (QTLs) contributing to quantitatively inherited DM resistance. With a combination of fine‐mapping and transcriptomics, we identified Amino Acid Permease 2A (CsAAP2A) as a candidate gene for QTL DM4.1.3. Whole‐genome and Sanger sequencing revealed the insertion of a Cucumis Mu‐like element (CUMULE) transposon in the allele of the resistant near‐isogenic line DM4.1.3. To confirm whether loss of CsAAP2A contributes to partial DM resistance, we performed targeting induced local lesions in genomes on a DM‐susceptible cucumber genotype to identify an additional csaap2a mutant, which indeed was partially DM resistant. In view of the loss of the putative function as amino acid transporter, we measured amino acids in leaves. We found that DM‐inoculated leaves of line DM4.1.3 (with the csaap2a mutation) contained significantly fewer amino acids than wild‐type cucumber. The decreased flow of amino acids towards infected leaves in csaap2a plants compared to the wild type might explain the resistant phenotype of the mutant, as this would limit the available nutrients for the pathogen and thereby its fitness. To examine whether AAP genes play a conserved role as susceptibility factors in plant–oomycete interactions, we made targeted mutations in two AAP genes from tomato and studied the effect on susceptibility to Phytophthora infestans. We conclude that not only CsAAP2A but also SlAAP5A/SlAAP5B are susceptibility genes for oomycete pathogens.

Manganese dioxide driven the carbon and nitrogen transformation by activating the complementary effects of core bacteria in composting

This study revealed core bacterial metabolic mechanisms involved in carbon (C) and nitrogen (N) in composting with adding MnO2. Two tests (control group (CK), adding MnO2 (M)) were performed. The results indicated that the MnO2 accelerated the transformation of carbon and nitrogen in composting. Core bacteria involved in the C and N conversion were identified, the complementarity effects of core bacteria were stimulated in M composting. Additionally, the influence of core bacteria on the C and N conversion could be divided into two pathways in M composting. One was that core bacteria promoted C and N conversion by accelerating the flow of amino acids into the tricarboxylic acid cycle. Another was that the complementarity effects of core bacteria increased the overall bacterial diversity, which contributed to C and N conversion. These findings showed that the addition of MnO2 to composting was a promising application to treat agricultural organic waste.

Microbial composition and omasal flows of bacterial, protozoal, and nonmicrobial amino acids in lactating dairy cows fed fresh perennial ryegrass (Lolium perenne L.) not supplemented or supplemented with rolled barley

The objective of this study was to evaluate the effect of rolled barley supplementation on microbial composition and omasal flows of bacterial, protozoal, and nonmicrobial AA in cows fed fresh perennial ryegrass (Lolium perenne L.; PRG). Ten ruminally cannulated multiparous Holstein cows averaging (mean ± standard deviation) 49 ± 23 d in milk and 513 ± 36 kg of body weight were assigned to 1 of 2 treatments in a switchback design. The treatment diets were PRG only or PRG plus 3.5 kg of dry matter rolled barley (G+RB). The study consisted of three 29-d periods where each period consisted of 21 d of diet adaptation and 8 d of data and sample collection. A double-marker system was used to quantify nutrient flow entering the omasal canal along with 15N-ammonium sulfate to label and measure the microbial and nonmicrobial omasal flow of AA. Overall, rolled barley supplementation had no effect on the AA composition of the omasal liquid-associated and particle-associated bacteria. Rolled barley supplementation affected the AA concentrations of omasal protozoa; however, the differences were nutritionally minor. Particle-associated bacteria AA flow was increased for all AA, except for Trp and Pro, in cows fed the G+RB diet. Rolled barley supplementation had no effect on protozoal AA flow. On average, protozoa accounted for 23% of the microbial essential AA flow, which ranged from 17 to 28% for Trp and Lys, respectively. The flow of all AA in omasal true digesta increased in cows fed the G+RB diet compared with the PRG-only diet, resulting in a 228 g/d increase in total AA flow in cows fed the G+RB diet. This increase in total AA flow in cows fed the G+RB diet was due to an increase in microbial AA flow. Rolled barley supplementation had no effect on nonmicrobial AA flow. The nonmicrobial AA flow modestly contributed to total AA flow, accounting for 15.6% on average. These results indicated that extensive ruminal degradation of PRG AA occurred (83.5%), and we demonstrated that cows consuming PRG-based diets exhibit a large dependence on microbial AA to support metabolizable AA supply. Rolled barley supplementation can increase the omasal flow of microbial AA in cows consuming PRG-based diets. However, further research is required to elucidate if this increased AA supply can support higher milk yield under such dietary conditions.

Effects of Dietary Crude Protein Levels on Fecal Crude Protein, Amino Acids Flow Amount, Fecal and Ileal Microbial Amino Acids Composition and Amino Acid Digestibility in Growing Pigs.

Simple SummaryThe purpose of this experiment was to evaluate a low protein corn-soybean meal-based diet with the same Lys, Met + Cys, Thr and Trp level as a high protein diet on fecal crude protein (CP), amino acid (AA) flow amount, AA digestibility and fecal and ileal microbial AA composition in growing pigs. Eighteen pigs with an initial body weight of (30 ± 1.35) kg were randomly divided into three groups, with six replicates in each group, and fed a corn-soybean meal-based diets with 12%, 15% and 18% CP levels, respectively. Our aim was to explain whether the addition of four crystalline essential AAs (EAA) to a low diet affected the digestibility of protein-bound NEAA (non-essential amino acid) and EAA and the composition of microbial AA in ileum and feces.The purpose of this experiment was to evaluate the effects of low protein corn-soybean meal-based diets on fecal CP, amino acid (AA) flow amount, AA digestibility and fecal and ileal microbial AA composition in growing pigs. Eighteen pigs (initial body weight = 30 ± 1.35) were randomly divided into three groups and fed with basal diets with CP levels of 12%, 15% and 18%, respectively. The Lys, Met + Cys, Thr and Trp level in the 12% CP and 15% CP groups is the same as 18% CP group by the addition of four crystalline Lys, Met + Cys, Thr and Trp to the diet. The results showed that with the decrease of dietary CP level from 18% to 12%, the fecal total nitrogen (N), CP and total AA (TAA) flow amount decreased linearly (p < 0.05). Dry matter (DM) digestibility, CP digestibility, TAA digestibility, essential amino acid (EAA) digestibility and non-essential amino acid (NEAA) digestibility increased linearly with the decrease of dietary CP concentration from 18% to 12%. Compared with 18% CP group, the flow amount of Asp, Ser, Glu, Gly, Tyr, Val, Leu and Phe in feces of pigs in the 15% CP group and 12% CP group decreased significantly, while the flow amount of Arg in the 15% CP group was lower than that in the 18% CP group and 12% CP group. The fecal microbial N and AA of the 15% CP group were higher than those of the 18% CP and 12% CP groups. Fecal TAA flow amount decreased linearly with the decrease of the dietary CP levels from 18% to 12%. Fecal TAA and NEAA flow amount also decreased linearly with the decrease of dietary CP level from 18% to 12%. Except for Glu, Gly, Met, Tyr, Thr and Phe, there were significant differences among the three groups in the composition of 17 kinds of AAs in fecal microorganisms. Among the 17 AA compositions of ileal microorganisms, except Tyr and Lys, the other AAs were significantly different among the three groups (p < 0.05)

Dietary supplementation with N-carbamoylglutamate initiated from the prepartum stage improves lactation performance of postpartum dairy cows

The objective of this study was to investigate the effects of supplementing N-carbamoylglutamate (NCG), an Arg enhancer, on amino acid (AA) supply and utilization and productive performance of early-lactating dairy cows. Thirty multiparous Chinese Holstein dairy cows were randomly divided into control (CON, n = 15) and NCG (CON diet supplemented with NCG at 20 g/d per cow, n = 15) groups at 4 wk before calving. Diets were offered individually in tie-stalls, and NCG was supplemented by top-dress feeding onto total mixed ration for the NCG group. The experiment lasted until wk 10 after calving. Dry matter intake tended to be higher (P = 0.06), and yields of milk (P < 0.01), milk protein (P < 0.01), and milk fat (P < 0.01) were higher in the NCG-cows than in the CON-cows. Plasma activities of aspartate aminotransferase (P < 0.01), alanine aminotransferase (P = 0.03), and plasma level of β-hydroxybutyrate (P = 0.04) were lower in the NCG-cows than in the CON-cows, whereas plasma glucose (P = 0.05) and nitric oxide (NO, P < 0.01) concentrations were higher. Coccygeal vein concentrations of Cys (P < 0.01), Pro (P < 0.01), Tyr (P = 0.05), most essential AA except Thr and His (P < 0.01), total essential AA (P < 0.01), and total AA (P < 0.01) were higher in the NCG-cows than in the CON-cows. The arterial supply of all AA was greater in the NCG-cows than in the CON-cows. The NCG-cows had higher mammary plasma flow of AA (P = 0.04) and clearance rate of Cys (P < 0.01), Pro (P < 0.01) and Asp (P < 0.01), and higher ratios of uptake to output of Met (P = 0.05), Lys (P < 0.01), Cys (P = 0.01), Pro (P = 0.03), and Asp (P = 0.01). In summary, addition of NCG initiated from the prepartum period improved the lactation performance of postpartum dairy cows, which might attribute to greater Arg and NO concentrations, as well as improved AA supply and utilization, liver function, and feed intake in these cows.

Essential amino acids and their need by lactating cows

Metabolic protein is the main source of truly absorbed amino acids. The determination of metabolic protein and metabolic acids is an important, but very difficult task. To do this, you need to know the exact number of incoming protein fractions in the small intestine as part of the duodenal fl ow of chymus, their digestibility, the fl ow of metabolism of essential amino acids from the intestine to the bloodstream and the coefficients of their use for milk protein biosynthesis and maintenance of life. The purpose of the research was to determine the need of lactating cows for metabolic essential amino acids by factorial method. During the researches, developed standards needs to metabolic essential amino acids for lactating cows (g/kg) milk with a content of 3,3 % (33 g/kg) crude protein and 3,15 % (31,5 g/kg) of pure protein and to maintain 1 kg/body weight 0,75, which allows to calculate the daily requirement in the absolute number of amino acids (g). The assessment of security of rations of lactating cows metabolic lysine and methionine has been shown coincidence with the norms of needs in the amount of 93 %; daily milk yield of natural milk under this provision of lysine and methionine were 38,7 % and 39,4. The availability of metabolic methionine was at the level of 77-85 %, which is explained by the unprocessed coefficients of transformation of metabolic methionine into pure methionine of milk protein. In the total duodenal fl ow of amino acids approximately 60-70 % is microbial protein and 30-40 % is non-degradable protein in the rumen. It has been found that the digestibility of dry and organic matter in the rumen and the general digestive tract in relation to UDP:DP 35: 65 % crude protein is 2-3 % (absolute) higher than for UDP:DP = 50:50 % crude protein.

Bioorthogonal Non-canonical Amino Acid Tagging Combined With Flow Cytometry for Determination of Activity in Aquatic Microorganisms.

In this study, we have combined bioorthogonal non-canonical amino acid tagging (BONCAT) and flow cytometry (FCM) analysis, and we demonstrate the applicability of the method for marine prokaryotes. Enumeration of active marine bacteria was performed by combining the DNA stain SYBR Green with detection of protein production with BONCAT. After optimization of incubation condition and substrate concentration on monoculture of Escherichia coli, we applied and modified the method to natural marine samples. We found that between 10 and 30% of prokaryotes in natural communities were active. The method is replicable, fast, and allow high sample throughput when using FCM. We conclude that the combination of BONCAT and FCM is an alternative to current methods for quantifying active populations in aquatic environments.

Assessing amino acid utilization in young Nellore steers fed high-concentrate diets with different sources and levels of nitrogen

The aim of this study was to evaluate the availability and utilization of individual amino acids (AA) in young Nellore steers fed high-concentrate diets with different levels and sources of nitrogen (N). Six castrated Nellore steers, cannulated in the rumen, duodenum and ileum were randomly assigned in a 6 × 6 Latin square design arranged in a 2 × 3 factorial, which consisted of 3 protein sources (soybean meal plus urea (SU), corn gluten meal (CGM) and dry distillers grains (DDG)) and 2 dietary crude protein (CP) levels (110 and 140 g/kg dry matter). Arginine concentration in rumen microbial crude protein (MICP) was greater in steers fed diets containing DDG (P = 0.014). Histidine concentration in MICP was greater (P = 0.015) in steers fed CGM or DDG diets. Lysine concentration in MICP was greater (P = 0.050) as the protein level (PL) increased in the CGM diets. Steers fed diets containing DDG had greater NEAA flow (P = 0.049) and tended (P ≤ 0.08) to have greater non-essential AA (NEAA), arginine, leucine, lysine, proline and serine flow from MICP compared to those fed SU diets. Histidine and glutamate from MICP had a greater supply (P = 0.01) by the dietary inclusion of CGM or DDG. Steers fed CGM or DDG tended (P ≤ 0.07) to have greater EAA, arginine, isoleucine and valine supply from rumen undegraded protein (RUP). The Leucine supply from RUP was greater (P = 0.03) in the sources CGM or DDG. Increasing the protein level (PL) from 110 to 140 g/kg CP in SU diets reduced (P ≤ 0.04) the digestibility of isoleucine, leucine, and tyrosine. In contrast, the digestibility of leucine, methionine, aspartic acid, glutamate, glycine, serine, and tyrosine was improved (P ≤ 0.05) by the increase in the PL in DDG diets. Plasma leucine and phenylalanine concentration was greater (P ≤ 0.047) in steers fed diets containing CGM or DDG. Plasma leucine and valine concentration was greater (P ≤ 0.042) in steers fed diets containing 140 g/kg of CP. Arginine and histidine utilization were greater (P ≤ 0.013) in steers fed diets containing 110 g/kg CP with DDG and 140 g/kg CP with CGM. Steers fed the diet containing 110 g/kg CP and DDG showed greater (P ≤ 0.04) isoleucine, lysine, phenylalanine, threonine, valine, alanine, aspartic, glutamate, serine, and tyrosine use efficiency than those fed other diets, except for those fed the diet containing 140 g/kg CP with CGM which did not differ. The AA use efficiency is affected by dietary protein levels and sources. Our results suggest that it is possible to increase the supply of essential AA by the use of CGM or DDG (RUP sources).

Prediction of the total and standardized ileal digestible amino acid contents from the chemical composition of soybean meals of different origin in broilers

The objective of this experiment was to determine total amino acid (TAA) content, standardized ileal digestibility (SID) of crude protein, and standardized ileal amino acid digestibility in 9 sources of soybean meal (SBM) of different origin and to subsequently establish equations for predicting the TAA content and concentration of standardized ileal digestible amino acids (SIDAA) based on their protein content and other proximate components. Concentration of SIDAA of the samples was also predicted using TAA values. A total of 160 1-day-old male broiler chicks were randomly assigned to 10 dietary treatments consisted of 9 semipurified diets containing one SBM (200 g of crude protein/kg) as the only source of dietary amino acid (AA) and one N-free diet to determine endogenous ileal AA flow. The birds were fed with a standard diet from 0 to 18 D of age, and experimental diets were fed from 19 to 24 D of age. The fitness of the models of the study was tested using the adjusted coefficient of determination (R2) value, P-value regression and coefficients, and standard error of prediction (SEP). The coefficient of SID for Lys and Cys among SBM varied from 86.7 to 96.3 and 74.1 to 89.3, respectively, with significant difference (P < 0.05). In equations based on protein content, the adjusted R2 value ranged from 40.7 (Ile) to 99.6 (Met) and 37.2 (Met + Cys) to 99.6 (Met) for TAA content and concentration of SIDAA, respectively. Inclusion of other proximate components of test samples (e.g., crude fiber, neutral detergent fiber, acid detergent fiber, ash, gross energy, and so on) into the regression equation increased the adjusted R2 value and decreased the SEP. The results of linear regression revealed that it is possible to satisfactorily estimate the TAA content and concentration of SIDAA of SBM through its protein content and other proximate components, but the prediction equations based on other proximate components were more accurate in terms of reflecting the measured results; however, additional time and costs were associated with this approach. It is also possible to estimate the concentration of SIDAA through TAA values with reasonable accuracy and lower SEP.

Interaction between tannin and urea on nitrogen utilization by beef cattle grazing during the dry season

Tannins are secondary plant products that may reduce ammonia in the rumen and increase amino acid flow to intestine, but a reduction in ruminal N availability can limits its use for beef cattle fed low-quality tropical forage. Two studies, in vivo and in vitro, were carried out with the objective of evaluating the effects of the inclusion of a blend of condensed and hydrolysable tannins on protein-energy supplements (PES) with or without urea on intake, total nutrient digestibility and protein dietary efficiency of grazing beef cattle during the dry season. In the in vitro study, the effects of the interaction of urea and tannin in a 2 × 3 factorial arrangement of treatments were evaluated. Urea was included at 2% of the dry matter (DM) of the PES. Tannins were supplied at 0, 1 and 2 g/kg DM of the PES. The substrate was a mixture of forage and PES (50:50, w/w). In vitro DM and neutral detergent fiber (NDF) digestibility as well as ruminal pH and ammonia were measured. In the in vivo study, four rumen-cannulated Nellore bulls (470 ± 8 kg) were randomly distributed in a 4 × 4 Latin square design. The treatments were in a 2 × 2 factorial arrangement: PES with or without the inclusion of urea x PES with or without the addition of tannin. Urea was included at 2% of the DM of the PES. Tannins were supplied at 1 g/kg DM intake estimated, totalizing 10 g/animal/day. In the in vitro study, gas production decreased with the addition of urea (P < 0.001). It was observed an interaction between urea x tannins (P < 0.01) for in vitro DM and NDF digestibility at 24 h. Increasing tannin concentration, linearly decreased (P < 0.05) in vitro DM digestibility, and quadratically decreased (P < 0.05) in vitro NDF digestibility in the PES with urea. In the in vivo study, lower digestibility was observed (P < 0.01) for DM and organic matter with inclusion of urea in PES. The addition of tannins increased (P < 0.05) the digestibility of crude protein when urea was provided. Lower fecal nitrogen excretion was observed in the PES containing urea and tannin (P < 0.05). The results indicate that the combination of the tannin blend with urea modified the route of N utilization, lowering its excretion in the feces.

Casein hydrolysate supplementation in low-crude protein diets increases feed intake and nitrogen retention without affecting nitrogen utilization of growing pigs.

An extreme reduction of the crude protein (CP) level in diets, even balanced with amino acids (AAs), is detrimental for intestinal nitrogen (N) metabolism and the growth of pigs. This study investigated the effects of casein hydrolysate supplementation in low-CP diets on growth performance, N balance, and intestinal N supply for pigs. A total of 24 barrows were randomly assigned to one of three dietary treatments of 160 g kg-1 CP (control), 130 g kg-1 CP (LAA), and 130 g kg-1 CP plus casein hydrolysate (LCH) for 28 days. The LCH group had a higher average daily feed intake (ADFI) and average daily gain (ADG) than the LAA group, and a higher ADG than the control (P < 0.05). Compared with the control, both the LAA and LCH decreased N intake, serum urea N, fecal N, and N excretion, and increased apparent N availability, with LCH having higher N intake and N retention than LAA group (P < 0.05). Compared with LAA, LCH increased ileal fluxes of CP and AA (P < 0.05), and with values similar to those of the control. However, ileal flows of CP and AA were similar between LCH and LAA, both of which were lower than those in the control (P < 0.05). Using protein hydrolysate to replace some crystalline AAs in low-CP diets increased feed intake, N retention and ADG without affecting N utilization. These findings point to the important impact of protein hydrolysate supplementation on improving growth for pigs fed low-CP diets. © 2019 Society of Chemical Industry.

Intact and hydrolyzed casein lead to similar ileal endogenous protein and amino acid flows in adult humans

Endogenous amino acids (AAs) contribute to terminal ileal digesta and must be corrected for in determining coefficients of true ileal digestibility. Such estimates are also needed for the factorial calculation of AA requirements. The effect of the form of delivery of dietary AAs on endogenous nitrogen and AA flows at the end of the ileum was studied. Isotopically labeled 15N-casein (33-34 g), in either a hydrolyzed (HC) or intact (C) form, was included as the sole source of nitrogen in a mixed meal (320 mmol N) consumed by healthy adult humans equipped with a triple-lumen sampling tube in the small intestine. Ileal endogenous AA flows were determined by isotope dilution. An additional meal (A) containing a free AA mixture (306 mmol N) simulating the AA composition of casein was included. Serine was omitted from the AA mixture to allow direct determination of its ileal endogenous flow. Endogenous N and AA flows did not differ (P>0.05) for diets C and HC, with mean respective N flows of 728 and 617 mg/8 h (± pooled SD: 144 mg/8 h). Endogenous serine flow was similar (P>0.05) for diets C, HC, and A [181, 169, and 191 mg/8 h (± 56 mg/8 h)]. Recycling of the 15N marker was determined to be ∼11%, suggesting that the 15N endogenous loss values could underestimate endogenous protein and AA losses by ∼6% (the proportion of recycled 15N divided by the sum of endogenous N and recycled 15N). The 3 different forms of dietary AA delivery (free AAs, HC, or C) elicited similar ileal endogenous AA flows in the adult human.This study was registered at clinicaltrials.gov as NCT00873951.

Abstract 2402: GITR cancer immunotherapy: Epitope swapping of anti-GITR TRX518 to inform functional translatability from mouse to human

Abstract Introduction: At 52.5% identity in their extracellular domains (ECDs), mouse and human GITR (CD357) sequences are only modestly conserved. Thus transitioning promising murine anti-tumor activity data with the anti-GITR DTA-1 and 2F8 Mabs to clinical trials with the non-crossreactive anti-human GITR Mab TRX518 poses a considerable limitation. Here we investigated three elements of the GITR mouse and human antibodies to address this limitation: 1) the functional epitopes targeted by anti-human GITR (TRX-518) and anti-murine GITR (DTA-1 and 2F8) antibodies; 2) their biological activities on primary T cells; and 3) the role of anti-GITR Fc receptor engagement. Results and Conclusions: Alanine scanning, amino acid substitutions and flow cytometry experiments were performed to define critical residues important for the binding of all three Mabs to their respective GITR targets. Swaps of 5-8 of these critical residues showed both the loss of murine and the gain of human directed Mab binding when residues critical for TRX18 binding were substituted into murine GITR. Likewise, the substitution of residues critical for DTA-1/2F8 binding into human GITR resulted in loss of TRX518 binding and the gain of 2F8/DTA-1 binding. We then mapped these critical residues to 3D structural models of the murine and human GITR ECDs built by contact-map guided fragment assembly simulations. Regions defined by these critical residues showed a high degree of overlap, indicating structural conservation even without amino acid identity. Thus the epitopes defined by both DTA-1 and 2F8 are homologous to those defined on human GITR by TRX518. In in vitro standard proliferation/suppression assays with effector T cells (Teff) and regulatory T cells (Tregs) isolated from human healthy donors and naïve mice respectively, TRX518, similarly to DTA-1, significantly increased proliferation, activation and pro-inflammatory cytokine production of human and murine Teff cultured alone or in the presence of Tregs. These observations indicate that Mabs directed to similar mouse and human GITR ECD epitopes result in similar biological activities. Lastly, we questioned the importance of a functional Fc domain in anti-GITR Mabs in order to translate these in vitro effects into anti-tumor activity in vivo. To this end, we compared the anti-tumor activity of DTA-1 and its aglycosyl version in the aggressive and poorly immunogenic B16 melanoma model. Both Fc and non-Fc binding DTA-1 were able to control tumor growth. This suggests that the aglycosyl-anti-GITR Mab TRX518 has the potential to be effective in the clinic despite the lack of Fc receptor engagement. These results align with our preliminary results in patients treated with TRX518 monotherapy showing reductions in Treg frequencies both in tumor biopsies and in the periphery. TRX518 would therefore appear to be well-poised for use in the treatment of solid tumor malignancies. Note: This abstract was not presented at the meeting. Citation Format: Roberta Zappasodi, Heidi Heath, Yang Zhang, Michael Haas, Min Yang, Christopher Mirabelli, Jedd D. Wolchok, Cyndi Sirard, Walter Newman, Taha Merghoub. GITR cancer immunotherapy: Epitope swapping of anti-GITR TRX518 to inform functional translatability from mouse to human [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2402.

Emulsifying and structural properties of polysaccharides extracted from Chinese yam by an enzyme-assisted method

Enzyme-assisted extraction is a powerful technology for polysaccharides from plant materials. In the present investigation, the emulsifying properties of Chinese yam polysaccharides extracted by enzyme-assisted methods were determined and compared to the properties of polysaccharide fractions isolated by conventional hot water method. The structural properties of the various polysaccharide fractions were also investigated to understand their relationships with emulsifying properties. Results showed that the polysaccharide fractions extracted by papain hydrolysis exhibited relatively better emulsifying activities and stability. According to particle size distribution and gravitational phase separation experiments, PA12 exhibited the best emulsifying capacity and stability among the various polysaccharide fractions. All of the polysaccharide fractions differed from each other with respect to monosaccharide composition, free amino acid distribution, molecular weight, and flow behavior; these differences influenced the emulsifying properties of the polysaccharide fractions. The results obtained provide important information for choosing appropriate polysaccharide-based emulsifiers for food processing and other applications.

Placental transport of nutrients and its implications for fetal growth

Placental growth during early and mid-pregnancy has a powerful, constraining influence on fetal growth during late pregnancy. Studies involving surgical and environmental reduction of placental size in sheep have shown an associated reduction in capacity to transport oxygen, glucose and amino acids. Oxygen transport is limited by placental blood flow but transport of glucose and amino acids is determined by the abundance and activity of specific transport proteins. Glucose transporters include the GLUT1 and GLUT3 isoforms previously identified in brain and other tissues; systems for active transport of amino acids have been inferred but not characterized. Placental metabolism of glucose and amino acids has major effects both on the quantity of carbon and nitrogen delivered to the fetus, and on the composition of substrates involved. For example, the uteroplacental tissues consume more than 60% of uterine glucose uptake during late pregnancy, and the placenta substantially modifies the pattern of amino acids delivered to fetal blood. The placenta also participates in the array of metabolic adaptations of maternal and conceptus tissues to altered maternal nutrient supply. Placental capacity for glucose transport in moderately undernourished ewes is upregulated, partly by increased expression of the GLUT3 transport protein. During more severe glucose deprivation, placental transfer and fetal uptake of glucose are constrained in proportion with maternal supply, leading to fetal growth retardation.

The feasibility of bridge-grafting to restore the flow of sucrose in girdled Ocotea bullata and Curtisia dentata medicinal trees

Ocotea bullata (Burch.) Baill. (Lauraceae) and Curtisia dentata (Burm.f.) C.A.Sm. (Curtisiaceae) are two South African indigenous medicinal tree species of which the bark is overexploited due to its high demand in the local medicinal plant trade. The bark of O. bullata is used for headaches, as an emetic for both nervous and urinary disorders, and for diarrhea, whereas the bark of C. dentata is taken for stomach ailments, diarrhea and sexually transmitted diseases. Curtisia dentata bark is further applied as an aphrodisiac, and to “purify the blood”. Due to uncontrolled harvesting of bark by informal commercial harvesters, these trees are often found girdled in natural areas. The removal of bark from a tree affects its foliage, flowering, fruiting, seeding, basal sprouting and rooting since the removal of phloem tissue during girdling affects both the basipetal flow of carbohydrates from the canopy of the tree to the root system and the acropetal flow of amino acids, sugars and hormones from the roots to the canopy. This study investigated whether the flow of sucrose can be restored in girdled O. bullata and C. dentata trees using the technique of bridge-grafting. A total of 300 bark samples were collected bi-monthly over a 12-month period to perform targeted 1H NMR based metabolomic analyses to determine the sucrose concentration of each sample. Fluctuations in sucrose concentrations occurred at every sampling date, however, sucrose dynamics in the bridge-grafted trees appeared to be restored after one year.

Amino Acid Metabolism in Dairy Cows and their Regulation in Milk Synthesis.

Reducing dietary Crude Protein (CP) and supplementing with certain Amino Acids (AAs) has been known as a potential solution to improve Nitrogen (N) efficiency in dairy production. Thus understanding how AAs are utilized in various sites along the gut is critical. AA flow from the intestine to Portal-drained Viscera (PDV) and liver then to the mammary gland was elaborated in this article. Recoveries in individual AA in PDV and liver seem to share similar AA pattern with input: output ratio in mammary gland, which subdivides essential AA (EAA) into two groups, Lysine (Lys) and Branchedchain AA (BCAA) in group 1, input: output ratio > 1; Methionine (Met), Histidine (His), Phenylalanine (Phe) etc. in group 2, input: output ratio close to 1. AAs in the mammary gland are either utilized for milk protein synthesis or retained as body tissue, or catabolized. The fractional removal of AAs and the number and activity of AA transporters together contribute to the ability of AAs going through mammary cells. Mammalian Target of Rapamycin (mTOR) pathway is closely related to milk protein synthesis and provides alternatives for AA regulation of milk protein synthesis, which connects AA with lactose synthesis via α-lactalbumin (gene: LALBA) and links with milk fat synthesis via Sterol Regulatory Element-binding Transcription Protein 1 (SREBP1) and Peroxisome Proliferatoractivated Receptor (PPAR). Overall, AA flow across various tissues reveals AA metabolism and utilization in dairy cows on one hand. While the function of AA in the biosynthesis of milk protein, fat and lactose at both transcriptional and posttranscriptional level from another angle provides the possibility for us to regulate them for higher efficiency.