A morphology-based assay such as immunohistochemistry (IHC) should be a highly effective means to define the expression of a target molecule of interest, especially if the target is a protein. However, over the past decade, IHC as a platform for biomarkers has been challenged by more quantitative molecular assays with reference standards but that lack morphologic context. For IHC to be considered a ''top-tier'' biomarker assay, it must provide truly quantitative data on par with non-morphologic assays, which means it needs to be run with reference standards. However, creating such standards for IHC will require optimizing all aspects of tissue collection, fixation, section thickness, morphologic criteria for assessment, staining processes, digitization of images, and image analysis. This will also require anatomic pathology to evolve from a discipline that is descriptive to one that is quantitative. A major step in this transformation will be replacing traditional ocular microscopes with computer monitors and whole slide images, for without digitization, there can be no accurate quantitation; without quantitation, there can be no standardization; and without standardization, the value of morphology-based IHC assays will not be realized.

Thirty-eight multiparous Holstein cows were utilized in a completely randomized design to examine the effect of feeding calcium salts of conjugated linoleic acid (CLA) and trans-octadecenoic acids (trans-C18:1) on animal performance and lipid and glucose metabolism during the transition to lactation. Dietary treatments were initiated approximately 28 d prior to expected calving dates and continued through d 49 postpartum. Prepartum treatments consisted of 1) a basal diet (Control), 2) basal diet + 150 g/d of CLA mix (CLA), and 3) basal diet + 150 g/d of trans-C18:1 mix (TRANS). Amounts of calcium salts of CLA and trans-C18:1 mixes were adjusted to 225 g/d during the 49-d postpartum treatment period. All diets were offered as a total mixed ration. Prepartum fat supplementation had no detectable effects on dry matter intake, body weight, or body condition score. After parturition, cows in the TRANS group consumed less dry matter at wk 4, 5, and 6 of lactation than did cows in the control group. Cows fed the trans-C18:1 supplement were in a more severe negative energy balance than those fed the control diet at 1 wk of lactation. Periparturient fat supplementation had no detectable effects on milk yield during wk 1 to 7 of lactation. Milk fat was not affected during wk 1 to 4, but was reduced after wk 4 of lactation by dietary CLA. Feeding calcium salts of CLA decreased short- to medium-chain fatty acid (C4 to C14) concentrations and increased both linoleic and linolenic acid concentrations in milk fat. Concentrations of nonesterified fatty acids and beta-hydroxybutyric acid in blood were greater in cows fed the CLA-supplemented diet than in those fed the control diet at 1 wk of lactation. In spite of small numerical tendencies, hepatic lipid and triacylglycerol concentrations did not vary significantly among dietary treatments. Periparturient fat supplementation had no detectable effects on plasma glucose and insulin concentrations. Steady-state concentrations of hepatic mRNA encoding pyruvate carboxylase and phosphoenolpyruvate carboxykinase were greater for the TRANS treatment group than the control and CLA groups. Results indicate that dietary CLA and trans-C18:1 fatty acids may affect lipid and glucose metabolism in early postpartum Holstein cows through distinct mechanisms.

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Background Substance use by youth living with HIV (YLWH) is a concern, given potential interactions with virus-associated immune suppression and adverse effects on risk behaviors, neurocognition, and adherence. Self-report substance use measures provide efficient cost-effective assessments. Analyses describe self-reported substance use among YLWH and examine agreement with toxicology assays. Methods Seventy-eight youth age 18–24 years (87% male, 71% African–American) with behaviorally acquired HIV-1 infection and 55 uninfected youth completed the Alcohol, Smoking, and Substance Involvement Screening Test to assess drug use frequency, including tobacco, marijuana, cocaine, and alcohol, over the prior three months. Elisa-based toxicology assays were used to detect 27 substances in plasma. Chi-square tests compared substance use between YLWH and uninfected youth; Kappa statistics compared agreement between self-report and toxicology. Results YLWH reported marijuana (49%), tobacco (56%), and alcohol (87%) use, with 20%, 28% and 3% reporting daily use of each substance, respectively; other substance use was uncommon. Uninfected youth reported less tobacco use but otherwise similar substance use. All youth who reported daily use of marijuana or tobacco had positive plasma toxicology results, while concordance decreased with less frequent self-reported use. Among youth reporting no substance use, few tested positive (4% YLWH, 2% uninfected youth for cannabis; 8%YLWH for tobacco). Conclusions Youth report high rates of marijuana, tobacco, and alcohol use. Concordance between self-report and toxicology for marijuana and tobacco use, particularly for daily users, supports self-report as a valid indicator of substance use in research studies of youth with or without HIV-1 infection.

Next generation sequencing (NGS) is superseding Sanger technology for analysing intra-host viral populations, in terms of genome length and resolution. We introduce two new empirical validation data sets and test the available viral population assembly software. Two intra-host viral population ‘quasispecies’ samples (type-1 human immunodeficiency and hepatitis C virus) were Sanger-sequenced, and plasmid clone mixtures at controlled proportions were shotgun-sequenced using Roche's 454 sequencing platform. The performance of different assemblers was compared in terms of phylogenetic clustering and recombination with the Sanger clones. Phylogenetic clustering showed that all assemblers captured a proportion of the most divergent lineages, but none were able to provide a high precision/recall tradeoff. Estimated variant frequencies mildly correlated with the original. Given the limitations of currently available algorithms identified by our empirical validation, the development and exploitation of additional data sets is needed, in order to establish an efficient framework for viral population reconstruction using NGS.