A Bifunctional Amino Acid Enables Both Covalent Chemical Capture and Isolation of in Vivo Protein–Protein Interactions

In vivo covalent chemical capture by using photoactivatable unnatural amino acids (UAAs) is a powerful tool for the identification of transient protein–protein interactions (PPIs) in their native environment. However, the isolation and characterization of the crosslinked complexes can be challenging. Here, we report the first in vivo incorporation of the bifunctional UAA BPKyne for the capture and direct labeling of crosslinked protein complexes through post‐crosslinking functionalization of a bioorthogonal alkyne handle. Using the prototypical yeast transcriptional activator Gal4, we demonstrate that BPKyne is incorporated at the same level as the commonly used photoactivatable UAA pBpa and effectively captures the Gal4–Gal80 transcriptional complex. Post‐crosslinking, the Gal4–Gal80 adduct was directly labeled by treatment of the alkyne handle with a biotin‐azide probe; this enabled facile isolation and visualization of the crosslinked adduct from whole‐cell lysate. This bifunctional amino acid extends the utility of the benzophenone crosslinker and expands our toolbox of chemical probes for mapping PPIs in their native cellular environment.Using the bifunctional unnatural amino acid, BPKyne, we have developed a strategy to capture and directly label transient protein–protein interactions (PPIs) in their native environment. Click chemical functionalization post‐crosslinking with a biotin–azide probe enabled the isolation of transcriptional protein complexes from yeast cells. This amino acid will expand the toolbox for the discovery of new PPIs in live cells.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/135955/1/cbic201600578.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/135955/2/cbic201600578_am.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/135955/3/cbic201600578-sup-0001-misc_information.pd

Dairy herd mastitis and reproduction: using simulation to aid interpretation of results from discrete time survival analysis

Probabilistic sensitivity analysis (PSA) is a simulation-based technique for evaluating the relative importance of different inputs to a complex process model. It is commonly employed in decision analysis and for evaluation of the potential impact of uncertainty in research findings on clinical practice, but has a wide variety of other possible applications. In this example, it was used to evaluate the association between herd-level udder health and reproductive performance in dairy herds. Although several recent studies have found relatively large associations between mastitis and fertility at the level of individual inseminations or lactations, the current study demonstrated that herd-level intramammary infection status is highly unlikely to have a clinically significant impact on the overall reproductive performance of a dairy herd under typical conditions. For example, a large increase in incidence rate of clinical mastitis (from 92 to 131 cases per 100 cows per year) would be expected to increase a herd's modified FERTEX score (a cost-based measure of overall reproductive performance) by just £4.501 per cow per year. The herd's background level of submission rate (proportion of eligible cows served every 21 days) and pregnancy risk (proportion of inseminations leading to a pregnancy) correlated strongly with overall reproductive performance and explained a large proportion of the between-herd variation in performance. PSA proved to be a highly useful technique to aid understanding of results from a complex statistical model, and has great potential for a wide variety of applications within the field of veterinary science

Autosomal dominant nanophthalmos and high hyperopia associated with a C-terminal frameshift variant in MYRF

Made available with a Creative Commons Attribution-NonCommercial-NoDerivatives License 3.0, or CC BY-NC-ND 3.0 (see http://creativecommons.org/licenses/by-nc-nd/3.0/ for license terms). Copyright (2019) The authors.Purpose: Nanophthalmos is a rare subtype of microphthalmia associated with high hyperopia and an increased risk of angle-closure glaucoma. We investigated the genetic cause of nanophthalmos and high hyperopia in an autosomal dominant kindred. Methods: A proband with short axial length, high hyperopia, and dextrocardia was subjected to exome sequencing. Human and rodent gene expression data sets were used to investigate the expression of relevant genes. Results: We identified a segregating heterozygous frameshift variant at the 3′ end of the penultimate exon of MYRF. Using Myc-MYRF chromatin immunoprecipitation data from rat oligodendrocytes, MYRF was found to bind immediately upstream of the transcriptional start site of Tmem98, a gene that itself has been implicated in autosomal dominant nanophthalmos. MYRF and TMEM98 were found to be expressed in the human retina, with a similar pattern of expression across several dissected human eye tissues. Conclusions: C-terminal variants in MYRF, which are expected to escape nonsense-mediated decay, represent a rare cause of autosomal dominant nanophthalmos with or without dextrocardia or congenital diaphragmatic hernia

Estimating survival in patients with gastrointestinal cancers and brain metastases: An update of the graded prognostic assessment for gastrointestinal cancers (GI-GPA).

BackgroundPatients with gastrointestinal cancers and brain metastases (BM) represent a unique and heterogeneous population. Our group previously published the Diagnosis-Specific Graded Prognostic Assessment (DS-GPA) for patients with GI cancers (GI-GPA) (1985-2007, n = 209). The purpose of this study is to update the GI-GPA based on a larger contemporary database.MethodsAn IRB-approved consortium database analysis was performed using a multi-institutional (18), multi-national (3) cohort of 792 patients with gastrointestinal (GI) cancers, with newly-diagnosed BM diagnosed between 1/1/2006 and 12/31/2017. Survival was measured from date of first treatment for BM. Multiple Cox regression was used to select and weight prognostic factors in proportion to their hazard ratios. These factors were incorporated into the updated GI-GPA.ResultsMedian survival (MS) varied widely by primary site and other prognostic factors. Four significant factors (KPS, age, extracranial metastases and number of BM) were used to formulate the updated GI-GPA. Overall MS for this cohort remains poor; 8 months. MS by GPA was 3, 7, 11 and 17 months for GPA 0-1, 1.5-2, 2.5-3.0 and 3.5-4.0, respectively. >30% present in the worst prognostic group (GI-GPA of ≤1.0).ConclusionsBrain metastases are not uncommon in GI cancer patients and MS varies widely among them. This updated GI-GPA index improves our ability to estimate survival for these patients and will be useful for therapy selection, end-of-life decision-making and stratification for future clinical trials. A user-friendly, free, on-line app to calculate the GPA score and estimate survival for an individual patient is available at brainmetgpa.com

Genetic inhibition of hepatic acetyl-CoA carboxylase activity increases liver fat and alters global protein acetylationa

Lipid deposition in the liver is associated with metabolic disorders including fatty liver disease, type II diabetes, and hepatocellular cancer. The enzymes acetyl-CoA carboxylase 1 (ACC1) and ACC2 are powerful regulators of hepatic fat storage; therefore, their inhibition is expected to prevent the development of fatty liver. In this study we generated liver-specific ACC1 and ACC2 double knockout (LDKO) mice to determine how the loss of ACC activity affects liver fat metabolism and whole-body physiology. Characterization of LDKO mice revealed unexpected phenotypes of increased hepatic triglyceride and decreased fat oxidation. We also observed that chronic ACC inhibition led to hyper-acetylation of proteins in the extra-mitochondrial space. In sum, these data reveal the existence of a compensatory pathway that protects hepatic fat stores when ACC enzymes are inhibited. Furthermore, we identified an important role for ACC enzymes in the regulation of protein acetylation in the extra-mitochondrial space

Novel Bone-Targeting Agent for Enhanced Delivery of Vancomycin to Bone

We examined the pharmacokinetic properties of vancomycin conjugated to a bone-targeting agent (BT) with high affinity for hydroxyapatite after systemic intravenous administration. The results confirm enhanced persistence of BT-vancomycin in plasma and enhanced accumulation in bone relative to vancomycin. This suggests that BT-vancomycin may be a potential carrier for the systemic targeted delivery of vancomycin in the treatment of bone infections, potentially reducing the reliance on surgical debridement to achieve the desired therapeutic outcome

The impact of dairy cows' bedding material and its microbial content on the quality and safety of milk: a cross sectional study of UK farms

The introduction of bedding dairy cows on recycled manure solids (RMS) in the UK led to concern by competent authorities that there could be an increased, unacceptable risk to animal and human health. A cross-sectional study was designed to evaluate the microbial content of different bedding materials, when used by dairy cows, and its impact on the microbial content of milk. Data were collected from farms bedding lactating cows on sand (n = 41), sawdust (n = 44) and RMS (n = 40). The mean duration of RMS use prior to sampling was 13 months. Total bacterial count, and counts of Streptococcus/Enterococcus spp., Staphylococcus spp., Bacillus cereus, thermophilic, thermoduric and psychrotrophic bacteria were determined in used bedding and milk. Samples were evaluated for the presence/absence of Listeria monocytogenes, Salmonella spp. and Yersinia enterocolitica. Data on milking practices were collected to investigate their potential to reduce microbial transfer from bedding to milk. There were substantial differences in bacterial counts both within and between bedding materials. However, there were no significant differences between bedding groups in counts in milk for any of the organisms studied, and no significant correlations between bacterial load in used bedding and milk. Fore-milking was associated with a reduced total bacterial count in milk. Dipping teats with disinfectant and drying, prior to milking, was associated with lower numbers of Streptococcus/Enterococcus spp. in milk. Disinfecting clusters between milking different cows was associated with a reduction in thermophilic and psychrotrophic counts in milk. This study did not provide evidence that use of RMS bedding increased the risk of presence of Y. enterocolitica, Salmonella spp. or L. monocytogenes in milk. However, the strength of this conclusion should be tempered by the relatively small number of farms on which Y. enterocolitica and Salmonella spp. were isolated. It is concluded that, despite the higher bacterial load of RMS, its use as bedding for lactating dairy cows need not be associated with a higher bacterial load in milk than the use of sand or sawdust. However, this finding must be interpreted in the light of the relatively recent introduction of RMS as a bedding material on the farms studied. Teat preparation provides a control point for the potential transfer of microorganisms from bedding to milk. The detection of zoonotic pathogens in a small proportion of milk samples, independent of bedding type, indicates that pasteurisation of milk prior to human consumption remains an important control measure

Large-scale proteomic analysis of human brain identifies proteins associated with cognitive trajectory in advanced age

In advanced age, some individuals maintain a stable cognitive trajectory while others experience a rapid decline. Such variation in cognitive trajectory is only partially explained by traditional neurodegenerative pathologies. Hence, to identify new processes underlying variation in cognitive trajectory, we perform an unbiased proteome-wide association study of cognitive trajectory in a discovery (n = 104) and replication cohort (n = 39) of initially cognitively unimpaired, longitudinally assessed older-adult brain donors. We find 579 proteins associated with cognitive trajectory after meta-analysis. Notably, we present evidence for increased neuronal mitochondrial activities in cognitive stability regardless of the burden of traditional neuropathologies. Furthermore, we provide additional evidence for increased synaptic abundance and decreased inflammation and apoptosis in cognitive stability. Importantly, we nominate proteins associated with cognitive trajectory, particularly the 38 proteins that act independently of neuropathologies and are also hub proteins of protein co-expression networks, as promising targets for future mechanistic studies of cognitive trajectory.Accelerating Medicine Partnership for AD [U01AG046161, U01 AG061357]; Emory Alzheimer's Disease Research Center [P50 AG025688]; NINDS Emory Neuroscience Core [P30 NS055077]; intramural program of the National Institute on Aging (NIA); Alzheimer's Association; Alzheimer's Research UK; Michael J. Fox Foundation for Parkinson's Research; Weston Brain Institute Biomarkers Across Neurodegenerative Diseases Grant [11060]; National Institute of Neurological Disorders and Stroke [U24 NS072026]; National Institute on Aging [P30 AG19610]; Arizona Department of Health Services [211002]; Arizona Biomedical Research Commission [4001, 0011, 05-901, 1001]; [R01 AG056533]; [R01 AG053960]; [U01 MH115484]; [I01 BX003853]; [IK2 BX001820]; [R01 AG061800]; [R01 AG057911]Open access journalThis item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]