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Excess collagen synthesis (fibrogenesis) in the liver plays a causal role in the progression of nonalcoholic fatty liver disease (NAFLD). Methods are needed to identify patients with more rapidly progressing disease and to demonstrate early response to treatment. We describe here a novel method to quantify hepatic fibrogenesis flux rates both directly in liver tissue and noninvasively in blood. Twenty-one patients with suspected NAFLD ingested heavy water (2 H2 O, 50-mL aliquots) two to three times daily for 3-5 weeks prior to a clinically indicated liver biopsy. Liver collagen fractional synthesis rate (FSR) and plasma lumican FSR were measured based on 2 H labeling using tandem mass spectrometry. Patients were classified by histology for fibrosis stage (F0-F4) and as having nonalcoholic fatty liver or nonalcoholic steatohepatitis (NASH). Magnetic resonance elastography measurements of liver stiffness were also performed. Hepatic collagen FSR in NAFLD increased with advancing disease stage (e.g., higher in NASH than nonalcoholic fatty liver, positive correlation with fibrosis score and liver stiffness) and correlated with hemoglobin A1C. In addition, plasma lumican FSR demonstrated a significant correlation with hepatic collagen FSR.ConclusionUsing a well-characterized cohort of patients with biopsy-proven NAFLD, this study demonstrates that hepatic scar in NASH is actively remodeled even in advanced fibrosis, a disease that is generally regarded as static and slowly progressive. Moreover, hepatic collagen FSR correlates with established risks for fibrotic disease progression in NASH, and plasma lumican FSR correlates with hepatic collagen FSR, suggesting applications as direct or surrogate markers, respectively, of hepatic fibrogenesis in humans. (Hepatology 2017;65:78-88).

Central nervous system (CNS) infection is a nearly universal facet of systemic HIV infection that varies in character and neurological consequences. While clinical staging and neuropsychological test performance have been helpful in evaluating patients, cerebrospinal fluid (CSF) biomarkers present a valuable and objective approach to more accurate diagnosis, assessment of treatment effects and understanding of evolving pathobiology. We review some lessons from our recent experience with CSF biomarker studies. We have used two approaches to biomarker analysis: targeted, hypothesis-driven and non-targeted exploratory discovery methods. We illustrate the first with data from a cross-sectional study of defined subject groups across the spectrum of systemic and CNS disease progression and the second with a longitudinal study of the CSF proteome in subjects initiating antiretroviral treatment. Both approaches can be useful and, indeed, complementary. The first is helpful in assessing known or hypothesized biomarkers while the second can identify novel biomarkers and point to broad interactions in pathogenesis. Common to both is the need for well-defined samples and subjects that span a spectrum of biological activity and biomarker concentrations. Previously-defined guide biomarkers of CNS infection, inflammation and neural injury are useful in categorizing samples for analysis and providing critical biological context for biomarker discovery studies. CSF biomarkers represent an underutilized but valuable approach to understanding the interactions of HIV and the CNS and to more objective diagnosis and assessment of disease activity. Both hypothesis-based and discovery methods can be useful in advancing the definition and use of these biomarkers.

Abstract Background Central nervous system (CNS) infection is a nearly universal feature of untreated systemic HIV infection with a clinical spectrum that ranges from chronic asymptomatic infection to severe cognitive and motor dysfunction. Analysis of cerebrospinal fluid (CSF) has played an important part in defining the character of this evolving infection and response to treatment. To further characterize CNS HIV infection and its effects, we applied advanced high-throughput proteomic methods to CSF to identify novel proteins and their changes with disease progression and treatment. Results After establishing an accurate mass and time (AMT) tag database containing 23,141 AMT tags for CSF peptides, we analyzed 91 CSF samples by LC-MS from 12 HIV-uninfected and 14 HIV-infected subjects studied in the context of initiation of antiretroviral therapy and correlated abundances of identified proteins a) within and between subjects, b) with all other proteins across the entire sample set, and c) with "external" CSF biomarkers of infection (HIV RNA), immune activation (neopterin) and neural injury (neurofilament light chain protein, NFL). We identified a mean of 2,333 +/- 328 (SD) peptides covering 307 +/-16 proteins in the 91 CSF sample set. Protein abundances differed both between and within subjects sampled at different time points and readily separated those with and without HIV infection. Proteins also showed inter-correlations across the sample set that were associated with biologically relevant dynamic processes. One-hundred and fifty proteins showed correlations with the external biomarkers. For example, using a threshold of cross correlation coefficient (Pearson's) ≤ -0.3 and ≥0.3 for potentially meaningful relationships, a total of 99 proteins correlated with CSF neopterin (43 negative and 56 positive correlations) and related principally to neuronal plasticity and survival and to innate immunity. Pathway analysis defined several networks connecting the identified proteins, including one with amyloid precursor protein as a central node. Conclusions Advanced CSF proteomic analysis enabled the identification of an array of novel protein changes across the spectrum of CNS HIV infection and disease. This initial analysis clearly demonstrated the value of contemporary state-of-the-art proteomic CSF analysis as a discovery tool in HIV infection with likely similar application to other neurological inflammatory and degenerative diseases.

Here, we have described and validated a strategy for monitoring skeletal muscle protein synthesis rates in rodents and humans over days or weeks from blood samples. We based this approach on label incorporation into proteins that are synthesized specifically in skeletal muscle and escape into the circulation. Heavy water labeling combined with sensitive tandem mass spectrometric analysis allowed integrated synthesis rates of proteins in muscle tissue across the proteome to be measured over several weeks. Fractional synthesis rate (FSR) of plasma creatine kinase M-type (CK-M) and carbonic anhydrase 3 (CA-3) in the blood, more than 90% of which is derived from skeletal muscle, correlated closely with FSR of CK-M, CA-3, and other proteins of various ontologies in skeletal muscle tissue in both rodents and humans. Protein synthesis rates across the muscle proteome generally changed in a coordinate manner in response to a sprint interval exercise training regimen in humans and to denervation or clenbuterol treatment in rodents. FSR of plasma CK-M and CA-3 revealed changes and interindividual differences in muscle tissue proteome dynamics. In human subjects, sprint interval training primarily stimulated synthesis of structural and glycolytic proteins. Together, our results indicate that this approach provides a virtual biopsy, sensitively revealing individualized changes in proteome-wide synthesis rates in skeletal muscle without a muscle biopsy. Accordingly, this approach has potential applications for the diagnosis, management, and treatment of muscle disorders., Introduction Disorders of muscle mass, quality, and function cause substantial and increasing morbidity and mortality. Sarcopenia, cachexia, and frailty are growing in importance in association with aging demographics world-wide (1, [...]

A novel method is described for G protein transducin (Gt) purification from native bovine rod photoreceptor membranes without subunit dissociation caused by exposure to photoactivated rhodopsin (Rho*). The results shed light on differences in conformation and heterotrimer complex formation between the two preparations or altered stability of the reconstituted Gt that assembles differently than the native protein.

Abstract: Metarhodopsin-II is the light-excited form of rhodopsin that triggers G protein function. Metarhodopsin-II is stabilized when the N-terminus of the carboxyl (340–350) tail peptide of the α-subunit of transducin (Gtα) is crosslinked to rhodopsin cysteine 140 or the 340–350 peptide C-terminus of Gtα is crosslinked to rhodopsin cysteine 316. When the N-terminus of the peptide is coupled to C316 the MI↔MII equilibrium is not affected. The evidence suggests that the N-terminus of the 340–350 region of Gtα is located near C140 when transducin stabilizes metarhodopsin-II and alternative explanations are suggested for the effectiveness of the 340–350 Gtα tail peptide when bound to C316. [Copyright &y& Elsevier]

Cultures of the cyanobacterial genus Cyanothece have been shown to produce high levels of biohydrogen. These strains are diazotrophic and undergo pronounced diurnal cycles when grown under N2-fixing conditions in light-dark cycles. We seek to better understand the way in which proteins respond to these diurnal changes, and we performed quantitative proteome analysis of Cyanothece sp. strains ATCC 51142 and PCC 7822 grown under 8 different nutritional conditions. Nitrogenase expression was limited to N2-fixing conditions, and in the absence of glycerol, nitrogenase gene expression was linked to the dark period. However, glycerol induced expression of nitrogenase during part of the light period, together with cytochrome c oxidase (Cox), glycogen phosphorylase (Glp), and glycolytic and pentose phosphate pathway (PPP) enzymes. This indicated that nitrogenase expression in the light was facilitated via higher levels of respiration and glycogen breakdown. Key enzymes of the Calvin cycle were inhibited in Cyanothece ATCC 51142 in the presence of glycerol under H2-producing conditions, suggesting a competition between these sources of carbon. However, in Cyanothece PCC 7822, the Calvin cycle still played a role in cofactor recycling during H2 production. Our data comprise the first comprehensive profiling of proteome changes in Cyanothece PCC 7822 and allow an in-depth comparative analysis of major physiological and biochemical processes that influence H2 production in both strains. Our results revealed many previously uncharacterized proteins that may play a role in nitrogenase activity and in other metabolic pathways and may provide suitable targets for genetic manipulation that would lead to improvement of large-scale H2 production. [ABSTRACT FROM AUTHOR]

The catalytic core of the phagocyte NADPH oxidase is a heterodimeric integral membrane protein (flavocytochrome b (Cyt b)) that generates superoxide and initiates a cascade of reactive oxygen species critical for the host inflammatory response. In order to facilitate structural characterization, the present study reports the first direct analysis of human phagocyte Cyt b by matrix-assisted laser desorption/ionization and nanoelectrospray mass spectrometry. Mass analysis of in-gel tryptic digest samples provided 73% total sequence coverage of the gp91phox subunit, including three of the six proposed transmembrane domains. Similar analysis of the p22phox subunit provided 72% total sequence coverage, including assignment of the hydrophobic N-terminal region and residues that are polymorphic in the human population. To initiate mass analysis of Cyt b post-translational modifications, the isolated gp91phox subunit was subject to sequential in-gel digestion with Flavobacterium meningosepticum peptide N-glycosidase F and trypsin, with matrix-assisted laser desorption/ionization and liquid chromatography-mass spectrometry/mass spectrometry used to demonstrate that Asn-132, -149, and -240 are genuinely modified by N-linked glycans in human neutrophils. Since the PLB-985 cell line represents an important model system for analysis of the NADPH oxidase, methods were developed for the purification of Cyt b from PLB-985 membrane fractions in order to confirm the appropriate modification of N-linked glycosylation sites on the recombinant gp91phox subunit. This study reports extensive sequence coverage of the integral membrane protein Cyt b by mass spectrometry and provides analytical methods that will be useful for evaluating post-translational modifications involved in the regulation of superoxide production. [ABSTRACT FROM AUTHOR]