Quantifying absolute gene expression profiles reveals distinct regulation of central carbon metabolism genes in yeast

In addition to controlled expression of genes by specific regulatory circuits, the abundance of proteins and transcripts can also be influenced by physiological states of the cell such as growth rate and metabolism. Here we examine the control of gene expression by growth rate and metabolism, by analyzing a multi-omics dataset consisting of absolute-quantitative abundances of the transcriptome, proteome, and amino acids in 22 steady-state yeast cultures. We find that transcription and translation are coordinately controlled by the cell growth rate via RNA polymerase II and ribosome abundance, but they are independently controlled by nitrogen metabolism via amino acid and nucleotide availabilities. Genes in central carbon metabolism, however, are distinctly regulated and do not respond to the cell growth rate or nitrogen metabolism as all other genes. Understanding these effects allows the confounding factors of growth rate and metabolism to be accounted for in gene expression profiling studies

Exosomes in the nose induce immune cell trafficking and harbour an altered protein cargo in chronic airway inflammation

Background: Exosomes are nano-sized extracellular vesicles participating in cell-to-cell communication both in health and disease. However, the knowledge about the functions and molecular composition of exosomes in the upper airways is limited. The aim of the current study was therefore to determine whether nasal exosomes can influence inflammatory cells and to establish the proteome of nasal lavage fluid-derived exosomes in healthy subjects, as well as its alterations in individuals with chronic airway inflammatory diseases [asthma and chronic rhinosinusitis (CRS)]. Methods: Nasal lavage fluid was collected from 14 healthy subjects, 15 subjects with asthma and 13 subjects with asthma/CRS. Exosomes were isolated with differential centrifugation and the proteome was analysed by LC-MS/MS with the application of two exclusion lists as well as using quantitative proteomics. Ingenuity Pathways Analysis and GO Term finder was used to predict the functions associated with the exosomal proteome and a migration assay was used to analyse the effect on immune cells by nasal exosomes. Results: Firstly, we demonstrate that nasal exosomes can induce migration of several immune cells, such as monocytes, neutrophils and NK cells in vitro. Secondly, a mass spectrometry approach, with the application of exclusion lists, was utilised to generate a comprehensive protein inventory of the exosomes from healthy subjects. The use of exclusion lists resulted in the identification of similar to 15 % additional proteins, and increased the confidence in similar to 20 % of identified proteins. In total, 604 proteins were identified in nasal exosomes and the nasal exosomal proteome showed strong associations with immune-related functions, such as immune cell trafficking. Thirdly, a quantitative proteomics approach was used to determine alterations in the exosome proteome as a result of airway inflammatory disease. Serum-associated proteins and mucins were more abundant in the exosomes from subjects with respiratory diseases compared to healthy controls while proteins with antimicrobial functions and barrier-related proteins had decreased expression. Conclusions: Nasal exosomes were shown to induce the migration of innate immune cells, which may be important as the airway epithelium is the first line of defence against pathogens and allergens. The decreased expression in barrier and antimicrobial exosomal proteins in subjects with airway diseases, could possibly contribute to an increased susceptibility to infections, which have important clinical implications in disease progression.11138Ysciescopu

Nitrogen limitation reveals large reserves in metabolic and translational capacities of yeast

Cells maintain reserves in their metabolic and translational capacities as a strategy to quickly respond to changing environments. Here we quantify these reserves by stepwise\ua0reducing nitrogen availability in yeast steady-state chemostat cultures, imposing severe restrictions on total cellular protein and transcript content. Combining multi-omics analysis with metabolic modeling, we find that seven metabolic superpathways maintain >50% metabolic capacity in reserve, with glucose metabolism maintaining >80% reserve capacity. Cells maintain >50% reserve in translational capacity for 2490 out of 3361 expressed genes (74%), with a disproportionately large reserve dedicated to translating metabolic proteins. Finally, ribosome reserves contain up to 30% sub-stoichiometric ribosomal proteins, with activation of reserve translational capacity associated with selective upregulation of 17 ribosomal proteins. Together, our dataset provides a quantitative link between yeast physiology and cellular economics, which could be leveraged in future cell engineering through targeted proteome streamlining

Investigation of human apoB48 metabolism using a new, integrated non-steady-state model of apoB48 and apoB100 kinetics

Background Triglyceride-rich lipoproteins and their remnants have emerged as major risk factors for cardiovascular disease. New experimental approaches are required that permit simultaneous investigation of the dynamics of chylomicrons (CM) and apoB48 metabolism and of apoB100 in very low-density lipoproteins (VLDL). Methods Mass spectrometric techniques were used to determine the masses and tracer enrichments of apoB48 in the CM, VLDL1 and VLDL2 density intervals. An integrated non-steady-state multicompartmental model was constructed to describe the metabolism of apoB48- and apoB100-containing lipoproteins following a fat-rich meal, as well as during prolonged fasting. Results The kinetic model described the metabolism of apoB48 in CM, VLDL1 and VLDL2. It predicted a low level of basal apoB48 secretion and, during fat absorption, an increment in apoB48 release into not only CM but also directly into VLDL1 and VLDL2. ApoB48 particles with a long residence time were present in VLDL, and in subjects with high plasma triglycerides, these lipoproteins contributed to apoB48 measured during fasting conditions. Basal apoB48 secretion was about 50 mg day?1, and the increment during absorption was about 230 mg day?1. The fractional catabolic rates for apoB48 in VLDL1 and VLDL2 were substantially lower than for apoB48 in CM. Discussion This novel non-steady-state model integrates the metabolic properties of both apoB100 and apoB48 and the kinetics of triglyceride. The model is physiologically relevant and provides insight not only into apoB48 release in the basal and postabsorptive states but also into the contribution of the intestine to VLDL pool size and kinetics.Peer reviewe

The chaperone protein clusterin may serve as a cerebrospinal fluid biomarker for chronic spinal cord disorders in the dog

Chronic spinal cord dysfunction occurs in dogs as a consequence of diverse aetiologies, including long-standing spinal cord compression and insidious neurodegenerative conditions. One such neurodegenerative condition is canine degenerative myelopathy (DM), which clinically is a challenge to differentiate from other chronic spinal cord conditions. Although the clinical diagnosis of DM can be strengthened by the identification of the Sod1 mutations that are observed in affected dogs, genetic analysis alone is insufficient to provide a definitive diagnosis. There is a requirement to identify biomarkers that can differentiate conditions with a similar clinical presentation, thus facilitating patient diagnostic and management strategies. A comparison of the cerebrospinal fluid (CSF) protein gel electrophoresis profile between idiopathic epilepsy (IE) and DM identified a protein band that was more prominent in DM. This band was subsequently found to contain a multifunctional protein clusterin (apolipoprotein J) that is protective against endoplasmic reticulum (ER) stress-mediated apoptosis, oxidative stress, and also serves as an extracellular chaperone influencing protein aggregation. Western blot analysis of CSF clusterin confirmed elevated levels in DM compared to IE (p < 0.05). Analysis of spinal cord tissue from DM and control material found that clusterin expression was evident in neurons and that the clusterin mRNA levels from tissue extracts were elevated in DM compared to the control. The plasma clusterin levels was comparable between these groups. However, a comparison of clusterin CSF levels in a number of neurological conditions found that clusterin was elevated in both DM and chronic intervertebral disc disease (cIVDD) but not in meningoencephalitis and IE. These findings indicate that clusterin may potentially serve as a marker for chronic spinal cord disease in the dog; however, additional markers are required to differentiate DM from a concurrent condition such as cIVDD

NIST interlaboratory study on glycosylation analysis of monoclonal antibodies : comparison of results from diverse analytical methods

Glycosylation is a topic of intense current interest in the development of biopharmaceuticals since it is related to drug safety and efficacy. This work describes results of an interlaboratory study on the glycosylation of the Primary Sample (PS) of NISTmAb, a monoclonal antibody reference material. Seventy‑six laboratories from industry, university, research, government, and hospital sectors in Europe, North America, Asia, and Australia submitted a total of 103 reports on glycan distributions. The principal objective of this study was to report and compare results for the full range of analytical methods presently used in the glycosylation  analysis of mAbs. Therefore, participation was unrestricted, with laboratories choosing their own measurement techniques. Protein glycosylation was determined in various ways, including at the level of intact mAb, protein fragments, glycopeptides, or released glycans, using a wide variety of methods for derivatization, separation, identification, and quantification. Consequently, the diversity of results was enormous, with the number of glycan compositions identified by each laboratory ranging from 4 to 48. In total, one hundred sixteen glycan compositions were reported, of which 57 compositions could be assigned consensus abundance values. These consensus medians provide community-derived values for NISTmAb PS. Agreement with the consensus medians did not depend on the specific method or laboratory type.. The study provides a view of the current state-of-the-art for biologic glycosylation measurement and suggests a clear need for harmonization of glycosylation analysis methods

Plasma Apolipoprotein Levels Are Associated with Cognitive Status and Decline in a Community Cohort of Older Individuals

<div><h3>Objectives</h3><p>Apolipoproteins have recently been implicated in the etiology of Alzheimer’s disease (AD). In particular, Apolipoprotein J (ApoJ or clusterin) has been proposed as a biomarker of the disease at the pre-dementia stage. We examined a group of apolipoproteins, including ApoA1, ApoA2, ApoB, ApoC3, ApoE, ApoH and ApoJ, in the plasma of a longitudinal community based cohort.</p> <h3>Methods</h3><p>664 subjects (257 with Mild Cognitive Impairment [MCI] and 407 with normal cognition), mean age 78 years, from the Sydney Memory and Aging Study (MAS) were followed up over two years. Plasma apolipoprotein levels at baseline (Wave 1) were measured using a multiplex bead fluorescence immunoassay technique.</p> <h3>Results</h3><p>At Wave 1, MCI subjects had lower levels of ApoA1, ApoA2 and ApoH, and higher levels of ApoE and ApoJ, and a higher ApoB/ApoA1 ratio. Carriers of the apolipoprotein E ε4 allele had significantly lower levels of plasma ApoE, ApoC3 and ApoH and a significantly higher level of ApoB. Global cognitive scores were correlated positively with ApoH and negatively with ApoJ levels. ApoJ and ApoE levels were correlated negatively with grey matter volume and positively with cerebrospinal fluid (CSF) volume on MRI. Lower ApoA1, ApoA2 and ApoH levels, and higher ApoB/ApoA1 ratio, increased the risk of cognitive decline over two years in cognitively normal individuals. ApoA1 was the most significant predictor of decline. These associations remained after statistically controlling for lipid profile. Higher ApoJ levels predicted white matter atrophy over two years.</p> <h3>Conclusions</h3><p>Elderly individuals with MCI have abnormal apolipoprotein levels, which are related to cognitive function and volumetric MRI measures cross-sectionally and are predictive of cognitive impairment in cognitively normal subjects. ApoA1, ApoH and ApoJ are potential plasma biomarkers of cognitive decline in non-demented elderly individuals.</p> </div