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[This corrects the article DOI: 10.1371/journal.pone.0307678.].

An approach for the agnostic identification and validation of aptamers for the prediction of a medical state from plasma analysis is presented in application to a key risk factor for Alzheimer’s disease. brain amyloid deposition. This method involved the use of a newly designed aptamer library with sixteen random nucleotides interspersed with fixed sequences called a Neomer library. The Neomer library approach enables the direct application of the same starting library on multiple plasma samples, without the requirement for pre-enrichment associated with the traditional approach. Eight aptamers were identified as a result of the selection process and screened across 390 plasma samples by qPCR assay. Results were analysed using multiple machine learning algorithms from the Scikit-learn package along with clinical variables including cognitive status, age and sex to create predictive models. An Extra Trees Classifier model provided the highest predictive power. The Neomer approach resulted in a sensitivity of 0.88. specificity of 0.76. and AUC of 0.79. The only clinical variables that were included in the model were age and sex. We conclude that the Neomer approach represents a clear improvement for the agnostic identification of aptamers (Aptamarkers) that bind to unknown biomarkers of a medical state.

Abstract Cattle need physically effective fiber to promote rumination and maintain rumen health, but economics favor the use of low-roughage feedlot diets. The study investigated the optimum barley silage proportion in barley-based finishing diets. Apparent total-tract digestibility (4-d total fecal collection), chewing behavior (6-d video recording), ruminal pH (6-d indwelling pH recording), and fermentation (1 day, sampling 0, 3, 6, 12, and 18 h postfeeding), short-chain fatty acid (SCFA) absorption (washed reticulo-rumen technique), gastrointestinal tract barrier function (marker infusion), and blood variables (catheters) were measured. Eight ruminally fistulated crossbred beef heifers (653 ± 44.2 kg; mean starting body weight [BW] ± SD) were used in a replicated 4 × 4 Latin square design with 28-d periods. Dietary treatments were 0%, 4%, 8%, and 12% of dietary dry matter (DM) as barley silage, with diets containing 80%, 76%, 72%, and 68% barley grain, respectively. Increasing silage proportion decreased dietary starch content from 49.0% to 43.1% DM, while neutral detergent content increased from 22.7% to 25.1% DM. Silage proportion had no effect on DM intake, but apparent DM digestibility decreased quadratically (86.0%, 82.1%, 81.1%, 79.5% for the four diets, respectively; P < 0.001). Although, silage proportion had no effect on eating activity, rumination time increased quadratically (246, 289, 302, 316 min/d; P = 0.04). Increased silage proportion increased minimum (5.07, 5.27, 5.29, 5.41; quadratic, P = 0.011) and mean (5.61, 5.87, 5.93, 5.95; quadratic, P = 0.007) ruminal pH, and there was a quadratic (P ≤ 0.047) decrease in duration and area under the pH acidosis threshold curves of 5.8, 5.5, and 5.2. Although increasing silage proportion decreased ruminal acidosis, it was not completely eliminated even with a diet containing 12% silage DM. SCFA concentration in ruminal fluid was not affected by diet, but silage proportion quadratically (P ≤ 0.088) increased ruminal acetate:propionate. There was no effect of diet on absolute or fractional rates of absorption of acetate, propionate, butyrate or total SCFA, and no effect on gastrointestinal barrier function or blood measurements. In conclusion, responses to roughage level were mostly quadratic with greatest improvements in acidosis variables between 0% and 4% barley silage, with incremental improvements with further increases in silage levels. The study showed a trade-off between maximizing digestibility and energy intake to promote animal performance and minimizing the risk of acidosis.

Abstract The objectives were to determine the effects of forage level and grain processing on whole-body urea kinetics, N balance, serosal-to-mucosal urea flux (Jsm-urea), and messenger ribonucleic acid (mRNA) abundance of urea transporter-B (UT-B; SLC14a1) and aquaporins (AQP) in ovine ruminal, duodenal, and cecal epithelia. Thirty-two wether lambs were blocked by body weight into groups of four and assigned to one of four diets (n = 8) in a 2 × 2 factorial design. Dietary factors were forage level (30% [LF] vs. 70% [HF]) and corn grain processing (whole-shelled [WS] vs. steam-flaked [SF]). Four blocks of lambs (n = 4) were used to determine urea kinetics and N balance using 4-d [15N15N]-urea infusions with concurrent fecal and urine collections. Lambs were killed after 23 d of dietary adaptation. Ruminal, duodenal, and cecal epithelia were collected to determine Jsm-urea and mRNA abundance of UT-B and AQP. Lambs fed LF had greater intakes of dry matter (DMI; 1.20 vs. 0.86 kg/d) and N (NI; 20.1 vs. 15.0 g/d) than those fed HF (P < 0.01). Lambs fed SF had greater DMI (1.20 vs. 0.86 kg/d) and NI (20.6 vs. 14.5 g/d) than those fed WS (P < 0.01). As a percentage of NI, total N excretion was greater in lambs fed HF compared with those fed LF (103% vs. 63.0%; P < 0.01) and was also greater in lambs fed WS compared with those fed SF (93.6% vs. 72.1%; P = 0.02). Retained N (% of NI) was greater in lambs fed LF compared with those fed HF (37.0% vs. −2.55%; P < 0.01). Lambs fed SF had a greater (P = 0.02) retained N (% of NI; 28.0% vs. 6.50%) compared with those fed WS. Endogenous urea production (UER) tended (P = 0.09) to be greater in lambs fed HF compared with those fed LF. As a proportion of UER, lambs fed HF had a greater urinary urea-N loss (0.38 vs. 0.22) and lower urea-N transferred to the gastrointestinal tract (GIT; 0.62 vs. 0.78) or urea-N used for anabolism (as a proportion of urea-N transferred to the GIT; 0.12 vs. 0.26) compared with lambs fed LF (P < 0.01). Ruminal Jsm-urea was unaffected by diet. Duodenal Jsm-urea was greater (P < 0.01) in lambs fed HF compared with LF (77.5 vs. 57.2 nmol/[cm2 × h]). Lambs fed LF had greater (P = 0.03) mRNA expression of AQP3 in ruminal epithelia and tended (P = 0.06) to have greater mRNA expression of AQP3 in duodenal epithelia compared with lambs fed HF. Expression of UT-B mRNA was unaffected by diet. Our results showed that feeding more ruminally available energy improved N utilization, partly through a greater proportion of UER being transferred to the GIT and being used for anabolic purposes.

Cardiovascular disease (CVD) remains the leading cause of death worldwide. Low-density lipoprotein cholesterol (LDL-C) is commonly used for CVD risk assessment; however, recent research has shown LDL particle (LDL-P) number to be a more sensitive indicator of CVD risk than both LDL-C and non-high-density lipoprotein cholesterol (HDL-C). Described herein are five single stranded DNA aptamers with dissociation constants in the low picomolar range specific to LDL-P and its subfractions. Furthermore, a set of antisense sequences have been developed and characterized that are capable of binding to the best aptamers and undergoing displacement by LDL-P for use in a simple, affordable diagnostic assay.

Abstract High grain diets are fed to finishing beef cattle to maximize animal performance in a cost-effective manner. However, a small amount of roughage is incorporated in finishing diets to help prevent ruminal acidosis, although few studies have examined optimum roughage inclusion level in barley-based diets. The objective of the study was to evaluate the effects of roughage proportion in barley-based finishing diets on growth performance, feeding behavior, and carcass traits of feedlot cattle. Crossbred beef steers (n = 160; mean body weight ± SD, 349.7 ± 21.4 kg) were allocated to 20 pens that were assigned randomly to four dietary treatments (five pens of eight steers per treatment). The treatment diets contained barley silage at 0%, 4%, 8%, and 12% of dietary dry matter (DM). The remainder of the diets (DM basis) consisted of 80%, 76%, 72%, and 68% barley grain, respectively, 15% corn dried distiller’s grains, 5% mineral and vitamin supplement, and 32 mg monensin/kg diet DM. The diets were fed as total mixed rations for ad libitum intake (minimum of 5% refusal) once per day. Cattle were weighed on 2 consecutive days at the start and end of the experiment and on 1 d every 3 wk throughout the experiment (124 d). Two pens for each treatment group were equipped with an electronic feeding system (GrowSafe Systems Ltd., Calgary, Alberta) to monitor feed intake and feeding behavior of individual cattle. The data for dry matter intake (DMI), average daily gain (ADG), gain:feed (G:F) ratio, and carcass traits were analyzed as a completely randomized design with fixed effect of barley silage proportion and pen replicate as experimental unit. Feeding behavior data were analyzed similarly, but with animal as experimental unit. Averaged over the study, DMI increased linearly (11.1, 11.3, 11.7, 11.8 kg/d; P = 0.001) as barley silage proportion increased from 0%, 4%, 8%, and 12% of DM, but ADG was not affected (carcass-adjusted,1.90, 1.85, 1.87, 1.89 kg/d; P ≥ 0.30). Consequently, G:F ratio decreased linearly (carcass-adjusted, 168.9, 163.8, 158.5, 160.6 g/kg DMI; P = 0.023). When averaged over the study, proportion of barley silage in the diet had no linear or quadratic effects (P > 0.10) on meal frequency, duration of meals, intermeal duration, or meal size, but eating rate decreased linearly with increasing silage proportion (P = 0.008). There was no diet effect on liver abscesses (P ≥ 0.92), and effects on carcass characteristics were minor or nonexistent. We conclude that increasing the proportion of barley silage in a feedlot finishing diet at the expense of barley grain to minimize the incidence of ruminal acidosis may decrease feed conversion efficiency.

Abstract The objective of this study was to determine effect of ruminal acidosis (RA) and low feed intake [LFI] on the regional barrier function of the gastrointestinal tract. Twenty-one Holstein steers were fed for ad libitum intake for 5 d (control [CON]), fed at 25% of ad libitum intake for 5 d (LFI), or provided 2 d of ad libitum intake followed by 1-d of feed restriction (25% of ad libitum intake), 1 d where 30% of ad libitum dry matter intake (DMI) was provided as pelleted barley followed by the full allocation (RA) and fed for ad libitum intake the following day. Tissues and digesta from the rumen, omasum, duodenum, jejunum, ileum, cecum, proximal, and distal colon were collected. Permeability was assessed using the mucosal-to-serosal flux of inulin (JMS-inulin) and mannitol (JMS-mannitol). Digesta pH was 0.81, 0.63, and 0.42 pH units less for RA than CON in the rumen, cecum, and proximal colon; while, LFI had pH that was 0.47 and 0.36 pH units greater in the rumen and proximal colon compared to CON. Total ruminal short-chain fatty acid (SCFA) concentration were less for LFI (92 mM; P = 0.010) and RA (87 mM; P = 0.007) than CON (172 mM) steers. In the proximal colon, the proportion of butyrate (P = 0.025 and P = 0.022) and isobutyrate (P = 0.019 and P = 0.019) were greater, and acetate (P = 0.028 and P = 0.028) was less for LFI and RA, respectively, when compared to CON steers. Ruminal papillae length, width, perimeter, and surface area were 1.21 mm, 0.78 mm, 3.84 mm, and 11.15 mm2 less for LFI than CON; while, RA decreased papillae width by 0.52 mm relative to CON. The JMS-mannitol was less for LFI steers than CON in the proximal colon (P = 0.041) and in the distal colon (P = 0.015). Increased gene expression for claudin 1, occludin, tight-cell junction protein 1 and 2, and toll-like receptor 4 were detected for LFI relative to CON in the rumen, jejunum, and proximal colon. For RA steers, expression of toll-like receptor 4 in the rumen, and occludin and tight-cell junction protein 1 were greater in the jejunum than CON. An acute RA challenge decreased pH in the rumen and large intestine but did not increase tissue permeability due to increases in the expression of genes related to barrier function within 1 d of the challenge. Steers exposed to LFI for 5 d had reduced ruminal SCFA concentrations, smaller ruminal papillae dimensions, and increased tissue permeability in the proximal and distal colon despite increases for genes related to barrier function and immune function.

The development of aptamers has been almost exclusively performed based on the SELEX method since their inception. While this method represents a powerful means of harnessing the in vitro evolution of sequences that bind to a given target, there are significant constraints in the design. The most significant constraint has been the reliance on counter selection on off-targets to drive specificity. Counter selection has not been as effective at driving aptamer specificity as the presence of immune tolerance, the capacity of the immune system to remove antibodies that bind to host targets, is for antibody development. This deficiency has constrained the commercial efficacy of aptamers to date. These limitations have been addressed with our design of a novel platform for aptamer identification. This new design is based on what we refer to as a Neomer library with sixteen random nucleotides interspersed with fixed sequences. The fixed sequences are designed to minimize the potential for hybridization, such that secondary structure is driven by the random nucleotides. The use of sixteen random nucleotides reduces the possible library sequences to 4.29 ×  109. This enables the application of the same sequences to either the same target or different targets while maintaining a high level of structural diversity. In effect, this introduces the capacity for reproducibility in aptamer selection and an in-silico approach to replicating immune tolerance. We provide here an overview of the new method and a description of the performance of aptamers selected for interleukin 6 developed using this approach.

The objective of this study was to investigate the effects of processing index (PI) of barley grain and dietary undigested neutral detergent fiber (uNDF) concentration on dry matter (DM) intake, chewing activity, ruminal pH and fermentation characteristics, total tract digestibility, gastrointestinal barrier function, and blood metabolites of finishing beef heifers. The PI was measured as the density after processing expressed as a percentage of the density before processing, and a smaller PI equates to a more extensively processed. Six ruminally cannulated heifers (average body weight, 715 ± 29 kg) were used in a 6 × 6 Latin square design with three PI (65%, 75%, and 85%) × 2 uNDF concentration (low and high; 4.6% vs. 5.6% of DM) factorial arrangement. The heifers were fed ad libitum a total mixed ration consisting of 10% barley silage (low uNDF), or 5% silage and 5% straw (high uNDF), 87% dry-rolled barley grain, and 3% mineral and vitamin supplements. Interactions (P < 0.01) of PI × uNDF were observed for DM intake, ruminating and total chewing time, and DM digestibility in the total digestive tract. Intake of DM, organic matter (OM), starch, and crude protein (CP) did not differ (P > 0.14) between low and high uNDF diets, but intakes of NDF and acid detergent fiber were greater (P = 0.01) for high uNDF diets regardless of barley PI. Heifers fed high uNDF diets had longer (P = 0.05) eating times (min/d or min/kg DM) and tended (P = 0.10) to have longer total chewing times (min/kg DM) than those fed low uNDF diets. Additionally, heifers sorted (P = 0.01) against long particles (>19 mm) for high uNDF diets but not for low uNDF diets. Altering PI of barley grain did not affect (P > 0.12) total volatile fatty acid (VFA) concentration, molar percentages of individual VFA, or duration of ruminal pH < 5.8 and <5.6. Total VFA concentration was less (P = 0.01), acetate percentage was greater (P = 0.01), and duration of ruminal pH < 5.8 and <5.6 was less (P = 0.05) for high compared with low uNDF diets. Digestibility of DM, OM, and CP was greater (P = 0.02) for low vs. high uNDF diets with PI of 65% and 75%, with no difference between low and high uNDF diets at PI of 85%. Blood metabolites and gastrointestinal tract barrier function were not affected (P ≥ 0.10) by the treatments. These results suggest that increasing dietary uNDF concentration is an effective strategy to improve ruminal pH status in finishing cattle, regardless of the extent of grain processing, whereas manipulating the extent of barley processing did not reduce the risk of ruminal acidosis.