ABSTRACT
We aimed to identify serum biomarkers that predict knee osteoarthritis (OA) before the appearance of radiographic abnormalities in a cohort of 200 women. As few as six serum peptides, corresponding to six proteins, reached AUC 77% probability to distinguish those who developed OA from age-matched individuals who did not develop OA up to 8 years later. Prediction based on these blood biomarkers was superior to traditional prediction based on age and BMI (AUC 51%) or knee pain (AUC 57%). These results identify a prolonged molecular derangement of joint tissue before the onset of radiographic OA abnormalities consistent with an unresolved acute phase response. Among all 24 protein biomarkers predicting incident knee OA, the majority (58%) also predicted knee OA progression, revealing the existence of a pathophysiological "OA continuum" based on considerable similarity in the molecular pathophysiology of the progression to incident OA and the progression of established OA.
Subject(s)
Biomarkers , Disease Progression , Osteoarthritis, Knee , Humans , Biomarkers/blood , Female , Osteoarthritis, Knee/metabolism , Osteoarthritis, Knee/diagnostic imaging , Osteoarthritis, Knee/physiopathology , Middle Aged , AgedABSTRACT
Objective: To further validate a serum proteomics panel for predicting radiographic (structural) knee OA progression. Design: Serum peptides were targeted by multiple-reaction-monitoring mass spectrometry in the New York University cohort (n â= â104). Knee OA progression was defined as joint space narrowing ≥1 in the tibiofemoral compartment of one knee per study participant over a 24-month follow-up. The discriminative ability of an 11-peptide panel was evaluated by multivariable logistic regression and area under the receiver operating characteristic curve (AUC), without and with demographic characteristics of age, sex, and body mass index. The association of each peptide with OA progression was assessed by odds ratios (OR) in multivariable logistic regression models adjusted for demographics. Results: The cohort included 46 (44%) knee OA progressors. The panel of 11 peptides alone yielded AUC â= â0.66 (95% CI [0.55, 0.77]) for discriminating progressors from non-progressors; demographic traits alone yielded AUC â= â0.66 (95% CI [0.55, 0.77]). Together the 11 peptides and demographics yielded AUC â= â0.72 (95% CI [0.62, 0.83]). CRAC1 had the highest odds for predicting OA progression (OR 2.014, 95% CI [0.996, 4.296], p â= â0.058). Conclusions: We evaluated a parsimonious serum proteomic panel and found it to be a good discriminator of knee radiographic OA progression from non-progression. Since these biomarkers are quantifiable in serum, they could be deployed relatively easily to provide a simple, cost-effective strategy for identifying and monitoring individuals at high risk of knee OA progression.
ABSTRACT
We aimed to identify markers in blood (serum) to predict clinically relevant knee osteoarthritis (OA) progression defined as the combination of both joint structure and pain worsening over 48 months. A set of 15 serum proteomic markers corresponding to 13 total proteins reached an area under the receiver operating characteristic curve (AUC) of 73% for distinguishing progressors from nonprogressors in a cohort of 596 individuals with knee OA. Prediction based on these blood markers was far better than traditional prediction based on baseline structural OA and pain severity (59%) or the current "best-in-class" biomarker for predicting OA progression, urinary carboxyl-terminal cross-linked telopeptide of type II collagen (58%). The generalizability of the marker set was confirmed in a second cohort of 86 individuals that yielded an AUC of 70% for distinguishing joint structural progressors. Blood is a readily accessible biospecimen whose analysis for these biomarkers could facilitate identification of individuals for clinical trial enrollment and those most in need of treatment.
Subject(s)
Biomarkers , Osteoarthritis, Knee , Humans , Biomarkers/blood , Disease Progression , Osteoarthritis, Knee/diagnosis , Osteoarthritis, Knee/metabolism , Pain , Proteomics , Clinical Trials as TopicABSTRACT
Cryptococcal Meningitis (CM) is uniformly fatal if not treated, and treatment options are limited. We previously reported on the activity of APX2096, the prodrug of the novel Gwt1 inhibitor APX2039, in a mouse model of CM. Here, we investigated the efficacy of APX2039 in mouse and rabbit models of CM. In the mouse model, the controls had a mean lung fungal burden of 5.95 log10 CFU/g, whereas those in the fluconazole-, amphotericin B-, and APX2039-treated mice were 3.56, 4.59, and 1.50 log10 CFU/g, respectively. In the brain, the control mean fungal burden was 7.97 log10 CFU/g, while the burdens were 4.64, 7.16, and 1.44 log10 CFU/g for treatment with fluconazole, amphotericin B, and APX2039, respectively. In the rabbit model of CM, the oral administration of APX2039 at 50 mg/kg of body weight twice a day (BID) resulted in a rapid decrease in the cerebrospinal fluid (CSF) fungal burden, and the burden was below the limit of detection by day 10 postinfection. The effective fungicidal activity (EFA) was -0.66 log10 CFU/mL/day, decreasing from an average of 4.75 log10 CFU/mL to 0 CFU/mL, over 8 days of therapy, comparing favorably with good clinical outcomes in humans associated with reductions of the CSF fungal burden of -0.4 log10 CFU/mL/day, and, remarkably, 2-fold the EFA of amphotericin B deoxycholate in this model (-0.33 log10 CFU/mL/day). A total drug exposure of the area under the concentration-time curve from 0 to 24 h (AUC0-24) of 25 to 50 mg · h/L of APX2039 resulted in near-maximal antifungal activity. These data support the further preclinical and clinical evaluation of APX2039 as a new oral fungicidal monotherapy for the treatment of CM. IMPORTANCE Cryptococcal meningitis (CM) is a fungal disease with significant global morbidity and mortality. The gepix Gwt1 inhibitors are a new class of antifungal drugs. Here, we demonstrated the efficacy of APX2039, the second member of the gepix class, in rabbit and mouse models of cryptococcal meningitis. We also analyzed the drug levels in the blood and cerebrospinal fluid in the highly predictive rabbit model and built a mathematical model to describe the behavior of the drug with respect to the elimination of the fungal pathogen. We demonstrated that the oral administration of APX2039 resulted in a rapid decrease in the CSF fungal burden, with an effective fungicidal activity of -0.66 log10 CFU/mL/day, comparing favorably with good clinical outcomes in humans associated with reductions of -0.4 log10 CFU/mL/day. The drug APX2039 had good penetration of the central nervous system and is an excellent candidate for future clinical testing in humans for the treatment of CM.
Subject(s)
Amphotericin B , Meningitis, Cryptococcal , Humans , Rabbits , Animals , Mice , Amphotericin B/therapeutic use , Meningitis, Cryptococcal/microbiology , Antifungal Agents/pharmacology , Fluconazole/therapeutic use , Drug Therapy, CombinationABSTRACT
Cellular recycling via autophagy-associated proteins is a key catabolic pathway critical to invasive fungal pathogen growth and virulence in the nutrient-limited host environment. Protein kinase A (PKA) is vital for the growth and virulence of numerous fungal pathogens. However, the underlying basis for its regulation of pathogenesis remains poorly understood in any species. Our Aspergillus fumigatus PKA-dependent whole proteome and phosphoproteome studies employing advanced mass spectroscopic approaches identified numerous previously undefined PKA-regulated proteins in catabolic pathways. Here, we demonstrate reciprocal inhibition of autophagy and PKA activity, and identify 16 autophagy-associated proteins as likely novel PKA-regulated effectors. We characterize the novel PKA-phosphoregulated sorting nexin Atg20, and demonstrate its importance for growth, cell wall stress response, and virulence of A. fumigatus in a murine infection model. Additionally, we identify physical and functional interaction of Atg20 with previously characterized sorting nexin Atg24. Furthermore, we demonstrate the importance of additional uncharacterized PKA-regulated putative autophagy-associated proteins to hyphal growth. Our data presented here indicate that PKA regulates the autophagy pathway much more extensively than previously known, including targeting of novel effector proteins with fungal-specific functions important for invasive disease.
ABSTRACT
OBJECTIVE: To design and establish a prospective biospecimen repository that integrates multi-omics assays with clinical data to study mechanisms of controlled injury and healing. BACKGROUND: Elective surgery is an opportunity to understand both the systemic and focal responses accompanying controlled and well-characterized injury to the human body. The overarching goal of this ongoing project is to define stereotypical responses to surgical injury, with the translational purpose of identifying targetable pathways involved in healing and resilience, and variations indicative of aberrant peri-operative outcomes. METHODS: Clinical data from the electronic medical record combined with large-scale biological data sets derived from blood, urine, fecal matter, and tissue samples are collected prospectively through the peri-operative period on patients undergoing 14 surgeries chosen to represent a range of injury locations and intensities. Specimens are subjected to genomic, transcriptomic, proteomic, and metabolomic assays to describe their genetic, metabolic, immunologic, and microbiome profiles, providing a multidimensional landscape of the human response to injury. RESULTS: The highly multiplexed data generated includes changes in over 28,000 mRNA transcripts, 100 plasma metabolites, 200 urine metabolites, and 400 proteins over the longitudinal course of surgery and recovery. In our initial pilot dataset, we demonstrate the feasibility of collecting high quality multi-omic data at pre- and postoperative time points and are already seeing evidence of physiologic perturbation between timepoints. CONCLUSIONS: This repository allows for longitudinal, state-of-the-art geno-mic, transcriptomic, proteomic, metabolomic, immunologic, and clinical data collection and provides a rich and stable infrastructure on which to fuel further biomedical discovery.
Subject(s)
Computational Biology , Proteomics , Genomics , Humans , Metabolomics , Prospective Studies , Proteomics/methodsABSTRACT
Immune-based metabolic reprogramming of arginine utilization in the brain contributes to the neuronal pathology associated with Alzheimer's disease (AD). To enable our long-term goals of differentiation of AD mouse model genotypes, ages, and sexes based on activity of this pathway, we describe here the novel dosing (using uniformly labeled (13C615N4) arginine) and analysis methods using capillary electrophoresis high-resolution accurate-mass mass spectrometry for isotope tracing of metabolic products of arginine. We developed a pseudoprimed infusion-dosing regimen, using repeated injections, to achieve a steady state of uniformly labeled arginine in 135-195 min post bolus dose. Incorporation of stable isotope labeled carbon and nitrogen from uniformly labeled arginine into a host of downstream metabolites was measured in vivo in mice using serially sampled dried blood spots from the tail. In addition to the dried blood spot time course samples, total isotope incorporation into arginine-related metabolites was measured in the whole brain and plasma after 285 min. Preliminary demonstration of the technique identified differences isotope incorporation in arginine metabolites between male and female mice in a mouse-model of sporadic Alzheimer's disease (APOE4/huNOS2). The technique described herein will permit arginine pathway activity differentiation between mouse genotypes, ages, sexes, or drug treatments in order to elucidate the contribution of this pathway to Alzheimer's disease.
Subject(s)
Alzheimer Disease/metabolism , Arginine/analysis , Electrophoresis, Capillary/methods , Mass Spectrometry/methods , Alzheimer Disease/blood , Animals , Apolipoprotein E4/genetics , Arginine/blood , Arginine/chemistry , Brain/metabolism , Carbon Isotopes/analysis , Carbon Isotopes/pharmacokinetics , Disease Models, Animal , Female , Humans , Isotope Labeling , Male , Mice, Transgenic , Nitric Oxide Synthase Type II/genetics , Nitrogen Isotopes/analysis , Nitrogen Isotopes/pharmacokinetics , Proof of Concept StudyABSTRACT
Branched-chain amino acids (BCAA) and their cognate α-ketoacids (BCKA) are elevated in an array of cardiometabolic diseases. Here we demonstrate that the major metabolic fate of uniformly-13C-labeled α-ketoisovalerate ([U-13C]KIV) in the heart is reamination to valine. Activation of cardiac branched-chain α-ketoacid dehydrogenase (BCKDH) by treatment with the BCKDH kinase inhibitor, BT2, does not impede the strong flux of [U-13C]KIV to valine. Sequestration of BCAA and BCKA away from mitochondrial oxidation is likely due to low levels of expression of the mitochondrial BCAA transporter SLC25A44 in the heart, as its overexpression significantly lowers accumulation of [13C]-labeled valine from [U-13C]KIV. Finally, exposure of perfused hearts to levels of BCKA found in obese rats increases phosphorylation of the translational repressor 4E-BP1 as well as multiple proteins in the MEK-ERK pathway, leading to a doubling of total protein synthesis. These data suggest that elevated BCKA levels found in obesity may contribute to pathologic cardiac hypertrophy via chronic activation of protein synthesis.
Subject(s)
Amino Acids, Branched-Chain/metabolism , Heart/physiology , Hemiterpenes/metabolism , Keto Acids/metabolism , Animals , Male , Mice , Mice, Inbred C57BL , Mitochondria/metabolism , Obesity/metabolism , Rats , Valine/metabolismABSTRACT
Selective serotonin reuptake inhibitors (SSRIs) are the first-line treatment for major depressive disorder (MDD), yet their mechanisms of action are not fully understood and their therapeutic benefit varies among individuals. We used a targeted metabolomics approach utilizing a panel of 180 metabolites to gain insights into mechanisms of action and response to citalopram/escitalopram. Plasma samples from 136 participants with MDD enrolled into the Mayo Pharmacogenomics Research Network Antidepressant Medication Pharmacogenomic Study (PGRN-AMPS) were profiled at baseline and after 8 weeks of treatment. After treatment, we saw increased levels of short-chain acylcarnitines and decreased levels of medium-chain and long-chain acylcarnitines, suggesting an SSRI effect on ß-oxidation and mitochondrial function. Amines-including arginine, proline, and methionine sulfoxide-were upregulated while serotonin and sarcosine were downregulated, suggesting an SSRI effect on urea cycle, one-carbon metabolism, and serotonin uptake. Eighteen lipids within the phosphatidylcholine (PC aa and ae) classes were upregulated. Changes in several lipid and amine levels correlated with changes in 17-item Hamilton Rating Scale for Depression scores (HRSD17). Differences in metabolic profiles at baseline and post-treatment were noted between participants who remitted (HRSD17 ≤ 7) and those who gained no meaningful benefits (<30% reduction in HRSD17). Remitters exhibited (a) higher baseline levels of C3, C5, alpha-aminoadipic acid, sarcosine, and serotonin; and (b) higher week-8 levels of PC aa C34:1, PC aa C34:2, PC aa C36:2, and PC aa C36:4. These findings suggest that mitochondrial energetics-including acylcarnitine metabolism, transport, and its link to ß-oxidation-and lipid membrane remodeling may play roles in SSRI treatment response.
Subject(s)
Depressive Disorder, Major , Amines/therapeutic use , Antidepressive Agents/therapeutic use , Carnitine/analogs & derivatives , Citalopram/therapeutic use , Depression , Depressive Disorder, Major/drug therapy , Humans , Lipids , Selective Serotonin Reuptake Inhibitors/therapeutic useABSTRACT
BACKGROUND: Postoperative cognitive dysfunction (POCD), a syndrome of cognitive deficits occurring 1-12 months after surgery primarily in older patients, is associated with poor postoperative outcomes. POCD is hypothesized to result from neuroinflammation; however, the pathways involved remain unclear. Unbiased proteomic analyses have been used to identify neuroinflammatory pathways in multiple neurologic diseases and syndromes but have not yet been applied to POCD. OBJECTIVE: To utilize unbiased mass spectrometry-based proteomics to identify potential neuroinflammatory pathways underlying POCD. METHODS: Unbiased LC-MS/MS proteomics was performed on immunodepleted cerebrospinal fluid (CSF) samples obtained before, 24 hours after, and 6 weeks after major non-cardiac surgery in older adults who did (nâ=â8) or did not develop POCD (nâ=â6). Linear mixed models were used to select peptides and proteins with intensity differences for pathway analysis. RESULTS: Mass spectrometry quantified 8,258 peptides from 1,222 proteins in >â50%of patient samples at all three time points. Twelve peptides from 11 proteins showed differences in expression over time between patients with versus withoutPOCD (qâ<â0.05), including proteins previously implicated in neurodegenerative disease pathophysiology. Additionally, 283 peptides from 182 proteins were identified with trend-level differences (qâ<â0.25) in expression over time between these groups. Among these, pathway analysis revealed that 50 were from 17 proteins mapping to complement and coagulation pathways (qâ=â2.44*10-13). CONCLUSION: These data demonstrate the feasibility of performing unbiased mass spectrometry on perioperative CSF samples to identify pathways associated with POCD. Additionally, they provide hypothesis-generating evidence for CSF complement and coagulation pathway changes in patients with POCD.
Subject(s)
Biomarkers/cerebrospinal fluid , Postoperative Cognitive Complications/cerebrospinal fluid , Aged , Case-Control Studies , Cohort Studies , Female , Humans , Male , Proteome/analysis , Tandem Mass SpectrometryABSTRACT
The benzdiimidazole NAB2 rescues α-synuclein-associated trafficking defects associated with early onset Parkinson's disease in a Nedd4-dependent manner. Despite identification of E3 ubiquitin ligase Nedd4 as a putative target of NAB2, its molecular mechanism of action has not been elucidated. As such, the effect of NAB2 on Nedd4 activity and specificity was interrogated through biochemical, biophysical, and proteomic analyses. NAB2 was found to bind Nedd4 (KDapp = 42 nM), but this binding is side chain mediated and does not alter its conformation or ubiquitination kinetics in vitro. Nedd4 co-localizes with trafficking organelles, and NAB2 exposure did not alter its co-localization. Ubiquitin enrichment coupled proteomics revealed that NAB2 stimulates ubiquitination of trafficking-associated proteins, most likely through modulating the substrate specificity of Nedd4, providing a putative protein network involved in the NAB2 mechanism and revealing trafficking scaffold protein TFG as a Nedd4 substrate.
Subject(s)
Nedd4 Ubiquitin Protein Ligases/antagonists & inhibitors , Parkinson Disease/drug therapy , Small Molecule Libraries/pharmacology , Cell Line, Tumor , Female , Humans , Nedd4 Ubiquitin Protein Ligases/isolation & purification , Nedd4 Ubiquitin Protein Ligases/metabolism , Parkinson Disease/metabolism , Signal Transduction/drug effectsABSTRACT
Protein kinase A (PKA) signaling plays a critical role in the growth and development of all eukaryotic microbes. However, few direct targets have been characterized in any organism. The fungus Aspergillus fumigatus is a leading infectious cause of death in immunocompromised patients, but the specific molecular mechanisms responsible for its pathogenesis are poorly understood. We used this important pathogen as a platform for a comprehensive and multifaceted interrogation of both the PKA-dependent whole proteome and phosphoproteome in order to elucidate the mechanisms through which PKA signaling regulates invasive microbial disease. Employing advanced quantitative whole-proteomic and phosphoproteomic approaches with two complementary phosphopeptide enrichment strategies, coupled to an independent PKA interactome analysis, we defined distinct PKA-regulated pathways and identified novel direct PKA targets contributing to pathogenesis. We discovered three previously uncharacterized virulence-associated PKA effectors, including an autophagy-related protein, Atg24; a CCAAT-binding transcriptional regulator, HapB; and a CCR4-NOT complex-associated ubiquitin ligase, Not4. Targeted mutagenesis, combined with in vitro kinase assays, multiple murine infection models, structural modeling, and molecular dynamics simulations, was employed to characterize the roles of these new PKA targets in growth, environmental and antimicrobial stress responses, and pathogenesis in a mammalian system. We also elucidated the molecular mechanisms of PKA regulation for these effectors by defining the functionality of phosphorylation at specific PKA target sites. We have comprehensively characterized the PKA-dependent phosphoproteome and validated PKA targets as direct regulators of infectious disease for the first time in any pathogen, providing new insights into PKA signaling and control over microbial pathogenesis.IMPORTANCE PKA is essential for the virulence of eukaryotic human pathogens. Understanding PKA signaling mechanisms is therefore fundamental to deciphering pathogenesis and developing novel therapies. Despite its ubiquitous necessity, specific PKA effectors underlying microbial disease remain unknown. To address this fundamental knowledge gap, we examined the whole-proteomic and phosphoproteomic impacts of PKA on the deadly fungal pathogen Aspergillus fumigatus to uncover novel PKA targets controlling growth and virulence. We also defined the functional consequences of specific posttranslational modifications of these target proteins to characterize the molecular mechanisms of pathogenic effector regulation by PKA. This study constitutes the most comprehensive analysis of the PKA-dependent phosphoproteome of any human pathogen and proposes new and complex roles played by PKA signaling networks in governing infectious disease.
Subject(s)
Aspergillosis/microbiology , Aspergillus fumigatus/enzymology , Aspergillus fumigatus/pathogenicity , Cyclic AMP-Dependent Protein Kinases/metabolism , Fungal Proteins/metabolism , Proteome/metabolism , Animals , Aspergillus fumigatus/genetics , Aspergillus fumigatus/metabolism , Cyclic AMP-Dependent Protein Kinases/genetics , Fungal Proteins/genetics , Humans , Mice , Phosphorylation , Proteome/genetics , Proteomics , VirulenceABSTRACT
BACKGROUND: As a pooled donor blood product, cryoprecipitate (cryo) carries risks of pathogen transmission. Pathogen inactivation (PI) improves the safety of cryoprecipitate, but its effects on haemostatic properties remain unclear. This study investigated protein expression in samples of pathogen inactivated cryoprecipitate (PI-cryo) using non-targeted quantitative proteomics and in vitro haemostatic capacity of PI-cryo. MATERIALS AND METHODS: Whole blood (WB)- and apheresis (APH)-derived plasma was subject to PI with INTERCEPT® Blood System (Cerus Corporation, Concord, CA, USA) and cryo was prepared from treated plasma. Protein levels in PI-cryo and paired controls were quantified using liquid chromatography-tandem mass spectrometry. Functional haemostatic properties of PI-cryo were assessed using a microparticle (MP) prothrombinase assay, thrombin generation assay, and an in vitro coagulopathy model subjected to thromboelastometry. RESULTS: Over 300 proteins were quantified across paired PI-cryo and controls. PI did not alter the expression of coagulation factors, but levels of platelet-derived proteins and platelet-derived MPs were markedly lower in the WB PI-cryo group. Compared to controls, WB (but not APH) cryo samples demonstrated significantly lower MP prothrombinase activity, prolonged clotting time, and lower clot firmness on thromboelastometry after PI. However, PI did not affect overall thrombin generation variables in either group. DISCUSSION: Data from this study suggest that PI via INTERCEPT® Blood System does not significantly impact the coagulation factor content or function of cryo but reduces the higher MP content in WB-derived cryo. PI-cryo products may confer benefits in reducing pathogen transmission without affecting haemostatic function, but further in vivo assessment is warranted.
Subject(s)
Blood Proteins/drug effects , Blood Proteins/radiation effects , Blood Safety , Blood-Borne Infections/prevention & control , Blood-Borne Pathogens/drug effects , Blood-Borne Pathogens/radiation effects , Microbial Viability , Plasma/drug effects , Plasma/radiation effects , Virus Inactivation , Blood Component Removal , Blood Platelets/chemistry , Blood Preservation , Blood Proteins/analysis , Cell-Derived Microparticles/enzymology , Cryopreservation , Furocoumarins/pharmacology , Furocoumarins/radiation effects , Humans , Microbial Viability/drug effects , Microbial Viability/radiation effects , Photochemistry , Photosensitizing Agents/pharmacology , Photosensitizing Agents/radiation effects , Plasma/microbiology , Plasma/virology , Thrombelastography , Thrombin/biosynthesis , Thromboplastin/analysis , Ultraviolet Rays , Virus Inactivation/drug effects , Virus Inactivation/radiation effectsABSTRACT
BACKGROUND: Advanced liver disease due to hepatitis C virus (HCV) is a leading cause of human immunodeficiency virus (HIV)-related morbidity and mortality. There remains a need to develop noninvasive predictors of clinical outcomes in persons with HIV/HCV coinfection. METHODS: We conducted a nested case-control study in 126 patients with HIV/HCV and utilized multiple quantitative metabolomic assays to identify a prognostic profile that predicts end-stage liver disease (ESLD) events including ascites, hepatic encephalopathy, hepatocellular carcinoma, esophageal variceal bleed, and spontaneous bacterial peritonitis. Each analyte class was included in predictive modeling, and area under the receiver operator characteristic curves (AUC) and accuracy were determined. RESULTS: The baseline model including demographic and clinical data had an AUC of 0.79. Three models (baseline plus amino acids, lipid metabolites, or all combined metabolites) had very good accuracy (AUC, 0.84-0.89) in differentiating patients at risk of developing an ESLD complication up to 2 years in advance. The all combined metabolites model had sensitivity 0.70, specificity 0.85, positive likelihood ratio 4.78, and negative likelihood ratio 0.35. CONCLUSIONS: We report that quantification of a novel set of metabolites may allow earlier identification of patients with HIV/HCV who have the greatest risk of developing ESLD clinical events.
Subject(s)
End Stage Liver Disease/metabolism , End Stage Liver Disease/virology , HIV Infections/complications , Hepatitis C/complications , Metabolome , Amino Acids/metabolism , Bile Acids and Salts/metabolism , Biomarkers/metabolism , Case-Control Studies , Coinfection , Fatty Acids/metabolism , Female , Humans , Male , Middle Aged , Models, Biological , Predictive Value of Tests , PrognosisABSTRACT
Late-onset Alzheimer's disease (AD) can, in part, be considered a metabolic disease. Besides age, female sex and APOE ε4 genotype represent strong risk factors for AD that also give rise to large metabolic differences. We systematically investigated group-specific metabolic alterations by conducting stratified association analyses of 139 serum metabolites in 1,517 individuals from the AD Neuroimaging Initiative with AD biomarkers. We observed substantial sex differences in effects of 15 metabolites with partially overlapping differences for APOE ε4 status groups. Several group-specific metabolic alterations were not observed in unstratified analyses using sex and APOE ε4 as covariates. Combined stratification revealed further subgroup-specific metabolic effects limited to APOE ε4+ females. The observed metabolic alterations suggest that females experience greater impairment of mitochondrial energy production than males. Dissecting metabolic heterogeneity in AD pathogenesis can therefore enable grading the biomedical relevance for specific pathways within specific subgroups, guiding the way to personalized medicine.
Subject(s)
Alzheimer Disease/blood , Alzheimer Disease/genetics , Apolipoproteins E/genetics , Blood/metabolism , Metabolome/genetics , Aged , Alzheimer Disease/diagnostic imaging , Biomarkers/blood , Biomarkers/cerebrospinal fluid , Cohort Studies , Female , Genotype , Humans , Male , Mitochondria/genetics , Mitochondria/metabolism , Positron-Emission Tomography , Sex FactorsABSTRACT
BACKGROUND: Acylcarnitines have important functions in mitochondrial energetics and ß-oxidation, and have been implicated to play a significant role in metabolic functions of the brain. This retrospective study examined whether plasma acylcarnitine profiles can help biochemically distinguish the three phenotypic subtypes of major depressive disorder (MDD): core depression (CD+), anxious depression (ANX+), and neurovegetative symptoms of melancholia (NVSM+). METHODS: Depressed outpatients (n = 240) from the Mayo Clinic Pharmacogenomics Research Network were treated with citalopram or escitalopram for eight weeks. Plasma samples collected at baseline and after eight weeks of treatment with citalopram or escitalopram were profiled for short-, medium- and long-chain acylcarnitine levels using AbsoluteIDQ®p180-Kit and LC-MS. Linear mixed effects models were used to examine whether acylcarnitine levels discriminated the clinical phenotypes at baseline or eight weeks post-treatment, and whether temporal changes in acylcarnitine profiles differed between groups. RESULTS: Compared to ANX+, CD+ and NVSM+ had significantly lower concentrations of short- and long-chain acylcarnitines at both baseline and week 8. In NVSM+, the medium- and long-chain acylcarnitines were also significantly lower in NVSM+ compared to ANX+. Short-chain acylcarnitine levels increased significantly from baseline to week 8 in CD+ and ANX+, whereas medium- and long-chain acylcarnitines significantly decreased in CD+ and NVSM+. CONCLUSIONS: In depressed patients treated with SSRIs, ß-oxidation and mitochondrial energetics as evaluated by levels and changes in acylcarnitines may provide the biochemical basis of the clinical heterogeneity of MDD, especially when combined with clinical characteristics.
Subject(s)
Depressive Disorder, Major , Carnitine/analogs & derivatives , Depressive Disorder, Major/drug therapy , Humans , Phenotype , Retrospective StudiesABSTRACT
The T helper 2 (Th2) inflammatory cytokine interleukin-13 (IL-13) has been associated with both obstructive and fibrotic lung diseases; however, its specific effect on the epithelial stem cells in the gas exchange compartment of the lung (alveolar space) has not been explored. Here, we used in vivo lung models of homeostasis and repair, ex vivo organoid platforms, and potentially novel quantitative proteomic techniques to show that IL-13 disrupts the self-renewal and differentiation of both murine and human type 2 alveolar epithelial cells (AEC2s). Significantly, we find that IL-13 promotes ectopic expression of markers typically associated with bronchiolar airway cells and commonly seen in the alveolar region of lung tissue from patients with idiopathic pulmonary fibrosis. Furthermore, we identify a number of proteins that are differentially secreted by AEC2s in response to IL-13 and may provide biomarkers to identify subsets of patients with pulmonary disease driven by "Th2-high" biology.
Subject(s)
Alveolar Epithelial Cells/metabolism , Interleukin-13/metabolism , Stem Cells/metabolism , Alveolar Epithelial Cells/drug effects , Animals , Cell Differentiation , Cytokines/metabolism , Epithelial Cells/metabolism , Female , Homeostasis , Humans , Idiopathic Pulmonary Fibrosis/metabolism , Interleukin-13/genetics , Interleukin-13/pharmacology , Lung/metabolism , Male , Mice , Mice, Inbred C57BL , Organoids/metabolism , Proteomics , Th2 Cells/metabolism , Uteroglobin/metabolismABSTRACT
Vendor-independent software tools for quantification of small molecules and metabolites are lacking, especially for targeted analysis workflows. Skyline is a freely available, open-source software tool for targeted quantitative mass spectrometry method development and data processing with a 10 year history supporting six major instrument vendors. Designed initially for proteomics analysis, we describe the expansion of Skyline to data for small molecule analysis, including selected reaction monitoring, high-resolution mass spectrometry, and calibrated quantification. This fundamental expansion of Skyline from a peptide-sequence-centric tool to a molecule-centric tool makes it agnostic to the source of the molecule while retaining Skyline features critical for workflows in both peptide and more general biomolecular research. The data visualization and interrogation features already available in Skyline, such as peak picking, chromatographic alignment, and transition selection, have been adapted to support small molecule data, including metabolomics. Herein, we explain the conceptual workflow for small molecule analysis using Skyline, demonstrate Skyline performance benchmarked against a comparable instrument vendor software tool, and present additional real-world applications. Further, we include step-by-step instructions on using Skyline for small molecule quantitative method development and data analysis on data acquired with a variety of mass spectrometers from multiple instrument vendors.
Subject(s)
Metabolomics , Proteomics , Amino Acid Sequence , Mass Spectrometry , SoftwareABSTRACT
Nontypeable Haemophilus influenzae (NTHI) is a human-restricted pathogen with an essential requirement for heme-iron acquisition. We previously demonstrated that microevolution of NTHI promotes stationary phase survival in response to transient heme-iron restriction. In this study, we examine the metabolic contributions to biofilm formation using this evolved NTHI strain, RM33. Quantitative analyses identified 29 proteins, 55 transcripts, and 31 metabolites that significantly changed within in vitro biofilms formed by RM33. The synthesis of all enzymes within the tryptophan and glycogen pathways was significantly increased in biofilms formed by RM33 compared with the parental strain. In addition, increases were observed in metabolite transport, adhesin production, and DNA metabolism. Furthermore, we observed pyruvate as a pivotal point in the metabolic pathways associated with changes in cAMP phosphodiesterase activity during biofilm formation. Taken together, changes in central metabolism combined with increased stores of nutrients may serve to counterbalance nutrient sequestration.
Subject(s)
Adaptation, Physiological , Biofilms/growth & development , Haemophilus influenzae/growth & development , Haemophilus influenzae/metabolism , Heme/metabolism , Microbial Viability , Gene Expression Profiling , Iron/metabolism , Metabolism , Metabolome , Proteome/analysisABSTRACT
Alzheimer's disease (AD) is the most common cause of dementia. The mechanism of disease development and progression is not well understood, but increasing evidence suggests multifactorial etiology, with a number of genetic, environmental, and aging-related factors. There is a growing body of evidence that metabolic defects may contribute to this complex disease. To interrogate the relationship between system level metabolites and disease susceptibility and progression, the AD Metabolomics Consortium (ADMC) in partnership with AD Neuroimaging Initiative (ADNI) is creating a comprehensive biochemical database for patients in the ADNI1 cohort. We used the Biocrates Bile Acids platform to evaluate the association of metabolic levels with disease risk and progression. We detail the quantitative metabolomics data generated on the baseline samples from ADNI1 and ADNIGO/2 (370 cognitively normal, 887 mild cognitive impairment, and 305 AD). Similar to our previous reports on ADNI1, we present the tools for data quality control and initial analysis. This data descriptor represents the third in a series of comprehensive metabolomics datasets from the ADMC on the ADNI.