Sensitive Method To Analyze Cell Surface GPI-Anchored Proteins Using DNA Hybridization Chain Reaction-Mediated Signal Amplification.

GPI-anchored proteins (GPI-APs) are ubiquitous and essential but exist in low abundances on the cell surface, making their analysis and investigation especially challenging. To tackle the problem, a new method to detect and study GPI-APs based upon GPI metabolic engineering and DNA-facilitated fluorescence signal amplification was developed. In this context, cell surface GPI-APs were metabolically engineered using azido-inositol derivatives to introduce an azido group. This allowed GPI-AP coupling with alkyne-functionalized multifluorophore DNA assemblies generated by hybridization chain reaction (HCR). It was demonstrated that this approach could significantly improve the detection limit and sensitivity of GPI-APs, thereby enabling various biological studies, including the investigation of live cells. This new, enhanced GPI-AP detection method has been utilized to successfully explore GPI-AP engineering, analyze GPI-APs, and profile GPI-AP expression in different cells.

Vitamin and mineral supplementation to beef heifers during gestation: impacts on morphometric measurements of the neonatal calf, vitamin and trace mineral status, blood metabolite and endocrine profiles, and calf organ characteristics at 30 h after birth.

To examine the effects of feeding a vitamin and mineral supplement to beef heifers throughout gestation on mineral status and hormone/endocrine profiles in the dam and calf, and morphometric characteristics and organ mass of the calf at 30 h after birth, Angus-based heifers (n = 72, 14 to 15 mo of age, initial body weight [BW] = 380.4 ±â€…50.56 kg) were estrus synchronized and artificially inseminated (AI) with female-sexed semen. Heifers were blocked by BW and randomly assigned to receive either a basal diet (CON; n = 36) or a basal diet plus a vitamin and mineral supplement (VTM; n = 36) via an individual feeding system beginning at breeding, with both diets targeting BW gains of 0.45 kg heifer-1·d-1. Heifers not pregnant after the first AI (CON, n = 19; VTM, n = 18) were rebred via AI 60 d after treatment initiation, and heifers gestating female fetuses (CON, n = 7; VTM, n = 7) received treatments throughout gestation and were experimental units for this study. Calves were separated from their dams and fed colostrum replacer within 2 h of birth and euthanized 30 h after the first feeding. Calf morphometrics were recorded, and tissues were weighed and sampled. Serum from the dam at calving and serum, liver, and muscle from the calf at 30 h were analyzed for concentrations of minerals. Serum from the dam and calf were analyzed for concentrations of leptin, vitamins A, D, and E, cortisol, growth hormone, and insulin-like growth factor 1. All response variables were analyzed using the MIXED procedure of SAS. Calf body morphometrics and BW of the dam at calving (P ≥ 0.32), calf organ weights (P ≥ 0.21), and calf ovarian follicle counts (P ≥ 0.13) were not affected by maternal treatment. Concentrations of Se and Co in calf serum and Se in calf liver were increased (P ≤ 0.02) in VTM. Serum concentrations of Co and vitamin A in the dam were greater (P ≤ 0.01) in supplemented compared with nonsupplemented dams, and serum concentrations of vitamin D were greater (P ≤ 0.0003) in supplemented dams and calves compared with the nonsupplemented cohort. Maternal supplementation supported vitamin and mineral status in the neonate, yet had no discernable impact on BW, organ mass, or circulating hormones/metabolites in the calf. Evaluating offspring at later postnatal time points is warranted to determine if prenatal vitamin and mineral supplementation affects performance, health, metabolism, and efficiency of energy utilization in key metabolic tissues in the calf.

Vitamins and minerals are essential for the reproduction, performance, skeletal support, and overall health of beef cattle. During pregnancy, vitamins and minerals are critical for proper fetal growth, development, and establishment of postnatal micronutrient reserves. The study objectives were to evaluate the impacts of vitamin and mineral supplementation to beef heifers throughout gestation on female offspring morphometric characteristics at birth, mineral status and blood metabolite/endocrine profiles of the dam and calf, histological evaluation of calf ovaries, and organ weights of the neonate at 30 h of age. We hypothesized that vitamin and mineral supplementation to the dam during pregnancy would increase calf size and organ masses, mineral status, and blood metabolite and hormone profiles. We observed no differences in calf body measurements, organ masses, and offspring ovarian reserve between calves from supplemented and nonsupplemented dams. However, Co, Se, and vitamin D status was increased in the supplemented dam and calf, and we propose that enhanced vitamin and mineral status at birth may support the underdeveloped immune system, growth performance, and overall health of the neonate in the postnatal period. Further research is warranted to investigate postnatal offspring health, performance, and efficiency of energy utilization in key metabolic tissues in the calf.

Influence of vasoactive intestinal polypeptide on growth performance, nutrient digestibility, nitrogen balance, and digestive enzyme activity in lambs.

This study evaluated if vasoactive intestinal polypeptide (VIP) influences growth performance, nutrient digestibility, nitrogen balance, and digestive enzyme activity. Sixteen wether lambs (69.6 ±â€…1.9 kg) were housed in individual pens, adapted to a corn grain-based diet, and randomly assigned to 2 treatment groups. Lambs were injected intraperitoneally every other day for 28 d with saline (0.9% NaCl) containing no VIP (n = 8; control) or containing VIP (n = 8; 1.3 nmol/kg body weight [BW]). All lambs were transferred to individual metabolic crates for the final 7 d of the experiment to measure nitrogen balance and nutrient digestibility. At the end of the treatment period, lambs were slaughtered, and pancreatic tissue, small intestinal tissue, and rumen fluid were collected for protein, digestive enzymes, ruminal pH, and volatile fatty acid (VFA) analyses. Lambs treated with VIP had greater final BW, average daily gain, and gain:feed (P = 0.01, 0.05, 0.03, respectively). No differences between treatment groups were observed (P ≥ 0.25) for nutrient intake, digestibility, nitrogen retention, ruminal pH, and VFA concentrations. Moreover, VIP treatment did not influence (P ≥ 0.19) plasma glucose, urea N, and insulin concentrations. Treatment with VIP increased (P = 0.03) relative cecum weight (g/kg BW) and decreased (P = 0.05) relative brain weight. Pancreatic and intestinal digestive enzyme activities, except for duodenal maltase (P = 0.02), were not influenced (P ≥ 0.09) by VIP treatment. These data suggest that the administration of VIP may have potential to improve average daily gain and gain:feed in lambs fed grain-based diets.

This research explored the influence of vasoactive intestinal polypeptide (VIP), an anti-inflammatory mediator, in lambs fed a high-concentrate finishing diet on growth performance, nutrient digestibility, nitrogen balance, and digestive enzyme activity. Wether lambs were fed a whole corn grain-based diet containing no added forage and randomly assigned to either the VIP or control group. Lambs received intraperitoneal saline injections with or without VIP every second day over a 28-d treatment period. Average daily gain and gain:feed ratio was positively influenced by VIP. However, treatment did not affect dry matter intake, nitrogen balance, nutrient digestibility, and digestive enzyme activity. These data indicate exogenous VIP treatment may influence growth in lambs fed a high-concentrate diet.

A single intranasal dose of essential oil spray confers modulation of the nasopharyngeal microbiota and short-term inhibition of Mannheimia in feedlot cattle: a pilot study.

Five essential oils (EOs) were previously characterized in vitro and identified as candidate EOs for the development of an intranasal EO spray to mitigate bovine respiratory disease (BRD) pathogens. In the present study, these EOs were evaluated for their potential to (i) reduce BRD pathogens, (ii) modulate nasopharyngeal microbiota, and (iii) influence animal performance, feeding behavior and immune response when a single dose administered intranasally to feedlot cattle. Forty beef steer calves (7-8 months old, Initial body weight = 284 ± 5 kg [SE]) received either an intranasal EO spray (ajowan, thyme, fennel, cinnamon leaf, and citronella) or PBS (Control; n = 20/group) on day 0. Deep nasopharyngeal swabs were collected on days (d) -1, 1, 2, 7, 14, 28, and 42 and processed for 16S rRNA gene sequencing, qPCR, and culturing. Significant effects of EO on community structure (d1), microbial richness and diversity, relative abundance of some dominant phyla (d1, d2, and d14), and the overall interaction network structure of the nasopharyngeal microbiota were detected. The relative abundance of Mannheimia was lower in the EO calves (4.34%) than in Control calves (10.4%) on d2, and M. haemolytica prevalence on d7 as compared to control calves. Feed intake, average daily gain, feeding behavior, and blood cell counts were not affected by EO treatment. Overall, a single intranasal dose of EO spray resulted in moderate modulation of nasopharyngeal microbiota and short-term inhibition of Mannheimia while not influencing animal performance, feeding behavior or immune response. Our study, for the first time, shows the potential use of intranasal EO to mitigate BRD in feedlot cattle.

Holistic View and Novel Perspective on Ruminal and Extra-Gastrointestinal Methanogens in Cattle.

Despite the extensive research conducted on ruminal methanogens and anti-methanogenic intervention strategies over the last 50 years, most of the currently researched enteric methane (CH4) abatement approaches have shown limited efficacy. This is largely because of the complex nature of animal production and the ruminal environment, host genetic variability of CH4 production, and an incomplete understanding of the role of the ruminal microbiome in enteric CH4 emissions. Recent sequencing-based studies suggest the presence of methanogenic archaea in extra-gastrointestinal tract tissues, including respiratory and reproductive tracts of cattle. While these sequencing data require further verification via culture-dependent methods, the consistent identification of methanogens with relatively greater frequency in the airway and urogenital tract of cattle, as well as increasing appreciation of the microbiome-gut-organ axis together highlight the potential interactions between ruminal and extra-gastrointestinal methanogenic communities. Thus, a traditional singular focus on ruminal methanogens may not be sufficient, and a holistic approach which takes into consideration of the transfer of methanogens between ruminal, extra-gastrointestinal, and environmental microbial communities is of necessity to develop more efficient and long-term ruminal CH4 mitigation strategies. In the present review, we provide a holistic survey of the methanogenic archaea present in different anatomical sites of cattle and discuss potential seeding sources of the ruminal methanogens.

Timing of maternal nutrient restriction during mid- to late-gestation influences net umbilical uptake of glucose and amino acids in adolescent sheep.

Previous research demonstrated that maternal nutrient restriction during mid- to late-gestation influenced net umbilical uptakes of glucose and amino acids in sheep. However, it is unclear how the timing and duration of nutrient restriction during mid- to late-gestation influences net uterine, uteroplacental, and fetal flux of glucose and amino acids. On day 50 of gestation, 41 adolescent ewe lambs carrying singletons were randomly assigned to one of six dietary treatments: 1) 100% of nutrient requirements from days 50 to 90 of gestation (CON; n = 7); 2) 60% of nutrient requirements (RES; n = 7) from days 50 to 90 of gestation; 3) 100% of nutrient requirements from days 50 to 130 of gestation (CON-CON; n = 6); 4) 100% of nutrient requirements from days 50 to 90 of gestation and 60% of nutrient requirements from days 90 to 130 of gestation (CON-RES; n = 7); 5) 60% of nutrient requirements from days 50 to 90 of gestation and 100% of nutrient requirements from days 90 to 130 of gestation (RES-CON; n = 7); or 6) 60% of nutrient requirements from days 50 to 130 of gestation (RES-RES; n = 7). On day 90 (n = 14) and day 130 (n = 27), intraoperative procedures were performed to evaluate uteroplacental blood flows, collect blood samples, and then ewes were euthanized. Net uterine, uteroplacental, and umbilical fluxes of glucose and amino acids were calculated by multiplying blood flow by the arterial-venous concentration difference. Data from days 90 and 130 were analyzed separately using ANOVA in SAS. Maternal nutrient restriction during mid-gestation increased (P = 0.04) net umbilical glucose uptake but, maternal nutrient restriction during late-gestation decreased (P = 0.02) net umbilical glucose uptake. Net umbilical essential amino acid uptake decreased (P = 0.03) with nutrient restriction during mid-gestation; however, net umbilical uptakes of Phe (P = 0.02), Thr (P = 0.05), Met (P = 0.09), and His (P = 0.08) increased or tended to increase after nutrient restriction during late-gestation. These data demonstrate that net umbilical glucose and amino acid uptakes were influenced by the timing of nutrient restriction during mid- to late-gestation. Elevated net umbilical glucose uptake after mid-gestational nutrient restriction was sustained throughout late-gestation, independent of late-gestational feeding level. Long-term adaptations in umbilical glucose uptake may have implications for prenatal and postnatal growth and development of the offspring.

Maternal undernutrition during gestation can lead to decreased fetal growth, decreased uteroplacental blood flow, and changes in nutrient supply to the fetus. However, it is unclear how the timing (mid-gestation vs. late-gestation) and duration (40 d vs. 80 d) of nutrient restriction influence nutrient supply to the fetus during mid- to late-gestation. Pregnant ewe lambs fed a pelleted diet to meet 100% of nutritional requirements or 60% of nutritional requirements during mid-gestation alone (days 50 to 90) or during mid- and late-gestation (days 50 to 130). At the end of mid-gestation and late-gestation, the net nutrient supply between the maternal, placental, and fetal compartments was measured. The results indicated that the timing of nutrient restriction influenced the net nutrient supply to the fetus but, the duration of nutrient restriction did not. Nutrient restriction during mid-gestation increased glucose to the fetus but nutrient restriction during late-gestation decreased glucose to the fetus. The opposite response occurred for fetal essential amino acid supply where nutrient restriction during mid-gestation decreased essential amino acid supply to the fetus but increased for several essential amino acids during late-gestational nutrient restriction. The timing of maternal undernutrition during mid- to late-gestation can affect the amount of nutrients delivered to the fetus and thus, could potentially impact postnatal growth and development.

Genetic Distinctness and Diversity of American Aberdeen Cattle Compared to Common Beef Breeds in the United States.

American Aberdeen (AD) cattle in the USA descend from an Aberdeen Angus herd originally brought to the Trangie Agricultural Research Centre, New South Wales, AUS. Although put under specific selection pressure for yearling growth rate, AD remain genomically uncharacterized. The objective was to characterize the genetic diversity and structure of purebred and crossbred AD cattle relative to seven common USA beef breeds using available whole-genome SNP data. A total of 1140 animals consisting of 404 purebred (n = 8 types) and 736 admixed individuals (n = 10 types) was used. Genetic diversity metrics, an analysis of molecular variance, and a discriminant analysis of principal components were employed. When linkage disequilibrium was not accounted for, markers influenced basic diversity parameter estimates, especially for AD cattle. Even so, intrapopulation and interpopulation estimates separate AD cattle from other purebred types (e.g., Latter's pairwise FST ranged from 0.1129 to 0.2209), where AD cattle were less heterozygous and had lower allelic richness than other purebred types. The admixed AD-influenced cattle were intermediate to other admixed types for similar parameters. The diversity metrics separation and differences support strong artificial selection pressures during and after AD breed development, shaping the evolution of the breed and making them genomically distinct from similar breeds.

Valproate regulates inositol synthesis by reducing expression of myo-inositol-3-phosphate synthase.

Inositol depletion is a hypothesized mechanism of action of mood stabilization drugs used in the treatment of bipolar disorder. It was previously reported that the mood stabilizer valproate (VPA) increased phosphorylation of myo-inositol-3-phosphate synthases (MIPS), the rate limiting enzyme of inositol synthesis. Phosphosites were identified and examination of site-directed mutants suggested that phosphorylation leads to decreased enzymatic activity. In this study, we examined the extent of MIPS phosphorylation in response to VPA and used two interaction screens to identify protein kinases that interact with MIPS. Using an epitope tagged MIPS construct, we determined the fraction of phosphorylated MIPS to be very low (less than 2% of total), and we could not detect phosphorylation of untagged MIPS in response to VPA. In vitro analyses of phosphorylation revealed that putative protein kinases, PKC and CKII, have low specificity toward MIPS. These findings suggest that VPA likely depletes inositol via a mechanism other than MIPS phosphorylation. Consistent with this, mRNA levels of the MIPS-encoding gene INO1 and MIPS protein levels were significantly reduced during the mid-log growth phase in response to VPA treatment. These findings suggest that the mechanism whereby VPA causes inositol depletion is by reducing expression of the rate-limiting enzyme MIPS.

Postruminal Casein Infusion and Exogenous Glucagon-Like Peptide 2 Administration Differentially Stimulate Pancreatic α-Amylase and Small Intestinal α-Glucosidase Activity in Cattle.

BACKGROUND: Increasing luminal carbohydrate flow decreases pancreatic α-amylase activity but can increase jejunal maltase activity, suggesting that regulation of carbohydrase activity is perhaps uncoordinated in response to luminal carbohydrate flow. Increasing luminal casein flow increases pancreatic α-amylase activity in cattle, and exogenous glucagon-like peptide 2 (GLP-2) has been shown to increase small intestinal α-glucosidase activity in nonruminants. OBJECTIVES: The objective was to evaluate the effects of postruminal casein infusion, exogenous GLP-2, or their combination on endogenous pancreatic and small intestinal carbohydrase activity in cattle postruminally infused with starch. METHODS: Holstein steers [n = 24; 250 ± 23 kg body weight (BW)] received a continuous abomasal infusion of 3.94 g raw corn starch/kg of BW combined with either 0 or 1.30 g casein/kg of BW. Steers received subcutaneous injections in 2 equal portions daily of excipient (0.5% bovine serum albumin) or 100 µg GLP-2/kg of BW per day. At the end of the 7-d treatment period, steers were slaughtered for tissue collection. Data were analyzed using the MIXED procedure of SAS version 9.4 (SAS Institute Inc.). RESULTS: Postruminal casein infusion increased (P ≤ 0.03) pancreatic mass by 12.6%, total pancreatic α-amylase activity by 50%, and postruminal starch disappearance from 96.7% to 99.3%. Exogenous GLP-2 increased (P < 0.01) total small intestinal and mucosal mass by 1.2 kg and 896 g, respectively. Relative to control, GLP-2 and casein + GLP-2 increased (P = 0.04) total small intestinal α-glucosidase activity by 83.5%. Total small intestinal maltase, isomaltase, and glucoamylase activity was 90%, 100%, and 66.7% greater for GLP-2 and casein + GLP-2 steers compared with control. CONCLUSIONS: Casein increased pancreatic α-amylase activity, GLP-2 increased small intestinal α-glucosidase activity, and the combination of casein and GLP-2 increased both pancreatic α-amylase activity and small intestinal α-glucosidase activity. This novel approach provides an in vivo model to evaluate effects of increasing endogenous carbohydrase activity on small intestinal starch digestion.

Whole-body microbiota of newborn calves and their response to prenatal vitamin and mineral supplementation.

Early life microbial colonization and factors affecting colonization patterns are gaining interest due to recent developments suggesting that early life microbiome may play a role in Developmental Origins of Health and Disease. In cattle, limited information exists on the early microbial colonization of anatomical sites involved in bovine health beyond the gastrointestinal tract. Here, we investigated 1) the initial microbial colonization of seven different anatomical locations in newborn calves and 2) whether these early life microbial communities and 3) serum cytokine profiles are influenced by prenatal vitamin and mineral (VTM) supplementation. Samples were collected from the hoof, liver, lung, nasal cavity, eye, rumen (tissue and fluid), and vagina of beef calves that were born from dams that either received or did not receive VTM supplementation throughout gestation (n = 7/group). Calves were separated from dams immediately after birth and fed commercial colostrum and milk replacer until euthanasia at 30 h post-initial colostrum feeding. The microbiota of all samples was assessed using 16S rRNA gene sequencing and qPCR. Calf serum was subjected to multiplex quantification of 15 bovine cytokines and chemokines. Our results indicated that the hoof, eye, liver, lung, nasal cavity, and vagina of newborn calves were colonized by site-specific microbiota, whose community structure differed from the ruminal-associated communities (0.64 ≥ R2 ≥ 0.12, p ≤ 0.003). The ruminal fluid microbial community was the only one that differed by treatment (p < 0.01). However, differences (p < 0.05) by treatment were detected in microbial richness (vagina); diversity (ruminal tissue, fluid, and eye); composition at the phylum and genus level (ruminal tissue, fluid, and vagina); and in total bacterial abundance (eye and vagina). From serum cytokines evaluated, concentration of chemokine IP-10 was greater (p = 0.02) in VTM calves compared to control calves. Overall, our results suggest that upon birth, the whole-body of newborn calves are colonized by relatively rich, diverse, and site-specific bacterial communities. Noticeable differences were observed in ruminal, vaginal, and ocular microbiota of newborn calves in response to prenatal VTM supplementation. These findings can derive future hypotheses regarding the initial microbial colonization of different body sites, and on maternal micronutrient consumption as a factor that may influence early life microbial colonization.

Feeding hempseed cake alters the bovine gut, respiratory and reproductive microbiota.

A growing number of studies have investigated the feasibility of utilizing hemp by-products as livestock feedstuffs; however, their impact on livestock microbiomes remains unexplored. Here, we evaluated the effects of feeding hempseed cake on the gastrointestinal, respiratory, and reproductive microbiota in beef heifers. Angus-crossbred heifers (19-months old, initial body weight = 494 ± 10 kg [SE]) were fed a corn-based finishing diet containing 20% hempseed cake as a substitute for 20% corn dried distillers' grains with solubles (DM basis; Control; n = 16/group) for 111 days until slaughter. Ruminal fluid and deep nasopharyngeal swabs (days 0, 7, 42, 70 and 98), and vaginal and uterine swabs (at slaughter) were collected, and the microbiota assessed using 16S rRNA gene sequencing. Diet affected the community structure of the ruminal (d 7-98; 0.06 ≤ R2 ≤ 0.12; P < 0.05), nasopharyngeal (d 98; R2 = 0.18; P < 0.001), and vaginal (R2 = 0.06; P < 0.01) microbiota. Heifers fed hempseed cake had increased microbial diversity in the rumen, reduced microbial richness in the vagina, and greater microbial diversity and richness in the uterus. In addition to the distinct microbial communities in the rumen, nasopharynx, vagina and uterus, we identified 28 core taxa that were shared (≥ 60% of all samples) across these sampling locations. Feeding hempseed cake appeared to alter the bovine gut, respiratory and reproductive microbiota. Our results suggest that future research aiming to evaluate the use of hemp by-products in livestock diet should consider their impact on animal microbiome and microbiome mediated animal health and reproductive efficiency. Our findings also highlight the need for research evaluating the impact of hemp-associated food and personal care products on the human microbiome.

Excretion and residue depletion of cannabinoids in beef cattle fed hempseed cake for 111 days.

Thirty-two crossbred heifers were fed either a control diet or 20% (dry matter basis) hempseed cake in a complete ration for 111 days; of the cattle fed hempseed cake, four each were harvested with 0, 1, 4, and 8-day withdrawal periods. Urine and plasma were collected during the feeding and withdrawal periods and liver, kidney, skeletal muscle, and adipose tissue were collected at harvest. Total cannabinoid (n = 10) concentration of hempseed cake averaged 11.3 ± 11.7 mg kg-1 across the feeding period with total cannabidiol and tetrahydrocannabinol (CBD/THC) concentrations of 1.3 ± 0.8 mg kg-1. Neutral cannabinoids (cannabinol [CBN], CBD/THC, and cannabidivarin [CBDV]) were not detected in plasma or urine, but CBD/THC was measured in adipose tissue at all withdrawal periods (6.3 ± 2.1 to 10.1 ± 2.5 ng g-1). In contrast, cannabinoid acids (cannabinolic acid [CBNA], cannabidiolic acid [CBDA]/tetrahydrocannabinolic acid [THCA], cannabichromenic acid [CBCA], and cannabidivarinic acid [CBDVA]) were sporadically detected (<15 ng mL-1) in plasma and urine of cattle fed hempseed cake. Cannabinoid acids were depleted from liver by withdrawal day 4, but could still be measured (<1 ng g-1) in kidney of some animals harvested on withdrawal day 8. Assessment of human exposures to CBD/THC residues through the consumption of beef fat from animals fed hempseed cake suggests that the probability of consuming the equivalent of an acute reference dose (ARfD) is remote, even with the use of a conservative reference dose ARfD (1 µg kg-1 body weight).

Labeling cell surface glycosylphosphatidylinositol-anchored proteins through metabolic engineering using an azide-modified phosphatidylinositol.

Glycosylphosphatidylinositol (GPI) anchorage is one of the most common mechanisms to attach proteins to the plasma membrane of eukaryotic cells. GPI-anchored proteins (GPI-APs) play a critical role in many biological processes but are difficult to study. Here, a new method was developed for the effective and selective metabolic engineering and labeling of cell surface GPI-APs with an azide-modified phosphatidylinositol (PI) as the biosynthetic precursor of GPIs. It was demonstrated that this azido-PI derivative was taken up by HeLa cells and incorporated into the biosynthetic pathway of GPIs to present azide-labeled GPI-APs on the live cell surface. The azido group was used as a molecular handle to install other labels through a biocompatible click reaction to enable various biological studies, e.g., fluorescent imaging and protein pull-down, which can help explore the functions of GPI-APs and discover new GPI-APs.

Effects of hempseed cake on ruminal fermentation parameters, nutrient digestibility, nutrient flow, and nitrogen balance in finishing steers.

Five ruminally and duodenally cannulated red angus steers (n = 5; initial body weight [BW] = 542 kg, SD = 40 kg) were used in a three-period Youden square design consisting of three 21-d periods, three treatments, and five steers (one or two steers per treatment within each period) to evaluate the effect of feeding hempseed cake on ruminal fermentation parameters, organic matter (OM) intake, total tract nutrient digestion, and nitrogen (N) balance in steers fed finishing diets. The control (CON) diet contained 75% dry-rolled corn, 20% corn silage, and 5% supplement (DM basis). The dried corn distillers grains plus solubles (DDGS) and hempseed cake (HEMP) diets contained 55% dry-rolled corn, 20% corn silage, 20% dried corn distillers grains plus solubles or hempseed cake, and 5% supplement (DM basis). Total ruminal volatile fatty acid concentration was greater (P < 0.01) in steers fed the HEMP diet than in steers fed the DDGS or CON diets. Ruminal fluid pH was not influenced (P = 0.93) by treatment. Organic matter intake tended (P = 0.07) to be greater and OM total tract digestibility was less (P = 0.03) in steers fed the HEMP diet compared with steers fed the DDGS or CON diets. Ruminal true and total tract apparent N digestibility was greater (P < 0.01) in steers fed the HEMP diet than steers fed the DDGS or CON diets. Duodenal flow of essential, nonessential, and total amino acids was not influenced (P ≥ 0.37) by dietary treatment, but the lack of response was likely because ruminally degradable protein (RDP) supply exceeded the RDP requirement. Steers fed the HEMP diet had greater (P < 0.01) N retention (g/d) than steers fed the DDGS diet, which was greater (P < 0.01) than steers fed the CON diet, suggesting that feeding hempseed cake improved utilization of N. Although inclusion of hempseed cake decreased total tract OM digestibility compared with dried corn distillers grains or corn, improvements in N utilization suggest that hempseed cake could be a useful alternative feed ingredient for finishing cattle diets.

This experiment evaluated the effects of dietary inclusion of hempseed cake or dried corn distillers grains plus solubles on ruminal fermentation parameters, organic matter (OM) intake, total tract nutrient digestion, and nitrogen (N) balance in steers fed finishing diets. Steers were assigned to one of three dietary treatments (no byproduct [CON], 20% dried corn distillers grains plus solubles [DDGS], or 20% hempseed cake [HEMP]; dry matter basis). Hempseed cake had greater acid detergent fiber concentrations, which resulted in greater acid detergent fiber flow to the small intestine and reduced total tract organic matter digestibility in steers fed the HEMP diet than in steers fed the DDGS or CON diets. Steers fed the HEMP diet had greater ruminal and total tract N digestibility, and greater ruminal ammonia concentrations than steers fed DDGS or CON diets, suggesting that the crude protein in hempseed cake is degraded to a greater extent in the rumen and total tract. Although inclusion of hempseed cake decreased total tract OM digestibility compared with dried corn distillers grains or no byproduct, the observed greater ruminal and total tract N digestibility suggest that it could be a useful alternative feed ingredient for finishing cattle diets.

Effects of long-term postgastric infusion of casein or glutamic acid on small intestinal starch digestion and energy balance in cattle.

The objective of this experiment was to evaluate effects of postruminal flows of casein or glutamic acid on small intestinal starch digestion and to quantify changes in energy and nutrient balance. Twenty-four steers (body weight = 179 ± 4 kg) were duodenally infused with raw cornstarch (1.46 ± 0.04 kg/d) and either 413 ± 7.0 g casein/d, 121 ± 3.6 g glutamic acid/d or water (control). Measures of small intestinal starch digestion and nutrient excretion were collected across 4 d after 42 d of infusion and measures of respiration via indirect calorimetry were collected across 2 d after 48 d of infusion. Ileal starch flow was least among calves provided casein, but ileal starch flow was not different between glutamic acid or control. Small intestinal starch digestion tended to be greatest among calves provided casein, least for glutamic acid and intermediate for control. Casein increased ileal flow of ethanol soluble oligosaccharides compared to glutamic acid and control. Large intestinal starch digestion was not different among treatments. By design, N intake was greatest among cattle provided casein, intermediate among calves provided glutamic acid and least for control. Nitrogen retention was greater in response to casein compared to control and glutamic acid. Intake of gross energy from feed was similar across treatments, and gross energy from infusate was greatest for casein, intermediate for glutamic acid and least for control. Variation in gross energy intake from feed resulted in no difference in overall gross energy intake across treatments. Similar to measures of small intestinal starch digestion and N retention, casein increased calories of digestible energy and metabolizable energy, compared to glutamic acid and control, which did not differ. Postruminal infusions did not influence methane production, but heat production was greatest in steers infused with casein, intermediate for steers provided glutamic acid, and least for control. Overall, amounts of energy retained by casein tended to be nearly 34% greater than control, but glutamic acid had no impact on energy balance. Improvement in small intestinal starch digestion in response to casein increased energy and N retained; however, glutamic acid did not influence small intestinal starch digestion and energy or N balance in cattle, which seems to suggest that responses in small intestinal starch digestion to greater postruminal flows of glutamic acid become refractory across greater durations of time.

Small intestinal digestion of starch can provide greater energy from dietary starch in comparison to ruminal fermentation, which can increase performance and improve feed efficiency in cattle fed starch-based diets; however, small intestinal starch digestion in cattle is restricted in comparison to nonruminant animals or to ruminal fermentation of starch. In this experiment, long-term increases in postruminal protein but not glutamic acid increased small intestinal starch digestion, and increases in small intestinal starch digestion resulted in improvements in nitrogen and energy balance. Responses in this study compared to previous reports may suggest effects of increased postgastric flows of glutamic acid in cattle are transient, but that effects of casein are not.

Profiling Glycosylphosphatidylinositol (GPI)-Interacting Proteins in the Cell Membrane Using a Bifunctional GPI Analogue as the Probe.

Glycosylphosphatidylinositol (GPI) anchorage of cell surface proteins to the membrane is biologically important and ubiquitous in eukaryotes. However, GPIs do not contain long enough lipids to span the entire membrane bilayer. To transduce binding signals, GPIs must interact with other membrane components, but such interactions are difficult to define. Here, a new method was developed to explore GPI-interacting membrane proteins in live cell with a bifunctional analogue of the glucosaminylphosphatidylinositol motif conserved in all GPIs as a probe. This probe contained a diazirine functionality in the lipid and an alkynyl group on the glucosamine residue to respectively facilitate the cross-linkage of GPI-binding membrane proteins with the probe upon photoactivation and then the installation of biotin to the cross-linked proteins via a click reaction for affinity-based protein isolation and analysis. Profiling the proteins pulled down from the Hela cells revealed 94 unique and 18 overrepresented proteins compared to the control, and most of them are membrane proteins and many are GPI-related. The results have proved not only the concept of using the new bifunctional GPI probe to investigate GPI-binding membrane proteins but also the important role of inositol in the biological functions of GPI anchors and GPI-anchored proteins.

Synthesis of a Bifunctionalized Glycosylphosphatidylinositol (GPI) Anchor Useful for the Study of GPI Biology.

A new, bifunctional glycosylphosphatidylinositol (GPI) derivative containing the highly conserved core structure of all natural GPI anchors with a photoactivable diazirine in the lipid chain and clickable alkynes in the glycan was synthesized by a convergent [3+2] glycosylation strategy with late stage protecting group manipulation and regioselective phosphorylation. The challenges of this synthesis were due to the presence of several distinctive functional groups in the synthetic target, which complicated the protection tactics, in addition to the inherent difficulties associated with GPI synthesis. This bifunctional GPI derivative can cross-react with molecules in proximity upon photoactivation and be subsequently labeled with other molecular tags via click reaction. Therefore, it should be a valuable probe for biological studies of GPIs, such as analysis of GPI-interacting membrane proteins, and gaining insights into their functional mechanisms.

Understanding the relationship between weather variables and intake in beef steers.

The relationship between weather variables and dry matter intake (DMI) in beef steers was examined using daily intake data from 790 beef steers collected through a computer-controlled feeding system in nonsummer months. Daily data were condensed into weekly averages (N = 13,895 steer-weeks). The variables considered to predict DMI (2.50 to 23.60 kg/d) were body weight (197 to 796 kg), dietary net energy for maintenance (NEm; 0.79 to 2.97 Mcal/kg), ambient temperature (-23.73 °C to 21.40 °C), range of temperature (2.79 °C to 19.43 °C), dew point (-27.84 °C to 14.34 °C), wind speed (2.08 to 6.49 m/s), solar radiation (30.8 to 297.1 W/m2), and 2-wk lag (average of previous 2 wk's values) and monthly lag (average of previous 4 wk's values) of each weather variable. Toeplitz variance-covariance structure for repeated measures was used to determine the model to predict DMI, while accounting for the effects of body weight, dietary NEm, and other variables in the model. Two-week lag of ambient temperature interacted (P ≤ 0.005) with 2-wk lag of range of temperature, monthly lag of wind speed, 2-wk lag of solar radiation, and dew point to predict DMI. Interactions (P = 0.0001) between 2-wk lag of range of temperature vs. dew point and monthly lag of wind speed vs. 2-wk lag of solar radiation were also detected. This study reports important weather variables associated with differences in DMI of growing and finishing steers and will help improve the accuracy of DMI prediction equations for beef cattle. Improvements in the accuracy of predicting DMI should give producers better tools to plan and execute efficient feeding management programs. The R2 of the overall model was 0.8891.

Dry matter intake (DMI) models for beef cattle in the Northern Great Plains may not be a good fit because of extreme weather conditions experienced in this region. The objective of this study is to include additional weather variables (temperature, dewpoint, wind speed, range of temperature, and solar radiation as well as 2-wk lag and monthly lag for each weather variable) that may influence DMI models to improve accuracy. Intake data (13,895 observations) collected from 790 beef steers using an automatic feeding system from 2011 to 2018 was utilized. It is well-established that body weight and dietary energy density influences DMI in cattle, therefore, both were included in the base model. Weather variables that contributed the most to the model were 2-wk lag of range of temperature, ambient temperature, and solar radiation. There were also two-way interactions between most of the weather variables. This study shows that weather variables interact and current DMI models should account for these interactions. Ultimately, this will improve DMI models in the Northern Great Plains.

Design and synthesis of 4-azido-phosphatidylinositol as a potential probe for metabolic engineering of glycosylphosphatidylinositol on cells.

A diacyl phosphatidylinositol (PI) derivative with an azide linked to its inositol C4-position was effectively synthesized in 19 steps for the longest linear sequence and in a ca. 1% overall yield from 1,2-distearoyl-sn-glycerol and D-glucose. This compound was designed as a biosynthetic precursor of glycosylphosphatidylinositol (GPI) anchors. Its azide would enable further modification to introduce other molecular tags by biocompatible click reaction. Therefore, it can be a useful probe for metabolic engineering of cell surface GPI anchors and GPI-anchored proteins.