Effect of feeding method on nutrient utilization and cow performance in limit-fed cow-calf systems.

Delivery of limit-fed, complete rations requires significant capital investment, and creates logistical challenges for producers. Deconstruction and separate delivery of roughage and concentrate portions of diets may decrease feeding cost. Two experiments were conducted to evaluate the potential of separately limit-feeding roughage and concentrate. In Experiment 1, 4 ruminally cannulated steers (371 ± 12 kg bodyweight) were used in a 4 × 4 Latin square to evaluate the effects of time of concentrate delivery in deconstructed diets. Intake was restricted to 80% of predicted NEm requirements of a diet consisting of wheat straw (35%), cracked corn (29%), and distillers' grains (27%) formulated to contain 1.58 Mcal NEm/kg. Treatments were: concentrate fed 2 h prior to wheat straw (-2S), concentrate and wheat straw fed as total mixed ration (TMR), concentrate fed 2 h after wheat straw (+2S), and concentrate fed 12 h after wheat straw (+12S). In Experiment 2, 95 mid- to late-gestation cows (503 ± 151 kg) were used in a 112-d trial to evaluate feeding system on cow performance. Cows were assigned to 1 of 12 pens. Treatments were limit-fed the complete diet from Experiment 1 (TMR), fed roughage and concentrate portions of the deconstructed TMR 12 h apart (SEP), and ad libitum bermudagrass hay (HAY). Bodyweight (BW), BCS, and back fat measures were collected every 28 d. In Experiment 1, treatment did not affect DM or OM digestion (P ≥ 0.88), rate of particulate passage (P ≥ 0.55), or ruminal DM fill (P ≥ 0.19). Fill averaged 3.8 kg DM. Nadir of ruminal pH occurred 4-8 h after concentrate was delivered, but mean ruminal pH was not different among treatments (P = 0.22) ranging from 6.4 to 6.6 for +2S and 12S, respectively. In Experiment 2, treatment did not affect final BW (518 kg; P = 0.72) or final BCS (5.6; P = 0.67), but limit-fed strategies tended (P = 0.06) to have greater final RE (137.1, 98.9, and -14.6 Mcal for TMR, SEP, and HAY, respectively). Delivering forage and concentrate separately did not change digestion, and timing of concentrate delivery had only minor effects on ruminal fermentation. Limit-feeding a TMR or separate delivery of roughage and concentrate sustained cow performance compared to ad libitum hay consumption.

The challenges of generalizability in artificial intelligence for ADME/Tox endpoint and activity prediction.

INTRODUCTION: Artificial intelligence (AI) has seen a massive resurgence in recent years with wide successes in computer vision, natural language processing, and games. The similar creation of robust and accurate AI models for ADME/Tox endpoint and activity prediction would be revolutionary to drug discovery pipelines. There have been numerous demonstrations of successful applications, but a key challenge remains: how generalizable are these predictive models? AREAS COVERED: The authors present a summary of current promising components of AI models in the context of early drug discovery where ADME/Tox endpoint and activity prediction is the main driver of the iterative drug design process. Following that is a review of applicability domains and dataset construction considerations which determine generalizability bottlenecks for AI deployment. Further reviewed is the role of promising learning frameworks - multitask, transfer, and meta learning - which leverage auxiliary data to overcome issues of generalizability. EXPERT OPINION: The authors conclude that the most promising direction toward integrating reliable and informative AI models into the drug discovery pipeline is a conjunction of learned feature representations, deep learning, and novel learning frameworks. Such a solution would address the sparse and incomplete datasets that are available for key endpoints related to drug discovery.

The development of a staphylococcus aureus four antigen vaccine for use prior to elective orthopedic surgery.

Staphylococcus aureus (S. aureus) is a challenging bacterial pathogen which can cause a range of diseases, from mild skin infections, to more serious and invasive disease including deep or organ space surgical site infections, life-threatening bacteremia, and sepsis. S. aureus rapidly develops resistance to antibiotic treatments. Despite current infection control measures, the burden of disease remains high. The most advanced vaccine in clinical development is a 4 antigen S. aureus vaccine (SA4Ag) candidate that is being evaluated in a phase 2b/3 efficacy study in patients undergoing elective spinal fusion surgery (STaphylococcus aureus suRgical Inpatient Vaccine Efficacy [STRIVE]). SA4Ag has been shown in early phase clinical trials to be generally safe and well tolerated, and to induce high levels of bactericidal antibodies in healthy adults. In this review we discuss the design of SA4Ag, as well as the proposed clinical development plan supporting licensure of SA4Ag for the prevention of invasive disease caused by S. aureus in elective orthopedic surgical populations. We also explore the rationale for the generalizability of the results of the STRIVE efficacy study (patients undergoing elective open posterior multilevel instrumented spinal fusion surgery) to a broad elective orthopedic surgery population due to the common pathophysiology of invasive S. aureus disease and commonalties of patient and procedural risk factors for developing postoperative S. aureus surgical site infections.

In-silico guided discovery of novel CCR9 antagonists.

Antagonism of CCR9 is a promising mechanism for treatment of inflammatory bowel disease, including ulcerative colitis and Crohn's disease. There is limited experimental data on CCR9 and its ligands, complicating efforts to identify new small molecule antagonists. We present here results of a successful virtual screening and rational hit-to-lead campaign that led to the discovery and initial optimization of novel CCR9 antagonists. This work uses a novel data fusion strategy to integrate the output of multiple computational tools, such as 2D similarity search, shape similarity, pharmacophore searching, and molecular docking, as well as the identification and incorporation of privileged chemokine fragments. The application of various ranking strategies, which combined consensus and parallel selection methods to achieve a balance of enrichment and novelty, resulted in 198 virtual screening hits in total, with an overall hit rate of 18%. Several hits were developed into early leads through targeted synthesis and purchase of analogs.

Glucose and acetate metabolism in bovine intramuscular and subcutaneous adipose tissues from steers infused with glucose, propionate, or acetate.

We hypothesized that abomasal infusion of glucose would promote de novo fatty acid biosynthesis from glucose in vitro in bovine intramuscular (i.m.) and subcutaneous (s.c.) adipose tissues to a greater extent than ruminal infusion of acetate, propionate, or glucose. Angus crossbred steers (n = 24), 22 mo of age, were fitted with ruminal cannulas, and steers were adapted to another corn/sorghum finishing diet over a 2-wk period while recovering from the placement of the cannulas. After the adaptation period, the steers were fed the second finishing diet at 130% of their voluntary intake and were infused with isocaloric amounts (3.76 Mcal/d) of glucose, propionate, or acetate for 35 d. Glucose was infused either into the rumen or into the abomasum, whereas propionate and acetate were infused into the rumen. Acetate infusion decreased DM and DE intakes (P < 0.05). The 5th to 8th longissimus muscle section was removed immediately and transported to the laboratory within 10 min post-exsanguination in 38 °C, oxygenated Krebs Henseleit buffer containing 5 mM glucose and 5 mM acetate. Intramuscular and s.c. adipose tissues were dissected from the muscle and incubated in vitro in 5 mM glucose plus 5 mM acetate (containing [U-14C]glucose or [1-14C]acetate). Lipid content was lower (P = 0.04) in i.m. adipose tissue of the acetate-infused steers than in the other treatment groups, and i.m. adipocytes from acetate-infused steers were smaller (P = 0.01) than those from propionate-infused steers. The rate of incorporation of acetate into glyceride-fatty acids (GFA) in i.m. and s.c. adipose tissues was greater (P < 0.03) in steers receiving ruminal or abomasal infusions of glucose than in adipose tissues from steers infused with acetate. The greatest rates of GFA synthesis were observed in s.c. adipose tissue from steers infused ruminally with propionate or abomasally infused with glucose (P < 0.001). In i.m. and s.c. adipose tissues, the proportion of acetyl units from acetate incorporated into GFA was greater in steers receiving glucose infusion in the rumen or abomasum than in steers receiving acetate or propionate infusion (P < 0.05). Contrary to our hypothesis, abomasal glucose infusion did not promote greater fatty acid biosynthesis from glucose in i.m. adipose tissue than ruminal glucose infusion. However, glucose infusion caused the greatest production of acetyl units from acetate in i.m. and s.c. adipose tissues.

Vaccine development to prevent Staphylococcus aureus surgical-site infections.

BACKGROUND: Staphylococcus aureus surgical-site infections (SSIs) are a major cause of poor health outcomes, including mortality, across surgical specialties. Despite current advances as a result of preventive interventions, the disease burden of S. aureus SSI remains high, and increasing antibiotic resistance continues to be a concern. Prophylactic S. aureus vaccines may represent an opportunity to prevent SSI. METHODS: A review of SSI pathophysiology was undertaken in the context of evaluating new approaches to developing a prophylactic vaccine to prevent S. aureus SSI. RESULTS: A prophylactic vaccine ideally would provide protective immunity at the time of the surgical incision to prevent initiation and progression of infection. Although the pathogenicity of S. aureus is attributed to many virulence factors, previous attempts to develop S. aureus vaccines targeted only a single virulence mechanism. The field has now moved towards multiple-antigen vaccine strategies, and promising results have been observed in early-phase clinical studies that supported the recent initiation of an efficacy trial to prevent SSI. CONCLUSION: There is an unmet medical need for novel S. aureus SSI prevention measures. Advances in understanding of S. aureus SSI pathophysiology could lead to the development of effective and safe prophylactic multiple-antigen vaccines to prevent S. aureus SSI.

Liver transplantation in recipients receiving renal replacement therapy: outcomes analysis and the role of intraoperative hemodialysis.

The Model for End-Stage Liver Disease (MELD) system has dramatically increased the number of recipients requiring pretransplant renal replacement therapy (RRT) prior to liver transplantation (LT). Factors affecting post-LT outcomes and the need for intraoperative RRT (IORRT) were analyzed in 500 consecutive recipients receiving pretransplant RRT, including comparisons among recipients not receiving IORRT (No-IORRT, n = 401), receiving planned IORRT (Pl-IORRT, n = 70), and receiving emergent, unplanned RRT after LT initiation (Em-IORRT, n = 29). Despite a median MELD of 39, overall 30-day, 1-, 3- and 5-year survivals were 93%, 75%, 68% and 65%, respectively. Em-IORRT recipients had significantly more intraoperative complications (arrhythmias, postreperfusion syndrome, coagulopathy) compared with both No-IORRT and Pl-IORRT and greater 30-day graft loss (28% vs. 10%, p = 0.004) and need for retransplantation (24% vs. 10%, p = 0.099) compared with No-IORRT. A risk score based on multivariate predictors of IORRT accurately identified recipients with chronic (sensitivity 84%, specificity 72%, concordance-statistic [c-statistic] 0.829) and acute (sensitivity 93%, specificity 61%, c-statistic 0.776) liver failure requiring IORRT. In this largest experience of LT in recipients receiving RRT, we report excellent survival and propose a practical model that accurately identifies recipients who may benefit from IORRT. For this select group, timely initiation of IORRT reduces intraoperative complications and improves posttransplant outcomes.

Pyroglutamate and O-linked glycan determine functional production of anti-IL17A and anti-IL22 peptide-antibody bispecific genetic fusions.

Protein biosynthesis and extracellular secretion are essential biological processes for therapeutic protein production in mammalian cells, which offer the capacity for correct folding and proper post-translational modifications. In this study, we have generated bispecific therapeutic fusion proteins in mammalian cells by combining a peptide and an antibody into a single open reading frame. A neutralizing peptide directed against interleukin-17A (IL17A) was genetically fused to the N termini of an anti-IL22 antibody, through either the light chain, the heavy chain, or both chains. Although the resulting fusion proteins bound and inhibited IL22 with the same affinity and potency as the unmodified anti-IL22 antibody, the peptide modality in the fusion scaffold was not active in the cell-based assay due to the N-terminal degradation. When a glutamine residue was introduced at the N terminus, which can be cyclized to form pyroglutamate in mammalian cells, the IL17A neutralization activity of the fusion protein was restored. Interestingly, the mass spectroscopic analysis of the purified fusion protein revealed an unexpected O-linked glycosylation modification at threonine 5 of the anti-IL17A peptide. The subsequent removal of this post-translational modification by site-directed mutagenesis drastically enhanced the IL17A binding affinity and neutralization potency for the resulting fusion protein. These results provide direct experimental evidence that post-translational modifications during protein biosynthesis along secretory pathways play critical roles in determining the structure and function of therapeutic proteins produced by mammalian cells. The newly engineered peptide-antibody genetic fusion is promising for therapeutically targeting multiple antigens in a single antibody-like molecule.

Automated generation of turn mimetics: proof of concept study for the MC4 receptor.

An algorithm has been devised for the automatic design of peptide turn mimetics, particularly applicable to peptide-activated GPCRs. The method is based on flexible alignments using a new design paradigm and scoring system that aims to reduce the molecular weight of the compound and preferentially lead to drug like molecules. The process can be applied either as a de novo design or a virtual screening tool. Its use has been demonstrated by the design of novel double digit nanomolar ligands for the melanocortin 4 receptor (MC4). The method is, in principle, applicable to any type of receptor, including orphan receptors.

Computation of 3D queries for ROCS based virtual screens.

Rapid overlay of chemical structures (ROCS) is a method that aligns molecules based on shape and/or chemical similarity. It is often used in 3D ligand-based virtual screening. Given a query consisting of a single conformation of an active molecule ROCS can generate highly enriched hit lists. Typically the chosen query conformation is a minimum energy structure. Can better enrichment be obtained using conformations other than the minimum energy structure? To answer this question a methodology has been developed called CORAL (COnformational analysis, Rocs ALignment). For a given set of molecule conformations it computes optimized conformations for ROCS screening. It does so by clustering all conformations of a chosen molecule set using pairwise ROCS combo scores. The best representative conformation is that which has the highest average overlap with the rest of the conformations in the cluster. It is these best representative conformations that are then used for virtual screening. CORAL was tested by performing virtual screening experiments with the 40 DUD (Directory of Useful Decoys) data sets. Both CORAL and minimum energy queries were used. The recognition capability of each query was quantified as the area under the ROC curve (AUC). Results show that the CORAL AUC values are on average larger than the minimum energy AUC values. This demonstrates that one can indeed obtain better ROCS enrichments with conformations other than the minimum energy structure. As a result, CORAL analysis can be a valuable first step in virtual screening workflows using ROCS.

GARD: a Generally Applicable Replacement for RMSD.

The root-mean-squared deviation (rmsd) is a widely used measure of distance between two aligned objects -- often chemical structures. However, rmsd has a number of known limitations including difficulty of interpretation, no limit on weighting for any portion of the alignment, and a lack of normalization. In this work, a Generally Applicable Replacement for rmsD (GARD) is proposed. In this implementation atomic contributions are weighted by their relative importance to binding, as determined statistically by Andrews et al. (1) , and as such this method is 'chemically aware'. This novel measure is normalized and does not have many of the failings of traditional rmsd. It is, thus, perfectly suited for a wide variety of uses, including the assessment of the quality of poses produced from molecular docking programs and the comparison of conformers. Rmsd and GARD are compared in their ability to assess docking software and multiple examples of the use of GARD to rescue essentially correct poses with a high rmsd are presented.

Comparison of several molecular docking programs: pose prediction and virtual screening accuracy.

Molecular docking programs are widely used modeling tools for predicting ligand binding modes and structure based virtual screening. In this study, six molecular docking programs (DOCK, FlexX, GLIDE, ICM, PhDOCK, and Surflex) were evaluated using metrics intended to assess docking pose and virtual screening accuracy. Cognate ligand docking to 68 diverse, high-resolution X-ray complexes revealed that ICM, GLIDE, and Surflex generated ligand poses close to the X-ray conformation more often than the other docking programs. GLIDE and Surflex also outperformed the other docking programs when used for virtual screening, based on mean ROC AUC and ROC enrichment values obtained for the 40 protein targets in the Directory of Useful Decoys (DUD). Further analysis uncovered general trends in accuracy that are specific for particular protein families. Modifying basic parameters in the software was shown to have a significant effect on docking and virtual screening results, suggesting that expert knowledge is critical for optimizing the accuracy of these methods.

The use of ligand-based de novo design for scaffold hopping and sidechain optimization: two case studies.

This paper describes the application of de novo design utilizing exclusively ligand information. In the current approach, ligand design criteria, including pharmacophores, similarity and desired properties are applied as part of a fitness function driving the design process, instead of using them as filters after the process. This allows relevant parts of chemical space to be explored more efficiently. Two case studies of successful ligand design are also presented, one aimed at scaffold hopping, the other for exploring substitution patterns around a novel scaffold.

Lead optimization via high-throughput molecular docking.

Structure-based lead optimization approaches are increasingly playing a role in the drug-discovery process. Recent advances in 'high-throughput' molecular docking methods and examples of their successful use in lead optimization are reviewed. Measures of docking accuracy, scoring function comparisons, and consensus approaches are discussed. Differences in docking protocols typically used for lead optimization versus lead generation are highlighted; this section includes a discussion of the latest methods for the incorporation of protein flexibility. New approaches developed specifically for the design of combinatorial libraries as well as those designed or used for 'fragment' versus lead optimization are presented. Finally, potential future improvements to the technology are outlined.

Novel 2D fingerprints for ligand-based virtual screening.

This paper describes the development of a set of new 2D fingerprints for the purposes of virtual screening in a pharmaceutical environment. The new fingerprints are based on established ones: the changes in their design included the introduction of overlapping pharmacophore feature types, feature counts for pharmacophore and structural fingerprints, as well as changes in the resolution in property description for property fingerprints. The effects of each of these changes on virtual screening performance were monitored using two types of training sets, emulating different stages in the drug discovery process. The results demonstrate that these changes all lead to an improvement in virtual screening performance.

The use of consensus scoring in ligand-based virtual screening.

A new consensus approach has been developed for ligand-based virtual screening. It involves combining highly disparate properties in order to improve performance in virtual screening. The properties include structural, 2D pharmacophore and property-based fingerprints, scores derived using BCUT descriptors, and 3D pharmacophore approaches. Different approaches for the combination of all or some of these methods have been tested. Logistic regression and sum ranks were found to be the most advantageous in different pharmaceutical applications. The three major reasons consensus scoring appears to enrich data sets better than single scoring functions are (1) using multiple scoring functions is similar to repeated samplings, in which case the mean is closer to the true value than any single value, (2) due to the better clustering of actives, multiple sampling will recover more actives than inactives, and (3) different methods seem to agree more on the ranking of the actives than on the inactives. Furthermore, consensus results are not only better but are also more consistent across receptor systems.

Predicting synthetic accessibility: application in drug discovery and development.

The estimation and use of synthetic accessibility in the drug discovery process is discussed. A distinction is drawn between synthetic feasibility and accessibility and the practical uses of an accessibility score are examined. Various techniques used in the estimation of accessibility are described and their utility and potential accuracy compared.

Analysis of slow interdomain motion of macromolecules using NMR relaxation data.

The interpretation of NMR relaxation data for macromolecules possessing slow interdomain motions is considered. It is shown how the "extended model-free approach" can be used to analyze (15)N backbone relaxation data acquired at three different field strengths for Xenopus Ca(2+)-ligated calmodulin. This protein is comprised of two domains connected by two rigid helices joined by a flexible segment. It is possible to uniquely determine all "extended model-free" parameters without any a priori assumptions regarding their magnitudes by simultaneously least-squares fitting the relaxation data measured at two different magnetic fields. It is found that the two connecting helices (and consequently the domains) undergo slow motions relative to the conformation in which the two helices are parallel. The time scales and amplitudes of these "wobbling" motions are characterized by effective correlation times and squared-order parameters of approximately 3 ns and 0.7, respectively. These values are consistent with independent estimates indicating that this procedure provides a useful first-order description of complex internal motions in macromolecules despite neglecting the coupling of overall and interdomain motions.

Chemical shift mapped DNA-binding sites and 15N relaxation analysis of the C-terminal KH domain of heterogeneous nuclear ribonucleoprotein K.

The K homology (KH) motif is one of the major classes of nucleic acid binding proteins. Some members of this family have been shown to interact with DNA while others have RNA targets. There have been no reports containing direct experimental evidence regarding the nature of KH module-DNA interaction. In this study, the interaction of the C-terminal KH domain of heterogeneous nuclear ribonucleoprotein K (KH3) with its cognate single-stranded DNA (ssDNA) are investigated. Chemical shift perturbation mapping indicates that the first two helices, the conserved GxxG loop, beta 1, and beta 2, are the primary regions involved in DNA binding for KH3. The nature of the KH3-ssDNA interaction is further illuminated by a comparison of backbone 15N relaxation data for the bound and unbound KH3. Relaxation data are also used to confirm that the backbone of wild-type KH3 is structurally identical to that of the G26R mutant KH3, which was previously published. Amide proton exchange experiments indicate that the two helices involved in DNA binding are less stable than other regions of secondary structure and that a large portion of KH3 backbone amide hydrogens are protected in some manner upon ssDNA binding. The major backbone dynamics features of KH3 are similar to those of the structurally comparable human papillomavirus-31 E2 DNA binding domain. Secondary structure information for ssDNA-bound wild-type KH3 is also presented and shows that binding results in no global changes in the protein fold.