Integrated plasma proteomics identifies tuberculosis-specific diagnostic biomarkers.

BACKGROUNDNovel biomarkers to identify infectious patients transmitting Mycobacterium tuberculosis are urgently needed to control the global tuberculosis (TB) pandemic. We hypothesized that proteins released into the plasma in active pulmonary TB are clinically useful biomarkers to distinguish TB cases from healthy individuals and patients with other respiratory infections.METHODSWe applied a highly sensitive non-depletion tandem mass spectrometry discovery approach to investigate plasma protein expression in pulmonary TB cases compared to healthy controls in South African and Peruvian cohorts. Bioinformatic analysis using linear modeling and network correlation analyses identified 118 differentially expressed proteins, significant through 3 complementary analytical pipelines. Candidate biomarkers were subsequently analyzed in 2 validation cohorts of differing ethnicity using antibody-based proximity extension assays.RESULTSTB-specific host biomarkers were confirmed. A 6-protein diagnostic panel, comprising FETUB, FCGR3B, LRG1, SELL, CD14, and ADA2, differentiated patients with pulmonary TB from healthy controls and patients with other respiratory infections with high sensitivity and specificity in both cohorts.CONCLUSIONThis biomarker panel exceeds the World Health Organization Target Product Profile specificity criteria for a triage test for TB. The new biomarkers have potential for further development as near-patient TB screening assays, thereby helping to close the case-detection gap that fuels the global pandemic.FUNDINGMedical Research Council (MRC) (MR/R001065/1, MR/S024220/1, MR/P023754/1, and MR/W025728/1); the MRC and the UK Foreign Commonwealth and Development Office; the UK National Institute for Health Research (NIHR); the Wellcome Trust (094000, 203135, and CC2112); Starter Grant for Clinical Lecturers (Academy of Medical Sciences UK); the British Infection Association; the Program for Advanced Research Capacities for AIDS in Peru at Universidad Peruana Cayetano Heredia (D43TW00976301) from the Fogarty International Center at the US NIH; the UK Technology Strategy Board/Innovate UK (101556); the Francis Crick Institute, which receives funding from UKRI-MRC (CC2112); Cancer Research UK (CC2112); and the NIHR Biomedical Research Centre of Imperial College NHS.

Functional glycoproteomics by integrated network assembly and partitioning.

The post-translational modification (PTM) of proteins by O-linked ß-N-acetyl-D-glucosamine (O-GlcNAcylation) is widespread across the proteome during the lifespan of all multicellular organisms. However, nearly all functional studies have focused on individual protein modifications, overlooking the multitude of simultaneous O-GlcNAcylation events that work together to coordinate cellular activities. Here, we describe Networking of Interactors and SubstratEs (NISE), a novel, systems-level approach to rapidly and comprehensively monitor O-GlcNAcylation across the proteome. Our method integrates affinity purification-mass spectrometry (AP-MS) and site-specific chemoproteomic technologies with network generation and unsupervised partitioning to connect potential upstream regulators with downstream targets of O-GlcNAcylation. The resulting network provides a data-rich framework that reveals both conserved activities of O-GlcNAcylation such as epigenetic regulation as well as tissue-specific functions like synaptic morphology. Beyond O-GlcNAc, this holistic and unbiased systems-level approach provides a broadly applicable framework to study PTMs and discover their diverse roles in specific cell types and biological states.

Adaptive exchange sustains cullin-RING ubiquitin ligase networks and proper licensing of DNA replication.

Cop9 signalosome (CSN) regulates the function of cullin-RING E3 ubiquitin ligases (CRLs) by deconjugating the ubiquitin-like protein NEDD8 from the cullin subunit. To understand the physiological impact of CSN function on the CRL network and cell proliferation, we combined quantitative mass spectrometry and genome-wide CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa) screens to identify factors that modulate cell viability upon inhibition of CSN by the small molecule CSN5i-3. CRL components and regulators strongly modulated the antiproliferative effects of CSN5i-3, and in addition we found two pathways involved in genome integrity, SCFFBXO5-APC/C-GMNN and CUL4DTL-SETD8, that contribute substantially to the toxicity of CSN inhibition. Our data highlight the importance of CSN-mediated NEDD8 deconjugation and adaptive exchange of CRL substrate receptors in sustaining CRL function and suggest approaches for leveraging CSN inhibition for the treatment of cancer.

Phosphodiesterase type 5 inhibitors enhance chemotherapy in preclinical models of esophageal adenocarcinoma by targeting cancer-associated fibroblasts.

The chemotherapy resistance of esophageal adenocarcinomas (EACs) is underpinned by cancer cell extrinsic mechanisms of the tumor microenvironment (TME). We demonstrate that, by targeting the tumor-promoting functions of the predominant TME cell type, cancer-associated fibroblasts (CAFs) with phosphodiesterase type 5 inhibitors (PDE5i), we can enhance the efficacy of standard-of-care chemotherapy. In ex vivo conditions, PDE5i prevent the transdifferentiation of normal fibroblasts to CAF and abolish the tumor-promoting function of established EAC CAFs. Using shotgun proteomics and single-cell RNA-seq, we reveal PDE5i-specific regulation of pathways related to fibroblast activation and tumor promotion. Finally, we confirm the efficacy of PDE5i in combination with chemotherapy in close-to-patient and in vivo PDX-based model systems. These findings demonstrate that CAFs drive chemotherapy resistance in EACs and can be targeted by repurposing PDE5i, a safe and well-tolerated class of drug administered to millions of patients world-wide to treat erectile dysfunction.

Integrated proteomics and transcriptomics analyses identify novel cell surface markers of HIV latency.

Elimination of the latent HIV cell reservoir may be possible, if the molecular identity of latently infected cells were fully elucidated. We conducted comprehensive molecular profiling, at the protein and RNA levels, of primary T cells latently infected with HIV in vitro. Isobaric labelling quantitative proteomics and RNA sequencing identified 1453 proteins and 618 genes, altered in latently infected cells compared to mock-infected controls (p < 0.05). Biomarker selection was based on results from integrated data analysis. Relative enrichment for latently infected cells was monitored using flow cytometric sorting and the HIV integrant assay. Antibodies against selected proteins, encoded by CEACAM1 and PLXNB2, enabled enrichment of latently infected cells from cell mixtures by 3-10 fold (5.8 average, p < 0.001), comparable to levels obtained with biomarkers reported previously. Individual biomarkers are likely linked to subsets of latently infected cells, and an extended antibody panel will be required to inclusively target the latent HIV reservoir.

Placental uptake and metabolism of 25(OH)vitamin D determine its activity within the fetoplacental unit.

Pregnancy 25-hydroxyvitamin D [25(OH)D] concentrations are associated with maternal and fetal health outcomes. Using physiological human placental perfusion and villous explants, we investigate the role of the placenta in regulating the relationships between maternal 25(OH)D and fetal physiology. We demonstrate active placental uptake of 25(OH)D3 by endocytosis, placental metabolism of 25(OH)D3 into 24,25-dihydroxyvitamin D3 and active 1,25-dihydroxyvitamin D [1,25(OH)2D3], with subsequent release of these metabolites into both the maternal and fetal circulations. Active placental transport of 25(OH)D3 and synthesis of 1,25(OH)2D3 demonstrate that fetal supply is dependent on placental function rather than simply the availability of maternal 25(OH)D3. We demonstrate that 25(OH)D3 exposure induces rapid effects on the placental transcriptome and proteome. These map to multiple pathways central to placental function and thereby fetal development, independent of vitamin D transfer. Our data suggest that the underlying epigenetic landscape helps dictate the transcriptional response to vitamin D treatment. This is the first quantitative study demonstrating vitamin D transfer and metabolism by the human placenta, with widespread effects on the placenta itself. These data demonstrate a complex interplay between vitamin D and the placenta and will inform future interventions using vitamin D to support fetal development and maternal adaptations to pregnancy.

Dyrk1a gene dosage in glutamatergic neurons has key effects in cognitive deficits observed in mouse models of MRD7 and Down syndrome.

Perturbation of the excitation/inhibition (E/I) balance leads to neurodevelopmental diseases including to autism spectrum disorders, intellectual disability, and epilepsy. Loss-of-function mutations in the DYRK1A gene, located on human chromosome 21 (Hsa21,) lead to an intellectual disability syndrome associated with microcephaly, epilepsy, and autistic troubles. Overexpression of DYRK1A, on the other hand, has been linked with learning and memory defects observed in people with Down syndrome (DS). Dyrk1a is expressed in both glutamatergic and GABAergic neurons, but its impact on each neuronal population has not yet been elucidated. Here we investigated the impact of Dyrk1a gene copy number variation in glutamatergic neurons using a conditional knockout allele of Dyrk1a crossed with the Tg(Camk2-Cre)4Gsc transgenic mouse. We explored this genetic modification in homozygotes, heterozygotes and combined with the Dp(16Lipi-Zbtb21)1Yey trisomic mouse model to unravel the consequence of Dyrk1a dosage from 0 to 3, to understand its role in normal physiology, and in MRD7 and DS. Overall, Dyrk1a dosage in postnatal glutamatergic neurons did not impact locomotor activity, working memory or epileptic susceptibility, but revealed that Dyrk1a is involved in long-term explicit memory. Molecular analyses pointed at a deregulation of transcriptional activity through immediate early genes and a role of DYRK1A at the glutamatergic post-synapse by deregulating and interacting with key post-synaptic proteins implicated in mechanism leading to long-term enhanced synaptic plasticity. Altogether, our work gives important information to understand the action of DYRK1A inhibitors and have a better therapeutic approach.

Liver Proteome Profile of Growth Restricted and Appropriately Grown Newborn Wistar Rats Associated With Maternal Undernutrition.

Background: Fetal growth restriction (FGR) has been associated with adverse perinatal outcomes and epigenetic modifications that impact gene expression leading to permanent changes of fetal metabolic pathways and thereby influence development of disease in childhood and adult life. In this study, we investigated the result of maternal food restriction on liver protein expression in Wistar male newborn pups. Materials & Methods: Ten (n = 10) timed pregnant Wistar rats on their 14th day of gestation were randomly assigned to either control (n = 4) or food restricted group (n = 6). The control group had ad libitum access to food. In the food restricted group, maternal diet was limited in a moderate fashion (50%) from day 15 of pregnancy until delivery. All rats delivered spontaneously on day 21 and newborn pups were immediately weighed. Pups born to normally nourished mothers were considered as controls, while pups born to food restricted mothers were subdivided into two groups, based on their birth weight: growth restricted (FGR) and appropriately grown (non-FGR). Rats were euthanized immediately after birth and liver tissues of 11 randomly selected male offspring (FGR n = 4, non-FGR n = 4, control n = 3) were collected and analyzed using quantitative proteomics. Results: In total 6,665 proteins were profiled. Of these, 451 and 751 were differentially expressed in FGR and non-FGR vs. control, respectively, whereas 229 proteins were commonly expressed. Bioinformatics analysis of the differentially expressed proteins (DEPs) in FGR vs. control revealed induction of the super-pathway of cholesterol biosynthesis and inhibition of thyroid hormone metabolism, fatty acid beta oxidation and apelin liver signaling pathway. Analysis of DEPs in non-FGR vs. control groups showed inhibition of thyroid hormone metabolism, fatty acid beta oxidation, and apelin liver signaling pathway. Conclusion: This study demonstrates the impact of prenatal food restriction on the proteomic liver profile of FGR and non-FGR offspring underlying the importance of both prenatal adversities and birth weight on liver-dependent postnatal disease.

N-acetylcysteine, xCT and suppression of Maxi-chloride channel activity in human placenta.

INTRODUCTION: Placental oxidative stress features in pregnancy pathologies but in clinical trials antioxidant supplementation has not improved outcomes. N-acetylcysteine (NAC) stimulates glutathione production and is proposed as a therapeutic agent in pregnancy. However, key elements of N-acetylcysteine biology, including its cellular uptake mechanism, remains unclear. This study explores how the cystine/glutamate transporter xCT may mediate N-acetylcysteine uptake and how N-acetylcysteine alters placental redox status. METHODS: The involvement of xCT in NAC uptake by the human placenta was studied in perfused placenta and Xenopus oocytes. The effect of short-term N-acetylcysteine exposure on the placental villous proteome was determined using LC-MS. The effect of N-acetylcysteine on Maxi-chloride channel activity was investigated in perfused placenta, villous fragments and cell culture. RESULTS: Maternoplacental N-acetylcysteine administration stimulated intracellular glutamate efflux suggesting a role of the exchange transporter xCT, which was localised to the microvillous membrane of the placental syncytiotrophoblast. Placental exposure to a bolus of N-acetylcysteine inhibited subsequent activation of the redox sensitive Maxi-chloride channel independently of glutathione synthesis. Stable isotope quantitative proteomics of placental villi treated with N-acetylcysteine demonstrated changes in pathways associated with oxidative stress, apoptosis and the acute phase response. DISCUSSION: This study suggests that xCT mediates N-acetylcysteine uptake into the placenta and that N-acetylcysteine treatment of placental tissue alters the placental proteome while regulating the redox sensitive Maxi-chloride channel. Interestingly N-acetylcysteine had antioxidant effects independent of the glutathione pathway. Effective placental antioxidant therapy in pregnancy may require maintaining the balance between normalising redox status without inhibiting physiological redox signalling.

The Effect of Prenatal Food Restriction on Brain Proteome in Appropriately Grown and Growth Restricted Male Wistar Rats.

BACKGROUND: Fetal growth restriction (FGR) has been associated with a higher risk of developing adverse perinatal outcomes and distinct neurodevelopmental and neurobehavioral disorders. The aim of the present study was to investigate the impact of prenatal food restriction on the brain proteome in both FGR and appropriately grown rats and to identify potential pathways connecting maternal malnutrition with altered brain development. METHODS: Ten time-dated pregnant Wistar rats were housed individually at their 12th day of gestation. On the 15th day of gestation, the rats were randomly divided into two groups, namely the food restricted one (n = 6) and the control group (n = 4). From days 15 to 21 the control group had unlimited access to food and the food restricted group was given half the amount of food that was on average consumed by the control group, based on measurements taken place the day before. On the 21st day of gestation, all rats delivered spontaneously and after birth all newborn pups of the food restricted group were weighed and matched as appropriately grown (non-FGR) or growth restricted (FGR) and brain tissues were immediately collected. A multiplex experiment was performed analyzing brain tissues from 4 FGR, 4 non-FGR, and 3 control male offspring. Differentially expressed proteins (DEPs) were subjected to bioinformatics analysis in order to identify over-represented processes. RESULTS: Proteomic analysis resulted in the profiling of 3,964 proteins. Gene ontology analysis of the common DEPs using DAVID (https://david.ncifcrf.gov/) showed significant enrichment for terms related to cellular morphology, learning, memory and positive regulation of NF-kappaB signaling. Ingenuity Pathway Analysis showed significant induction of inflammation in FGR pups, whereas significant induction of cell migration and cell spreading were observed in non-FGR pups. CONCLUSION: This study demonstrated that in both FGR and non-FGR neonates, a range of adaptive neurodevelopmental processes takes place, which may result in altered cellular morphology, chronic stress, poor memory and learning outcomes. Furthermore, this study highlighted that not only FGR, but also appropriately grown pups, which have been exposed to prenatal food deprivation may be at increased risk for impaired cognitive and developmental outcomes.

Myogenesis modelled by human pluripotent stem cells: a multi-omic study of Duchenne myopathy early onset.

BACKGROUND: Duchenne muscular dystrophy (DMD) causes severe disability of children and death of young men, with an incidence of approximately 1/5000 male births. Symptoms appear in early childhood, with a diagnosis made mostly around 4 years old, a time where the amount of muscle damage is already significant, preventing early therapeutic interventions that could be more efficient at halting disease progression. In the meantime, the precise moment at which disease phenotypes arise-even asymptomatically-is still unknown. Thus, there is a critical need to better define DMD onset as well as its first manifestations, which could help identify early disease biomarkers and novel therapeutic targets. METHODS: We have used both human tissue-derived myoblasts and human induced pluripotent stem cells (hiPSCs) from DMD patients to model skeletal myogenesis and compared their differentiation dynamics with that of healthy control cells by a comprehensive multi-omic analysis at seven time points. Results were strengthened with the analysis of isogenic CRISPR-edited human embryonic stem cells and through comparisons against published transcriptomic and proteomic datasets from human DMD muscles. The study was completed with DMD knockdown/rescue experiments in hiPSC-derived skeletal muscle progenitor cells and adenosine triphosphate measurement in hiPSC-derived myotubes. RESULTS: Transcriptome and miRnome comparisons combined with protein analyses demonstrated that hiPSC differentiation (i) leads to embryonic/foetal myotubes that mimic described DMD phenotypes at the differentiation endpoint and (ii) homogeneously and robustly recapitulates key developmental steps-mesoderm, somite, and skeletal muscle. Starting at the somite stage, DMD dysregulations concerned almost 10% of the transcriptome. These include mitochondrial genes whose dysregulations escalate during differentiation. We also describe fibrosis as an intrinsic feature of DMD skeletal muscle cells that begins early during myogenesis. All the omics data are available online for exploration through a graphical interface at https://muscle-dmd.omics.ovh/. CONCLUSIONS: Our data argue for an early developmental manifestation of DMD whose onset is triggered before the entry into the skeletal muscle compartment, data leading to a necessary reconsideration of dystrophin roles during muscle development. This hiPSC model of skeletal muscle differentiation offers the possibility to explore these functions as well as find earlier DMD biomarkers and therapeutic targets.

Impact of Maternal Food Restriction on Heart Proteome in Appropriately Grown and Growth-Restricted Wistar-Rat Offspring.

OBJECTIVE: Fetal growth restriction is associated with increased postnatal cardiovascular morbidity. The alterations in heart physiology and structure caused by in utero nutrient deprivation have not been extensively studied. We aim to investigate the impact of maternal food restriction on the cardiac proteome of newborn rats with normal (non-fetal growth-restricted (FGR)) and reduced (FGR) birth weight. METHODS: On day 14 of gestation, 10 timed pregnant rats were randomized into two nutritional groups: (a) Standard laboratory diet and (b) 50% global food restriction. Pups born to food-restricted mothers were subdivided, based on birthweight, into fetal growth-restricted (FGR) and non-FGR, while pups born from normally nourished mothers were considered controls. Rat neonates were euthanized immediately after birth and the hearts of 11 randomly selected male offspring (n = 4 FGR, n = 4 non-FGR, n = 3 control group) were analyzed using quantitative proteomics. RESULTS: In total, 7422 proteins were quantified (q < 0.05). Of these, 1175 were differentially expressed in FGR and 231 in non-FGR offspring vs. control with 151 common differentially expressed proteins (DEPs) between the two groups. Bioinformatics analysis of DEPs in FGR vs. control showed decreased integrin and apelin cardiac fibroblast signaling, decreased muscle contraction and glycolysis, and over-representation of a protein network related to embryonic development, and cell death and survival. CONCLUSION: Our study illustrates the distinct proteomic profile of FGR and non-FGR offspring of food-restricted dams underlying the importance of both prenatal adversities and birth weight in cardiac physiology and development.

LONP1 and mtHSP70 cooperate to promote mitochondrial protein folding.

Most mitochondrial precursor polypeptides are imported from the cytosol into the mitochondrion, where they must efficiently undergo folding. Mitochondrial precursors are imported as unfolded polypeptides. For proteins of the mitochondrial matrix and inner membrane, two separate chaperone systems, HSP60 and mitochondrial HSP70 (mtHSP70), facilitate protein folding. We show that LONP1, an AAA+ protease of the mitochondrial matrix, works with the mtHSP70 chaperone system to promote mitochondrial protein folding. Inhibition of LONP1 results in aggregation of a protein subset similar to that caused by knockdown of DNAJA3, a co-chaperone of mtHSP70. LONP1 is required for DNAJA3 and mtHSP70 solubility, and its ATPase, but not its protease activity, is required for this function. In vitro, LONP1 shows an intrinsic chaperone-like activity and collaborates with mtHSP70 to stabilize a folding intermediate of OXA1L. Our results identify LONP1 as a critical factor in the mtHSP70 folding pathway and demonstrate its proposed chaperone activity.

Identification of new OPA1 cleavage site reveals that short isoforms regulate mitochondrial fusion.

OPA1, a large GTPase of the dynamin superfamily, mediates fusion of the mitochondrial inner membranes, regulates cristae morphology, and maintains respiratory chain function. Inner membrane-anchored long forms of OPA1 (l-OPA1) are proteolytically processed by the OMA1 or YME1L proteases, acting at cleavage sites S1 and S2, respectively, to produce short forms (s-OPA1). In both mice and humans, half of the mRNA splice forms of Opa1 are constitutively processed to yield exclusively s-OPA1. However, the function of s-OPA1 in mitochondrial fusion has been debated, because in some stress conditions, s-OPA1 is dispensable for fusion. By constructing cells in which the Opa1 locus no longer produces transcripts with S2 cleavage sites, we generated a simplified system to identify the new YME1L-dependent site S3 that mediates constitutive and complete cleavage of OPA1. We show that mitochondrial morphology is highly sensitive to the ratio of l-OPA1 to s-OPA1, indicating that s-OPA1 regulates mitochondrial fusion.

Comprehensive plasma proteomic profiling reveals biomarkers for active tuberculosis.

BACKGROUNDTuberculosis (TB) kills more people than any other infection, and new diagnostic tests to identify active cases are required. We aimed to discover and verify novel markers for TB in nondepleted plasma.METHODSWe applied an optimized quantitative proteomics discovery methodology based on multidimensional and orthogonal liquid chromatographic separation combined with high-resolution mass spectrometry to study nondepleted plasma of 11 patients with active TB compared with 10 healthy controls. Prioritized candidates were verified in independent UK (n = 118) and South African cohorts (n = 203).RESULTSWe generated the most comprehensive TB plasma proteome to date, profiling 5022 proteins spanning 11 orders-of-magnitude concentration range with diverse biochemical and molecular properties. We analyzed the predominantly low-molecular weight subproteome, identifying 46 proteins with significantly increased and 90 with decreased abundance (peptide FDR ≤ 1%, q ≤ 0.05). Verification was performed for novel candidate biomarkers (CFHR5, ILF2) in 2 independent cohorts. Receiver operating characteristics analyses using a 5-protein panel (CFHR5, LRG1, CRP, LBP, and SAA1) exhibited discriminatory power in distinguishing TB from other respiratory diseases (AUC = 0.81).CONCLUSIONWe report the most comprehensive TB plasma proteome to date, identifying novel markers with verification in 2 independent cohorts, leading to a 5-protein biosignature with potential to improve TB diagnosis. With further development, these biomarkers have potential as a diagnostic triage test.FUNDINGColciencias, Medical Research Council, Innovate UK, NIHR, Academy of Medical Sciences, Program for Advanced Research Capacities for AIDS, Wellcome Centre for Infectious Diseases Research.

Activin-A limits Th17 pathogenicity and autoimmune neuroinflammation via CD39 and CD73 ectonucleotidases and Hif1-α-dependent pathways.

In multiple sclerosis (MS), Th17 cells are critical drivers of autoimmune central nervous system (CNS) inflammation and demyelination. Th17 cells exhibit functional heterogeneity fostering both pathogenic and nonpathogenic, tissue-protective functions. Still, the factors that control Th17 pathogenicity remain incompletely defined. Here, using experimental autoimmune encephalomyelitis, an established mouse MS model, we report that therapeutic administration of activin-A ameliorates disease severity and alleviates CNS immunopathology and demyelination, associated with decreased activation of Th17 cells. In fact, activin-A signaling through activin-like kinase-4 receptor represses pathogenic transcriptional programs in Th17-polarized cells, while it enhances antiinflammatory gene modules. Whole-genome profiling and in vivo functional studies revealed that activation of the ATP-depleting CD39 and CD73 ectonucleotidases is essential for activin-A-induced suppression of the pathogenic signature and the encephalitogenic functions of Th17 cells. Mechanistically, the aryl hydrocarbon receptor, along with STAT3 and c-Maf, are recruited to promoter elements on Entpd1 and Nt5e (encoding CD39 and CD73, respectively) and other antiinflammatory genes, and control their expression in Th17 cells in response to activin-A. Notably, we show that activin-A negatively regulates the metabolic sensor, hypoxia-inducible factor-1α, and key inflammatory proteins linked to pathogenic Th17 cell states. Of translational relevance, we demonstrate that activin-A is induced in the CNS of individuals with MS and restrains human Th17 cell responses. These findings uncover activin-A as a critical controller of Th17 cell pathogenicity that can be targeted for the suppression of autoimmune CNS inflammation.

Increased plasma CD14 levels 1 year postpartum in women with pre-eclampsia during pregnancy: a case-control plasma proteomics study.

Epidemiological data suggest that pre-eclampsia (PE) is associated with an increased risk of post-delivery metabolic dysregulation. The aim of the present case-control observational study was to examine the global plasma proteomic profile 1 year postpartum in women who developed PE during pregnancy (n = 5) compared to controls (n = 5), in order to identify a novel predictive marker linking PE with long-term metabolic imbalance. Key findings were verified with enzyme-linked immunosorbent assay (ELISA) in a separate cohort (n = 17 women with PE and n = 43 controls). One hundred and seventy-two proteins were differentially expressed in the PE vs. control groups. Gene ontology analysis showed that Inflammatory|Immune responses, Blood coagulation and Metabolism were significantly enriched terms. CD14, mapping to the inflammatory response protein network, was selected for verification based on bibliographic evidence. ELISA measurements showed CD14 to be significantly increased 1 year postpartum in women with PE during pregnancy compared to controls [PE group (median ± SD): 296.5 ± 113.6; control group (median ± SD): 128.9 ± 98.5; Mann-Whitney U test p = 0.0078]. Overall, the identified proteins could provide insight into the long-term disease risk among women with PE during pregnancy and highlight the need for their postpartum monitoring. CD14 could be examined in larger cohorts as a predictive marker of insulin resistance and type II diabetes mellitus among women with PE.

Stable Isotope Analysis of Intact Oxyanions Using Electrospray Quadrupole-Orbitrap Mass Spectrometry.

The stable isotopes of sulfate, nitrate, and phosphate are frequently used to study geobiological processes of the atmosphere, ocean, as well as land. Conventionally, the isotopes of these and other oxyanions are measured by isotope-ratio sector mass spectrometers after conversion into gases. Such methods are prone to various limitations on sensitivity, sample throughput, or precision. In addition, there is no general tool that can analyze several oxyanions or all the chemical elements they contain. Here, we describe a new approach that can potentially overcome some of these limitations based on electrospray hyphenated with Quadrupole Orbitrap mass spectrometry. This technique yields an average accuracy of 1-2‰ for sulfate δ34S and δ18O and nitrate δ15N and δ18O, based on in-house and international standards. Less abundant variants such as δ17O, δ33S, and δ36S, and the 34S-18O "clumped" sulfate can be quantified simultaneously. The observed precision of isotope ratios is limited by the number of ions counted. The counting of rare ions can be accelerated by removing abundant ions with the quadrupole mass filter. Electrospray mass spectrometry (ESMS) exhibits high-throughput and sufficient sensitivity. For example, less than 1 nmol sulfate is required to determine 18O/34S ratios with 0.2‰ precision within minutes. A purification step is recommended for environmental samples as our proposed technique is susceptible to matrix effects. Building upon these initial provisions, new features of the isotopic anatomy of mineral ions can now be explored with ESMS instruments that are increasingly available to bioanalytical laboratories.

Quantitative proteomic profiling of white matter in cases of cerebral amyloid angiopathy reveals upregulation of extracellular matrix proteins and clusterin.

Aims: Cerebral amyloid angiopathy (CAA) is the accumulation of amyloid beta (Aß) in the walls of cerebral arterioles, arteries and capillaries. Changes in the white matter in CAA are observed as hyperintensities and dilated perivascular spaces on MRI suggesting impairment of fluid drainage but the pathophysiology behind these changes is poorly understood. We tested the hypothesis that proteins associated with clearance of Aß peptides are upregulated in the white matter in cases of CAA. Methods: In this study, we compare the quantitative proteomic profile of white matter from post-mortem brains of patients with CAA and age-matched controls in order to gain insight into the cellular processes and key molecules involved in the pathophysiology of CAA. Results: Our proteomic analysis resulted in the profiling of 3,734 proteins (peptide FDR p<0.05). Of these, 189 were differentially expressed in CAA vs. control. Bioinformatics analysis of these proteins showed significant enrichment of proteins related to cell adhesion | cell-matrix interaction, mitochondrial dysfunction and hypoxia. Upregulated proteins in CAA included EMILIN2, COL4A2, TLN1, CLU, HSPG2. Downregulated proteins included DSP, IDE, HBG1. Conclusions: The present study reports an in-depth quantitative proteomic profiling of white matter from patients with CAA, highlighting extracellular matrix proteins and clusterin as key molecules in the pathophysiology of white matter changes in cases of CAA.