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Background and aims: The accumulation of fat increases the formation of lipid perox-ides, which are partly scavenged by alpha-tocopherol (a-TOH). Here, we aimed to investigate the associations between different measures of (abdominal) fat and levels of urinary a-TOH metab-olites in middle-aged individuals. Methods and results: In this cross-sectional analysis in the Netherlands Epidemiology of Obesity study (N Z 511, 53% women; mean [SD] age of 55 [6.1] years), serum a-TOH and a-TOH metab-olites from 24-h urine were measured as alpha-tocopheronolactone hydroquinone (a-TLHQ, oxidized) and alpha-carboxymethyl-hydroxychroman (a-CEHC, enzymatically converted) using liquid-chromatography-tandem mass spectrometry. Body mass index and total body fat were measured, and abdominal subcutaneous and visceral adipose tissue (aSAT and VAT) were as-sessed using magnetic resonance imaging. Using multivariable-adjusted linear regression ana -lyses, we analysed the associations of BMI, TBF, aSAT and VAT with levels of urinary a-TOH metabolites, adjusted for confounders. We observed no evidence for associations between body fat measures and serum a-TOH. Higher BMI and TBF were associated with lower urinary levels of TLHQ (0.95 [95%CI: 0.90, 1.00] and 0.94 [0.88, 1.01] times per SD, respectively) and with lower TLHQ relative to CEHC (0.93 [0.90, 0.98] and 0.93 [0.87, 0.98] times per SD, respectively). We observed similar associations for VAT (TLHQ: 0.94 [0.89, 0.99] times per SD), but not for aSAT. Conclusions: Opposite to our research hypothesis, higher abdominal adiposity was moderately associated with lower levels of oxidized a-TOH metabolites, which might reflect lower vitamin E antioxidative activity in individuals with higher abdominal fat instead. (c) 2021 The Author(s). Published by Elsevier B.V. on behalf of The Italian Diabetes Society, the Italian Society for the Study of Atherosclerosis, the Italian Society of Human Nutrition and the Department of Clinical Medicine and Surgery, Federico II University. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

BACKGROUND Multiple registries have reported that >40% of high-risk atrial fibrillation patients are not taking oral anticoagulants. The purpose of our study was to determine the presence or absence of active atrial fibrillation and CHA2DS2-VASc (Congestive heart failure, Hypertension, Age ≥75 y, Diabetes mellitus, prior Stroke [or transient ischemic attack or thromboembolism], Vascular disease, Age 65-74 y, Sex category) risk factors to accurately identify high-risk atrial fibrillation (CHA2DS2-VASc ≥2) patients requiring oral anticoagulants and the magnitude of the anticoagulant treatment gap. METHODS We retrospectively adjudicated 6514 patients with atrial fibrillation documented by at least one of: billing diagnosis, electronic medical record encounter diagnosis, electronic medical record problem list, or electrocardiogram interpretation. RESULTS After review, 4555/6514 (69.9%) had active atrial fibrillation, while 1201 had no documented history of atrial fibrillation and 758 had a history of atrial fibrillation that was no longer active. After removing the 1201 patients without a confirmed atrial fibrillation diagnosis, oral anticoagulant use in high-risk patients increased to 71.1% (P < .0001 compared with 62.9% at baseline). Oral anticoagulant use increased to 79.7% when the 758 inactive atrial fibrillation patients were also eliminated from the analysis (P < .0001 compared with baseline). In the active high-risk atrial fibrillation group, there was no significant difference in the use of oral anticoagulants between men (80.7%) and women (78.8%) with a CHA2DS2-VASc ≥2, or in women with a CHA2DS2-VASc ≥3 (79.9%). CONCLUSIONS Current registries and health system health records with unadjudicated diagnoses over-report the number of high-risk atrial fibrillation patients not taking oral anticoagulants. Expert adjudication identifies a smaller treatment gap than previously described.

Background Genetic changes in the LIM homeobox transcription factor 1 beta (LMX1B) have been associated with focal segmental glomerulosclerosis (FSGS) without the extra-renal or ultrastructural manifestations of Nail-patella syndrome (NPS) known as Nail-patella-like renal disease (NPLRD). Fabry disease (FD) is an X-linked lysosomal disease caused by the deficiency of alpha-galactosidase A. The classic form of the disease is characterized by acroparesthesia, angiokeratomas, cornea verticillata, hypertrophic cardiomyopathy, strokes, and chronic kidney disease. Podocyte myelin bodies on ultrastructural examination of kidney tissue are very characteristic of FD; however some medications and other conditions may mimic this finding. Case presentation Here, we report on a female patient with chronic kidney disease (CKD), positive family history for kidney disease and kidney biopsy showing a FSGS lesion and presence of focal myelin figures within podocytes concerning for FD. However, genetic testing for FD was negative. After comprehensive clinical, biochemical, and genetic evaluation, including whole exome and RNA sequencing, she was ultimately diagnosed with NPLRD. Conclusions This case illustrates the difficulties of diagnosing atypical forms of rare Mendelian kidney diseases and the role of a multidisciplinary team in an individualized medicine clinic setting in combination with state-of-the-art sequencing technologies to reach a definitive diagnosis.

BACKGROUND: Maintaining adequate oxygen delivery (DO(2)) after major surgery is associated with minimising organ dysfunction. Skin is particularly vulnerable to reduced DO(2). We tested the hypothesis that reduced perioperative DO(2) fuels inflammation in metabolically compromised skin after major surgery. METHODS: Participants undergoing elective oesophagectomy were randomised immediately after surgery to standard of care or haemodynamic therapy to achieve their individualised preoperative DO(2). Abdominal punch skin biopsies were snap-frozen before and 48 h after surgery. On-line two-dimensional liquid chromatography and ultra-high-definition label-free mass spectrometry was used to characterise the skin proteome. The primary outcome was proteomic changes compared between normal (≥preoperative value before induction of anaesthesia) and low DO(2) (

Fabry disease (FD) is an X-linked lysosomal storage disorder caused by deficiency of α-galactosidase-A, which results in accumulation of the glycosphingolipid (GSL) globotriaosylceramide (Gb3). Gb3 and globotriaosylsphingosine (lyso-Gb3) levels in plasma and urine are used routinely for diagnosis and treatment monitoring. FD female patients are problematic to diagnose and to predict when to begin treatment. Further biomarkers are needed to detect pre-symptomatic females that will develop the chronic symptoms associated with FD. A LC-MS/MS glycosphingolipidomic assay was developed to measure lyso-Gb3 and GSLs from the lysosomal GSL degradation pathway, including globoside (Gb4), Gb3, ceramide dihexosides (CDH) and ceramide monohexosides (CMH). We analysed plasma and urine from a cohort of Fabry patients, grouped according to clinical symptoms and independent of treatment status (asymptomatic females n = 18, symptomatic females n = 18, males n = 27 and control urines n = 16 and control plasmas n = 58). Multivariate and subsequent univariate analysis showed urine GSLs which had highest significance in identifying asymptomatic females were total levels of CDH, in particular the long chain isoforms C22:1,C22:0,C22:1-OH,C22:0-OH,C24:2,C24:0,C24:2-OH,C24:1-OH,C24:0-OH,C26:0 which likely represent Galabiosylceramide (Ga2) and not lactosylceramide. These long chain Ga2 isoforms were found to be 5-fold elevated and more statistically significant (p

BackgroundFabry disease is a progressive multisystemic disease, which affects the kidney and cardiovascular systems. Various treatments exist but decisions on how and when to treat are contentious. The current marker for monitoring treatment is plasma globotriaosylsphingosine (lyso-Gb3), but it is not informative about the underlying and developing disease pathology.MethodsWe have created a urine proteomic assay containing a panel of biomarkers designed to measure disease-related pathology which include the inflammatory system, lysosome, heart, kidney, endothelium and cardiovascular system. Using a targeted proteomic-based approach, a series of 40 proteins for organ systems affected in Fabry disease were multiplexed into a single 10 min multiple reaction monitoring Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS) assay and using only 1 mL of urine.ResultsSix urinary proteins were elevated in the early-stage/asymptomatic Fabry group compared with controls including albumin, uromodulin, α1-antitrypsin, glycogen phosphorylase brain form, endothelial protein receptor C and intracellular adhesion molecule 1. Albumin demonstrated an increase in urine and could indicate presymptomatic disease. The only protein elevated in the early-stage/asymptomatic patients that continued to increase with progressive multiorgan involvement was glycogen phosphorylase brain form. Podocalyxin, fibroblast growth factor 23, cubulin and Alpha-1-Microglobulin/Bikunin Precursor (AMBP) were elevated only in disease groups involving kidney disease. Nephrin, a podocyte-specific protein, was elevated in all symptomatic groups. Prosaposin was increased in all symptomatic groups and showed greater specificity (pConclusionThis work indicates that protein biomarkers could be helpful and used in conjunction with plasma lyso-Gb3 for monitoring of therapy or disease progression in patients with Fabry disease.

Background & aims: Damage induced by lipid peroxidation has been associated with impaired glucose homeostasis. Vitamin E (alpha-tocopherol, alpha-TOH) competitively reacts with lipid peroxyl radicals to mitigate oxidative damage, and forms oxidized vitamin E metabolites. Accordingly, we aimed to investigate the associations between alpha-TOH metabolites (oxidized and enzymatic) in both circulation and urine and measures of glucose homeostasis in the general middle-aged population. Methods: This cross-sectional study was embedded in the population-based Netherlands Epidemiology of Obesity (NEO) Study. alpha-TOH metabolites in blood (alpha-TOH and alpha-CEHC-SO3) and urine [sulfate (SO3) and glucuronide (GLU) of both alpha-TLHQ (oxidized) and alpha-CEHC (enzymatic)] were quantified by liquid chromatography coupled with tandem mass spectrometry (LC/MSeMS). Measures of glucose homeostasis (HOMA-B, HOMA-IR, Insulinogenic index and Matsuda index) were obtained from fasting and postprandial blood samples. Multivariable linear regression analyses were performed to assess the associations of alpha-TOH metabolites and measures of glucose homeostasis. Results: We included 498 participants (45% men) with mean (SD) age of 55.8 (6.1) years who did not use glucose-lowering medication. While blood alpha-TOH was not associated with measures of glucose homeostasis, urinary oxidized metabolites (alpha-TLHQ-SO3/GLU) were associated with HOMA-IR and Matsuda index. For example, a one-SD higher alpha-TLHQ-SO3 was associated with 0.92 (95% CI: 0.87, 0.97) fold lower HOMA-IR and 1.06 (1.01, 1.11) fold higher Matsuda index, respectively. Similar results were obtained for the urinary alpha-TLHQ to alpha-CEHC ratio as a measure of oxidized-over-enzymatic conversion of alpha-TOH. Conclusion: Higher urinary levels of oxidized alpha-TOH metabolites as well as higher oxidized-to-enzymatic alpha-TOH metabolite ratio, but not circulating alpha-TOH or enzymatic metabolites, were associated with lower insulin resistance. Rather than circulating alpha-TOH, estimates of the conversion of alpha-TOH might be informative in relation to health and disease. (c) 2021 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/

Introduction: Guidelines recommend oral anticoagulants (OAC) in high risk atrial fibrillation (HRAF) patients. It has been reported that >40% of such patients are not taking an OAC. We observed that active AF often was not present in patients labeled as having AF (either because of wrong diagnosis or no AF in last 5 years). Hypothesis: We undertook a systematic electronic medical record (EMR) review to determine the presence or absence of active AF to accurately identify HRAF patients requiring an OAC. Methods: We reviewed 6514 patients with presumed AF and 2 or more outpatient visits documented by at least one of: billing diagnosis, EMR encounter diagnosis, EMR problem list, or EKG interpretation. Results: Active AF was noted in 4555/6514 patients (69.7%) after 1959 patients with no prior history of AF or inactive AF were withdrawn from further analysis. In active AF patients, 3869 had HRAF with CHA 2 DS 2 -VASc ≥2. OAC use was statistically higher (p2 DS 2 -VASc ≥3 (79.9%). In HRAF patients not taking an OAC (20.1%), expert review frequently disagreed with that recommendation suggesting that OACs can be taken in up to 92% of HRAF patients. Conclusions: 1) Current registries with un-adjudicated EMR diagnoses over-report the number of HRAF patients not taking an OAC; 2) Expert adjudication of active AF diagnosis identifies a smaller OAC treatment gap than previously described; 3) absolute contraindication or repeated refusal to take an OAC exists in less than 10% of HRAF patients; and, 4) there were no gender differences in OAC use.

ObjectiveMaintaining adequate oxygen delivery after major surgery is associated with minimizing organ dysfunction, although the precise molecular mechanisms remain unclear.BackgroundSkin, the largest organ in the body, is vulnerable to reduced oxygen delivery. We hypothesized that the skin proteome, assessed before and after surgery, would reveal molecular differences in patients randomized to receive cardiovascular therapy aimed at maintaining preoperative oxygen delivery (DO2).MethodsAbdominal punch skin biopsies were snap frozen or fixed in paraformaldehyde immediately before and 48h after elective esophageal or liver resection. Immediately after surgery, patients were randomized to standard of care or therapy to maintain preoperative DO2. On-line two-dimensional liquid chromatography, followed by ultra-high definition label-free mass spectrometry analysis, and/or immunoblots quantified significant proteomic changes. Selected proteins identified by mass spectrometry were confirmed by immunohistochemistry and immunoblot. In a murine hepatic resection model, confirmation of specific proteomic signatures identified in patients was sought by immunoblotting.ResultsPaired biopsies were analyzed from 35 patients (mean age:68±9y; 31% female). We identified 2096 proteins, of which 157 were differentially expressed after surgery. Similar results for selected proteins were found using immunohistochemistry (n=6 patients), immunoblots (n=12 patients) and murine abdominal skin obtained after liver resection (n=14). After surgery, 14 proteins distinguished esophagectomy patients with normal (n=10) versus low (n=7) DO2.values. Failure to maintain preoperative DO2 was associated with upregulation of proteins counteracting oxidative stress and.ConclusionsSerial skin biopsies afford mechanistic insight into end-organ injury by quantifying proteomic changes associated with impaired oxygen delivery during high-risk surgery.Trial registrationISRCTN76894700FundingAcademy of Medical Sciences/Health Foundation Clinician Scientist Award [GLA]; British Oxygen Company research chair grant in Anesthesia [GLA]; Great Ormond Street Hospital Biomedical Research Centre [EB,WH,KM]; British Heart Foundation:PG/17/40/33028 [MC]; UK NIHR [GLA]; Barts Charity [TJ]Mini-AbstractSkin, one of the largest organs in the body, is vulnerable to reduced oxygen delivery. Proteomic analysis of skin biopsies obtained before and after surgery show distinct metabolic and inflammatory changes related to perioperative oxygen delivery. Mirrored by complementary laboratory data in mice, skin proteomics reveal new insights into perioperative organ dysfunction.

Advanced age is the major risk factor for idiopathic Parkinson's disease (PD), but to date the biological relationship between PD and ageing remains elusive. Here we describe the rationale and the design of the H2020 funded project "PROPAG-AGEING", whose aim is to characterize the contribution of the ageing process to PD development. We summarize current evidences that support the existence of a continuum between ageing and PD and justify the use of a Geroscience approach to study PD. We focus in particular on the role of inflammaging, the chronic, low-grade inflammation characteristic of elderly physiology, which can propagate and transmit both locally and systemically. We then describe PROPAG-AGEING design, which is based on the multi-omic characterization of peripheral samples from clinically characterized drug-naive and advanced PD, PD discordant twins, healthy controls and "super-controls", i.e. centenarians, who never showed clinical signs of motor disability, and their offspring. Omic results are then validated in a large number of samples, including in vitro models of dopaminergic neurons and healthy siblings of PD patients, who are at higher risk of developing PD, with the final aim of identifying the molecular perturbations that can deviate the trajectories of healthy ageing towards PD development.

Summary The neuronal ceroid lipofuscinoses (NCL) are a group of 13 rare neurodegenerative disorders characterized by accumulation of cellular storage bodies. There are few therapeutic options, and existing tests do not monitor disease progression and treatment response. However, urine biomarkers could address this need. Proteomic analysis of CLN2 patient urine revealed activation of immune response pathways and pathways associated with the unfolded protein response. Analysis of CLN5 and CLN6 sheep model urine showed subtle changes. To confirm and investigate the relevance of candidate biomarkers a targeted LC-MS/MS proteomic assay was created. We applied this assay to additional CLN2 samples as well as other patients with NCL (CLN1, CLN3, CLN5, CLN6, and CLN7) and demonstrated that hexosaminidase-A, aspartate aminotransferase-1, and LAMP1 are increased in NCL samples and betaine-homocysteine S-methyltransferase-1 was specifically increased in patients with CLN2. These proteins could be used to monitor the effectiveness of future therapies aimed at treating systemic NCL disease., Graphical Abstract, Highlights • The urine proteome is altered in humans and animals with NCL • Hexosaminidase A and LAMP1 are increased in patients with NCL • Betaine-homocysteine S-methyltransferase 1 is elevated in CLN2 patients • Proteins altered in CLN5 and CLN6 sheep models are not affected in humans, Disease; Systems Biology; Proteomics

Vitamin E (alpha-tocopherol [alpha-TOH] ) is transported in lipoprotein particles in blood, but little is known about the transportation of its oxidized metabolites. In the Netherlands Epidemiology of Obesity Study, we aimed to investigate the associations of 147 circulating metabolomic measures obtained through targeted nuclear magnetic resonance with serum alpha-TOH and its urinary enzymatic (alpha-CEHC) and oxidized (alpha-TLHQ) metabolites from 24-h urine quantified by liquid chromatography with tandem mass spectrometry. Multivariable linear regression analyses, in which multiple testing was taken into account, were performed to assess associations between metabolomic measures (determinants; standardized to mean = 0, SD = 1) and vitamin E metabolites (outcomes), adjusted for demographic factors. We analyzed 474 individuals (55% women, 45% men) with a mean (SD) age of 55.7 (6.0) y. Out of 147 metabolomic measures, 106 were associated (P < 1.34 x 10(3)) with serum alpha-TOH (median beta [interquartile range] = 0.416 [0.383-0.466]), predominantly lipoproteins associated with higher alpha-TOH. The associations of metabolomic measures with urinary alpha-CEHC have directions similar to those with alpha-TOH, but effect sizes were smaller and non-significant (median beta [interquartile range] = 0.065 [0.047-0.084]). However, associations of metabolomic measures with urinary alpha-TLHQ were markedly different from those with both serum alpha-TOH and urinary alpha-CEHC, with negative and small-to-null relations to most very-low-density lipoproteins and amino acids. Therefore, our results highlight the differences in the lipoproteins involved in the transportation of circulating alpha-TOH and oxidized vitamin E metabolites. This indicates that circulating alpha-TOH may be representative of the enzymatic but not the antioxidative function of vitamin E. (C) 2021 The Author( s). Published by Elsevier Inc.

Classic late infantile neuronal ceroid lipofuscinosis (CLN2 disease) is caused by a deficiency of tripeptidyl-peptidase-1. In 2017, the first CLN2 enzyme replacement therapy (ERT) cerliponase alfa (Brineura) was approved by the FDA and EMA. The CLN2 disease clinical rating scale (CLN2 CRS) was developed to monitor loss of motor function, language and vision as well as frequency of generalised tonic clonic seizures. Using CLN2 CRS in an open label clinical trial it was shown that Brineura slowed down the progression of CLN2 symptoms. Neurofilament light chain (NfL) is a protein highly expressed in myelinated axons. An increase of cerebrospinal fluid (CSF) and blood NfL is found in a variety of neuroinflammatory, neurodegenerative, traumatic, and cerebrovascular diseases. We analysed CSF NfL in CLN2 patients treated with Brineura to establish whether it can be used as a possible biomarker of response to therapy. Newly diagnosed patients had CSF samples collected and analysed at first treatment dose and up to 12 weeks post-treatment to look at acute changes. Patients on a compassionate use programme who were already receiving ERT for approximately 1yr had CSF samples collected and NfL analysed over the following 1.3 years (2.3 years post-initiation of ERT) to look at long-term changes. All newly diagnosed patients we investigated with classical late infantile phenotype had high NfL levels >2000 pg/ml at start of treatment. No significant change was observed in NfL up to 12 weeks post-treatment. After one year of ERT, two out of six patients still had high NfL levels, but all patients showed a continued decrease, and all had low NfL levels after two years on ERT. NfL levels appear to correspond and predict improved clinical status of patients on ERT and could be useful as a biomarker to monitor neurodegeneration and verify disease modification in CLN2 patients on ERT.

Lamins A and C are intermediate filament nuclear envelope proteins encoded by theLMNAgene. Mutations inLMNAcause autosomal dominant severe heart disease, accounting for 10% of dilated cardiomyopathy (DCM). Characterised by progressive conduction system disease, arrhythmia and systolic impairment, lamin A/C heart disease is more malignant than other common DCMs due to high event rates even when the left ventricular impairment is mild. It has several phenotypic mimics, but overall it is likely to be an under-recognised cause of DCM. In certain clinical scenarios, particularly familial DCM with early conduction disease, the pretest probability of finding anLMNAmutation may be quite high.Recognising lamin A/C heart disease is important because implantable cardioverter defibrillators need to be implanted early. Promising oral drug therapies are within reach thanks to research into the mitogen-activated protein kinase (MAPK) and affiliated pathways. Personalised heart failure therapy may soon become feasible forLMNA, alongside personalised risk stratification, as variant-related differences in phenotype severity and clinical course are being steadily elucidated.Genotyping and family screening are clinically important both to confirm and to excludeLMNAmutations, but it is the three-pronged integration of such genetic information with functional data from in vivo cardiomyocyte mechanics, and pathological data from microscopy of the nuclear envelope, that is properly reshaping ourLMNAknowledge base, one variant at a time. This review explains the biology of lamin A/C heart disease (genetics, structure and function of lamins), clinical presentation (diagnostic pointers, electrocardiographic and imaging features), aspects of screening and management, including current uncertainties, and future directions.

Background Polycystic ovarian syndrome (PCOS) is common, affecting up to one-fifth of females and is associated with significant comorbidity. Despite this, it is poorly understood, and diagnosis and management remain challenging in adolescents. Proteomics enables the better understanding of disease mechanisms and facilitates the identification of novel biomarkers. Objectives To describe the clinical phenotype of PCOS in adolescents and undertake discovery proteomic urine profiling using ultra-performance liquid chromatography-mass spectrometry (UPLC-MS/MS) to identify novel non-invasive biomarkers of PCOS. Method This prospective longitudinal study recruited adolescent females meeting NIH diagnostic criteria for PCOS. The following were measured at baseline and annual follow-up: hormonal and metabolic markers including an oral glucose tolerance test, psychological, pubertal and anthropometric parameters, and pelvic ultrasounds. We have undertaken UPLC-MS/MS and developed new methods for discovery proteomic profiling of urine in an attempt to identify new disease mechanisms, drug targets and potential biomarkers. Results We recruited 40 participants (median age 15.0 years, range 12.5-18.3), with two-thirds completing annual follow-up. Clinical signs at presentation included acne (89%), hirsutism (78%) and acanthosis nigricans (49%). Two-thirds of participants had depressive or anxiety symptoms yet only one-third were known to mental health services. Metabolic dysfunction was common at baseline; overweight/obesity (86%), elevated body fat (88%) and dyslipidaemia (35%). These parameters persisted at follow-up. Insulin resistance was almost universal at baseline and follow-up (91%). Impaired glucose metabolism was common but improved from baseline (29%) to follow-up (10%; p=0.11). Over two-thirds had elevated anti-Mullerian hormone, three-quarters had an elevated free androgen index. Raised inflammatory markers (CRP/ESR) were present in 40% participants. Only three participants had definitive ultrasonographic evidence of PCOS. Interventions included lifestyle advice (27%), combined oral contraceptive pill (COCP) ± anti-androgen (16%), metformin (30%) or metformin + COCP ± anti-androgen (27%). Conclusion and future directions Adolescents withPCOS are at high risk of metabolic dysfunction, inflammation and mental health disorders. Therefore, early diagnosis and intervention are imperative. However, current diagnostic and surveillance methods are suboptimal. We have used urinary proteomics to study metabolic pathways affected in PCOS and aim to identify novel non-invasive biomarkers. Subsequently, we will create a clinically translatable assay to aid diagnosis and stratify management of this common adolescent condition.

Among other mechanisms, oxidative stress has been postulated to play an important role in the rate of ageing. Oxidative damage contributes to the hallmarks of ageing and essential components in pathological pathways which are thought to drive multiple age-related diseases. Nonetheless, results from studies testing the hypothesis of oxidative stress in ageing and diseases showed controversial results. While observational studies mainly found detrimental effects of high oxidative stress levels on disease status, randomized clinical trials examining the effect of antioxidant supplementation on disease status generally showed null effects. However, re-evaluations of these counterinitiative observations are required considering the lack of reliability and specificity of traditionally used biomarkers for measuring oxidative stress. To facilitate these re-evaluations, this review summarizes the basic knowledge of oxidative stress and the present findings regarding the role of oxidative damage in ageing and age-related diseases. Meanwhile, two approaches are highlighted, namely proper participants selection, together with the development of reliable biomarkers. We propose that oxidized vitamin E metabolites may be used to accurately monitor individual functional antioxidant level, which might serve as promising key solutions for future elucidating the impact of oxidative stress on ageing and age-related diseases.

Background Polycystic ovarian syndrome (PCOS) is the most common hormone disorder in females, affecting 4–20% of the population. PCOS is associated with metabolic dysfunction, pro-inflammation and mood disorders. Despite this, it is poorly understood, and diagnosis and management remain challenging in adolescents. Proteomics enables a better understanding of disease mechanisms and facilitates the identification of novel biomarkers. Aims 1. To better understand the clinical phenotype of PCOS in adolescents. 2. To undertake discovery proteomic urine profiling using ultra-performance liquid chromatography-mass spectrometry (UPLC-MS/MS) to identify novel non-invasive biomarkers of PCOS. Method In this prospective longitudinal study, females aged 12–19 years meeting NIH diagnostic criteria for PCOS were recruited from adolescent endocrine and gynaecology clinics. At baseline and annual follow-up, the following were measured: pituitary, adrenal and ovarian hormones, anti-Mullerian hormone, inflammatory and metabolic markers including an oral glucose tolerance test, psychometric questionnaires, menstrual records, pubertal assessment, anthropometric parameters and pelvic ultrasounds. We have undertaken UPLC-MS/MS and developed new methods for discovery proteomic profiling of urine samples in an attempt to identify new disease mechanisms, drug targets and potential biomarkers. Results To date, 37 participants have been recruited (median age 15.0 years, range 12.6–18.3), and 22 have completed annual follow-up. Clinical signs at presentation included acne (89%), hirsutism (78%), acanthosis nigricans (49%) and overweight/obesity (81%). Two-thirds of participants had depressive or anxiety symptoms. Only one-third were known to mental health services. Metabolic dysfunction was common; elevated body fat (88%), dyslipidaemia (24%), insulin resistance (62%), and impaired fasting glucose, impaired glucose tolerance or type 2 diabetes (40%). AMH was elevated in one-third of participants and three-quarters had an elevated free androgen index. Elevated inflammatory markers (CRP/ESR) were present in 40% participants. Only three participants had definitive ultrasonographic evidence of PCOS. Interventions included lifestyle advice only (27%), combined oral contraceptive pill (COCP) ± anti-androgen (16%), metformin (30%) or metformin + COCP ± anti-androgen (27%). Conclusion and Future Directions Diagnosing PCOS in adolescents remains challenging; acne and irregular menstrual cycles are common and ultrasonographic diagnosis of PCOS is suboptimal. Given the high prevalence of metabolic and mental health disorders, early diagnosis and intervention are imperative. We describe the use of urinary proteomics to study metabolic pathways affected in PCOS and the potential identification of novel non-invasive biomarkers. Subsequently, we will use this hypothesis-generating data-set to create a non-invasive and clinically translatable assay to aid diagnosis and stratify management of this common adolescent condition.

Hypertrophic cardiomyopathy (HCM) is defined by pathological left ventricular hypertrophy (LVH). It is the commonest inherited cardiac condition and a significant number of high risk cases still go undetected until a sudden cardiac death (SCD) event. Plasma biomarkers do not currently feature in the assessment of HCM disease progression, which is tracked by serial imaging, or in SCD risk stratification, which is based on imaging parameters and patient/family history. There is a need for new HCM plasma biomarkers to refine disease monitoring and improve patient risk stratification. To identify new plasma biomarkers for patients with HCM, we performed exploratory myocardial and plasma proteomics screens and subsequently developed a multiplexed targeted liquid chromatography-tandem/mass spectrometry-based assay to validate the 26 peptide biomarkers that were identified. The association of discovered biomarkers with clinical phenotypes was prospectively tested in plasma from 110 HCM patients with LVH (LVH+ HCM), 97 controls, and 16 HCM sarcomere gene mutation carriers before the development of LVH (subclinical HCM). Six peptides (aldolase fructose-bisphosphate A, complement C3, glutathione S-transferase omega 1, Ras suppressor protein 1, talin 1, and thrombospondin 1) were increased significantly in the plasma of LVH+ HCM compared with controls and correlated with imaging markers of phenotype severity: LV wall thickness, mass, and percentage myocardial scar on cardiovascular magnetic resonance imaging. Using supervised machine learning (ML), this six-biomarker panel differentiated between LVH+ HCM and controls, with an area under the curve of ≥ 0.87. Five of these peptides were also significantly increased in subclinical HCM compared with controls. In LVH+ HCM, the six-marker panel correlated with the presence of nonsustained ventricular tachycardia and the estimated five-year risk of sudden cardiac death. Using quantitative proteomic approaches, we have discovered six potentially useful circulating plasma biomarkers related to myocardial substrate changes in HCM, which correlate with the estimated sudden cardiac death risk.

Background Polycystic ovarian syndrome (PCOS) is the most common hormone disorder in adolescent and young adult females, affecting 4%–20% of the population. PCOS is associated with metabolic dysfunction, pro-inflammation and mood disorders. Despite this, it is poorly understood in younger adolescents, and diagnosis and management remain challenging. Aims To better understand the clinical phenotype of PCOS in adolescents. To undertake discovery proteomic urine profiling to identify novel non–invasive biomarkers of PCOS. Method In this prospective longitudinal study, females aged 12–19 years meeting NIH diagnostic criteria for PCOS were recruited from adolescent endocrine and gynaecology clinics. The following were measured at baseline and annual follow-up: pituitary, adrenal and ovarian hormones, anti-mullerian hormone, inflammatory and metabolic markers including an oral glucose tolerance test. Psychometric questionnaires, menstrual records, pubertal assessment, anthropometric parameters and trans-abdominal pelvic ultrasounds were also undertaken. Urine samples were obtained for mass spectrometry discovery proteomic profiling. Results To date, 37 participants have been recruited (median age 15.0 years, range 12.6–18.3), and 22 have completed annual follow-up. Clinical signs at presentation included acne (89%), hirsutism (78%), acanthosis nigricans (49%) and overweight/obesity (81%). Two-thirds of participants had depressive or anxiety symptoms. Only one-third were known to mental health services. Metabolic dysfunction was common; elevated body fat (88%), dysplidaemia (24%), insulin resistance (62%), and impaired fasting glucose, impaired glucose tolerance or type 2 diabetes (40%). AMH was elevated in one-third of participants and three-quarters had an elevated free androgen index. Elevated inflammatory markers (CRP/ESR) were present in 40% participants. Only three participants had definitive ultrasonographic evidence of PCOS. Interventions included lifestyle advice only (27%), combined oral contraceptive pill (COCP) ±anti androgen (16%), metformin (30%) or metformin +COCP ± anti-androgen (27%). Conclusionand future directions Diagnosing PCOS in adolescents remains challenging; acne and irregular menstrual cycles are common and ultrasonographic diagnosis of PCOS is suboptimal. Given the high prevalence of metabolic and mental health disorders, early diagnosis and intervention are imperative. We are currently undertaking discovery urine proteomic analysis to identify novel non-invasive biomarkers of PCOS. Subsequently, we will create a clinically translatable assay to aid diagnosis and management of this common adolescent condition.

The antioxidant vitamin E (&alpha, tocopherol, &alpha, TOH) protects lipids from oxidation by reactive oxygen species. We hypothesized that lifestyle factors associate with vitamin E metabolism marked by urinary &alpha, tocopheronolactone hydroquinone (&alpha, TLHQ) and &alpha, carboxymethyl-hydroxychroman (&alpha, CEHC levels), as potential reflection of lipid oxidation. We conducted a cross-sectional study in the Netherlands Epidemiology of Obesity Study. Serum &alpha, TOH, and urinary &alpha, TLHQ and &alpha, CEHC were quantified by liquid chromatography coupled with tandem mass spectrometry. Information on the lifestyle factors (sleep, physical activity (PA), smoking and alcohol) were collected through questionnaires. Multivariable linear regression analyses were performed to assess the associations between the lifestyle factors and &alpha, TOH measures. A total of 530 participants (46% men) were included with mean (SD) age of 56 (6) years. Of the examined lifestyle factors, only poor sleep was associated with a higher serum &alpha, TOH (mean difference: 4% (95% CI: 1, 7%)). Current smoking was associated with higher urinary &alpha, CEHC (32%: (14%, 53%)), with evidence of a dose&ndash, response relationship with smoking intensity (low pack years, 24% (2, 52%), high pack years, 55% (25, 93%)). Moderate physical activity was associated with a lower &alpha, TLHQ relative to &alpha, CEHC (&minus, 17%: (&minus, 26, &minus, 6%), compared with low PA). Only specific lifestyle factors associate with vitamin E metabolism. Examining serum &alpha, TOH does not provide complete insight in vitamin E antioxidant capacity.

‘Long Covid’, or medical complications associated with post SARS-CoV-2 infection, is a significant post-viral complication that is being more and more commonly reported in patients. Therefore, there is an increasing need to understand the disease mechanisms, identify drug targets and inflammatory processes associated with a SARS-CoV-2 infection. To address this need, we created a targeted mass spectrometry based multiplexed panel of 96 immune response associated proteins. We applied the multiplex assay to a cohort of serum samples from asymptomatic and moderately affected patients. All patients had tested positive for a SARS-CoV-2 infection by PCR and were determined to be subsequently positive for antibodies. Even 40-60 days post-viral infection, we observed a significant remaining inflammatory response in all patients. Proteins that were still affected were associated with the anti-inflammatory response and mitochondrial stress. This indicates that biochemical and inflammatory pathways within the body can remain perturbed long after SARS-CoV-2 infections have subsided even in asymptomatic and moderately affected patients.

The neuronal ceroid lipofuscinoses are a group of severe and progressive neurodegenerative disorders, which generally present during childhood. With new treatments emerging on the horizon, there is a growing need to understand the specific disease mechanisms as well as identify prospective biomarkers for use to stratify patients and monitor treatment. The use of Omics technologies to NCLs have the potential to address this need. We discuss the recent use and outcomes of Omics to various forms of NCL including identification of interactomes, affected biological pathways and potential biomarker candidates. We also identify commonpathways affected in NCL across the reviewed studies.

Mutations of the glucocerebrosidase gene, GBA1 (OMIM 606463), are the most important risk factor for Parkinson disease (PD). In vitro and in vivo studies have reported that ambroxol increases β-glucocerebrosidase (GCase) enzyme activity and reduces α-synuclein levels. These observations support a potential role for ambroxol therapy in modifying a relevant pathogenetic pathway in PD.To assess safety, tolerability, cerebrospinal fluid (CSF) penetration, and target engagement of ambroxol therapy with GCase in patients with PD with and without GBA1 mutations.An escalating dose of oral ambroxol to 1.26 g per day.This single-center open-label noncontrolled clinical trial was conducted between January 11, 2017, and April 25, 2018, at the Leonard Wolfson Experimental Neuroscience Centre, a dedicated clinical research facility and part of the University College London Queen Square Institute of Neurology in London, United Kingdom. Participants were recruited from established databases at the Royal Free London Hospital and National Hospital for Neurology and Neurosurgery in London. Twenty-four patients with moderate PD were evaluated for eligibility, and 23 entered the study. Of those, 18 patients completed the study; 1 patient was excluded (failed lumbar puncture), and 4 patients withdrew (predominantly lumbar puncture-related complications). All data analyses were performed from November 1 to December 14, 2018.Primary outcomes at 186 days were the detection of ambroxol in the CSF and a change in CSF GCase activity.Of the 18 participants (15 men [83.3%]; mean [SD] age, 60.2 [9.7] years) who completed the study, 17 (8 with GBA1 mutations and 9 without GBA1 mutations) were included in the primary analysis. Between days 0 and 186, a 156-ng/mL increase in the level of ambroxol in CSF (lower 95% confidence limit, 129 ng/mL; P .001) was observed. The CSF GCase activity decreased by 19% (0.059 nmol/mL per hour; 95% CI, -0.115 to -0.002; P = .04). The ambroxol therapy was well tolerated, with no serious adverse events. An increase of 50 pg/mL (13%) in the CSF α-synuclein concentration (95% CI, 14-87; P = .01) and an increase of 88 ng/mol (35%) in the CSF GCase protein levels (95% CI, 40-137; P = .002) were observed. Mean (SD) scores on part 3 of the Movement Disorders Society Unified Parkinson Disease Rating Scale decreased (ie, improved) by 6.8 (7.1) points (95% CI, -10.4 to -3.1; P = .001). These changes were observed in patients with and without GBA1 mutations.The study results suggest that ambroxol therapy was safe and well tolerated; CSF penetration and target engagement of ambroxol were achieved, and CSF α-synuclein levels were increased. Placebo-controlled clinical trials are needed to examine whether ambroxol therapy is associated with changes in the natural progression of PD.ClinicalTrials.gov identifier: NCT02941822; EudraCT identifier: 2015-002571-24.

Extracellular matrix (ECM) disruption is known to be an early pathological feature of the Mucopolysaccharidoses (MPS). Collagen is the main component of the ECM and its metabolism could act as a useful indicator of ECM disruption. We have measured the specific collagen breakdown products; urinary free hydroxylated (Lys-OH) and glycosylated hydroxylysines (Lys-O-Gal and Lys-O-GalGlc) in MPS patients using a tandem liquid chromatography tandem mass spectrometry assay. A pilot study cohort analysis indicated that concentrations of lysine and Lys-OH were raised significantly in MPS I (Hurler) disease patients. Lys-O-GalGlc was raised in MPS II and MPS VI patients and demonstrated a significant difference between MPS I Hurler and an MPS I Hurler-Scheie group. Further analysis determined an age association for glycosylated hydroxylysine in control samples similar to that observed for the glycosaminoglycans. Using defined age ranges and treatment naive patient samples we confirmed an increase in glycosylated hydroxylysines in MPS I and in adult MPS IVA. We also looked at the ratio of Lys-O-Gal to Lys-O-GalGlc, an indicator of the source of collagen degradation, and noticed a significant change in the ratio for all pediatric MPS I, II, and IV patients, and a small significant increase in adult MPS IV. This indicated that the collagen degradation products were coming from a source other than bone such as cartilage or connective tissue. To see how specific the changes in glycosylated hydroxylysine were to MPS patients we also looked at levels in patients with other inherited metabolic disorders. MPS patients showed a trend towards increased glycosylated hydroxylysines and an elevated ratio compared to other metabolic disorders that included Battens disease, Fabry disease, Pyridoxine-dependent epilepsy (due to mutations in ALDH7A1), and Niemann Pick C disease.

Background: Hypertrophic cardiomyopathy (HCM) is characterized by a complex phenotype that is only partly explained by the biological effects of individual genetic variants. The aim of this study was to use proteomic analysis of myocardial tissue to explore the postgenomic phenotype. Methods: Label-free proteomic analysis was used initially to compare protein profiles in myocardial samples from 11 patients with HCM undergoing surgical myectomy with control samples from 6 healthy unused donor hearts. Differentially expressed proteins of interest were validated in myocardial samples from 65 unrelated individuals (HCM [n=51], controls [n=7], and aortic stenosis [n=7]) by the development and use of targeted multiple reaction monitoring-based triple quadrupole mass spectrometry. Results: In this exploratory study, 1586 proteins were identified with 151 proteins differentially expressed in HCM samples compared with controls ( P P P P =0.015), late gadolinium enhancement on cardiac magnetic resonance imaging ( P =0.03) and the presence of a pathogenic sarcomere mutation ( P =0.04). Conclusions: The myocardial proteome of HCM provides supporting evidence for dysregulation of metabolic and structural proteins. The finding that lumican is raised in HCM hearts provides insight into the myocardial fibrosis that characterizes this disease.

Ciaran Carson's writing plays with and renders strange all kinds of literary and generic classifications. The subject of numerous short critical accounts, a monograph and at least one collection of essays, his work, especially the poetry, has attracted a good deal of scholarly attention. ‘After Carson’ offers a hybridic, ‘creative-critical’ response to a genre-busting idiom. Focussing on just two of Carson's works, the well-known and much-discussed Belfast Confetti (1989), and the complex but less carefully read prose work The Star Factory (1997), the formal self-consciousness of this treatment is intended to problematise the conceptual and textual bounds of conventional literary scholarship. The intention is to figure, as well as justify, the disruption of conventional linear argument with the help of the subversive energies to be discovered in collision, lapse, detour, ambush, and / or other kinds of discursive misconduct. Informed partly by dissatisfaction with most theories of reading, our cri...

Cardiomyopathies are an important cause of sudden cardiac death in young people and are responsible for arrhythmias and premature heart failure in all age groups. Although many cardiomyopathies are inherited, biochemical markers are a fundamental part of the diagnostic work-up and are useful in the prognostic assessment of disease. In this article, the authors review the rationale and evidence for important biomarkers according to their role in patient management. Emerging biomarkers are briefly reviewed, alongside modern scientific approaches to biomarker discovery.

Although peri-conceptional folic acid (FA) supplementation can prevent a proportion of neural tube defects (NTD), there is increasing evidence that many NTD are FA non-responsive. The vitamin-like molecule inositol may offer a novel approach to preventing FA-non-responsive NTD. Inositol prevented NTD in a genetic mouse model, and was well tolerated by women in a small study of NTD recurrence. In the present study, we report the Prevention of Neural Tube Defects by Inositol (PONTI) pilot study designed to gain further experience of inositol usage in human pregnancy as a preliminary trial to a future large-scale controlled trial to evaluate efficacy of inositol in NTD prevention. Study subjects were UK women with a previous NTD pregnancy who planned to become pregnant again. Of 117 women who made contact, ninety-nine proved eligible and forty-seven agreed to be randomised (double-blind) to peri-conceptional supplementation with inositol plus FA or placebo plus FA. In total, thirty-three randomised pregnancies produced one NTD recurrence in the placebo plus FA group (n 19) and no recurrences in the inositol plus FA group (n 14). Of fifty-two women who declined randomisation, the peri-conceptional supplementation regimen and outcomes of twenty-two further pregnancies were documented. Two NTD recurred, both in women who took only FA in their next pregnancy. No adverse pregnancy events were associated with inositol supplementation. The findings of the PONTI pilot study encourage a large-scale controlled trial of inositol for NTD prevention, but indicate the need for a careful study design in view of the unwillingness of many high-risk women to be randomised.

Background The discovery that heterozygous missense mutations in the gene encoding triggering receptor expressed on myeloid cells 2 (TREM2) are risk factors for Alzheimer’s disease (AD), with only the apolipoprotein E (APOE) ε4 gene allele conferring a higher risk, has led to increased interest in immune biology in the brain. TREM2 is expressed on microglia, the resident immune cells of the brain and has been linked to phagocytotic clearance of amyloid β (Aβ) plaques. Soluble TREM2 (sTREM2) has previously been measured in cerebrospinal fluid (CSF) by ELISA but in our hands commercial kits have proved unreliable, suggesting that other methods may be required. We developed a mass spectrometry method using selected reaction monitoring for the presence of a TREM2 peptide, which can be used to quantify levels of sTREM2 in CSF. Findings We examined CSF samples from memory clinics in Sweden and the UK. For all samples the following were available: clinical diagnosis, age, sex, and measurements of the CSF AD biomarkers Aβ42, T-tau and P-tau181. AD patients (n = 37) all met biomarker (IWG2) criteria for AD. Control individuals (n = 22) were cognitively normal without evidence for AD in CSF. We found significantly higher sTREM2 concentration in AD compared to control CSF. There were significant correlations between CSF sTREM2 and T-tau as well as P-tau181. CSF sTREM2 increase in AD was replicated in a second, independent cohort consisting of 24 AD patients and 16 healthy volunteers. Conclusion CSF concentrations of sTREM2 are higher in AD than in controls, and correlate with markers of neurodegeneration. CSF sTREM2 may be used to quantify glial activation in AD. Electronic supplementary material The online version of this article (doi:10.1186/s13024-016-0071-x) contains supplementary material, which is available to authorized users.

Item does not contain fulltext Cerebrospinal fluid (CSF) biomarkers for Alzheimer's disease (AD) are increasingly used in clinical settings, research and drug trials. However, their broad-scale use on different technology platforms is hampered by the lack of standardization at the level of sample handling, determination of concentrations of analytes and the absence of well-defined performance criteria for in vitro diagnostic or companion diagnostic assays, which influences the apparent concentration of the analytes measured and the subsequent interpretation of the data. There is a need for harmonization of CSF AD biomarker assays that can reliably, across centers, quantitate CSF biomarkers with high analytical precision, selectivity and stability over long time periods. In this position paper, we discuss reference procedures for the measurement of CSF AD biomarkers, especially amyloid beta42 and tau. We describe possible technical approaches, focusing on a selected reaction monitoring mass spectrometry assay as a candidate reference method for quantification of CSF amyloid beta42. 01 augustus 2012

Introduction: Fabry disease (FD) is an X-linked lysosomal storage disorder resulting from mutations in the gene encoding α-galactosidase A (GLA). It is characterised by the abnormal accumulation of neutral glycosphingolipids (GSL), predominantly Globotriaosylceramide (Gb3), in the lysosomes of multiple cell types including vascular endothelial cells. It is associated with early-onset stroke, cardiomyopathy, and progression to end-stage renal failure. Current standard of care for FD is enzyme replacement therapy but alternatives including allogeneic stem cell transplantation have also been explored. Fabry disease is a good model for liver-targeted gene therapy, as relatively low levels of enzyme correction may suffice to reduce storage. Additionally, a broad therapeutic window exists, and mouse models of FD enabling preclinical proof-of-concept studies are readily available. Here we report the preclinical results of adeno-associated virus (AAV) mediated, liver-targeted gene transfer of a self-complementary (sc) vector encoding a codon-optimised GLA transgene under transcriptional control of a hepatocyte-specific promoter pseudotyped with AAV8 capsid (scAAV2/8-LP1-GLAco). Methods: In this study a single dose of scAAV2/8-LP1-GLAco vectors were administered into the tail vein of homozygote GLA knockout Fabry mice at either 1 month or 3 months of age. Two doses were tested for each age group; 2x1012 vg/kg and 2x1013 vg/kg, and an additional age-matched group left as untreated to serve as an experimental control for the effects of treatment. Mice were followed in the treatment for up to 11 months before sacrificing at 11 or 14 months of age. To assess the kinetics and durability of transgene expression, plasma GLA levels were measured at various time intervals post injection. Results: Long-term follow-up of Fabry mice injected with scAAV2/8-LP1-GLAco vector demonstrated sustained, durable levels of plasma GLA throughout the typical lifespan of a mouse. Plasma GLA activity levels at 10 or 11-months post vector administration to 1 and 3-month-old mice respectively, demonstrated a dose response from 2 x 1012 to 2 x 1013 vg/kg of >1 log increase in expression. These GLA activity levels represent supra-physiological expression (x 4000 of normal levels) which resulted in uptake of GLA from the bloodstream into kidney and cardiac tissues leading to dose-dependent clearance of lipid deposits in these tissues as analysed by electron microscopy. Most importantly, sustained expression of GLA resulted in complete normalisation of Gb3 and lyso-Gb3 levels in plasma and liver tissues as quantified by mass spectrometry. Interestingly, these preclinical studies in Fabry mice at the age of 1 and 3 months demonstrated not only prevention but also reversal of lysosomal storage deposits through liver-specific expression of GLA. Finally, in these in vivo mouse studies no adverse clinical signs were observed, despite the increased plasma exposure to GLA. Conclusions: Collectively, these data provide strong evidence that our liver-directed AAV-mediated gene therapy approach holds considerable therapeutic potential for the treatment of Fabry disease. We anticipate that a single dose IV procedure will pose minimal burden to Fabry patients and will be a viable alternative to biweekly enzyme infusions, potentially reducing treatment-related morbidity whislt improving patient quality of life and potentially providing them with a functional long-term cure. Disclosures Kia: Freeline: Employment, Equity Ownership. McIntosh:Freeline: Consultancy. Hosseini:Freeline: Employment, Equity Ownership. Sheridan:Freeline: Employment, Equity Ownership. Corbau:Freeline: Employment, Equity Ownership. Nathwani:BioMarin: Consultancy, Patents & Royalties; UniQure: Patents & Royalties; Freeline: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.

James (bv) Thomson's insomnia is well documented. Bertram Dobell's memoir of the poet makes several mentions of it, as do critics of his work such as Robert Crawford, Edwin Morgan, and William Sharpe.1 Although widely acknowledged and noted as a significant biographical detail by critics, Thomson's insomnia has not been examined in terms of its relation to the specifically apocalyptic tone in some of his work. It is this curious relationship and its implications for the reading of his long poem The City of Dreadful Night which form the focus of this paper. Thomson often wrote about sleep the lack of it and the desire for it. "The Sleeper," for example, is an envious and touching description of a woman drowsing and then falling into "slumber deep," "Deep sleep, so holy in its calm." The final stanza is particularly telling

Aim: The aim of this study was to determine whether globotriaosylceramide (Gb 3 ) is a useful biomarker in Fabry disease. Methods: The levels of Gb 3 were measured in plasma and urine by tandem mass spectrometry in untreated hemizygotes and heterozygotes with Fabry disease and in healthy controls, and the levels were monitored in patients on treatment with enzyme replacement therapy (ERT). Results: Hemizygotes with classic Fabry disease showed elevated levels of Gb 3 in both plasma and urine and could readily be distinguished from normal controls. Male patients with the N215S mutation had normal levels in their plasma but 50% had marginally elevated levels in their urine. Thirty-three percent of proven heterozygotes had elevated Gb 3 concentrations in plasma but 97% of those without the N215S mutation (36/37) had an elevated level in urine. The four heterozygotes with the N215S mutation had normal Gb 3 levels in urine. The level of Gb 3 in plasma initially fell following the start of ERT in all patients who had an elevated level before treatment. However, in a few patients the level subsequently rose. Similar results were found for the levels of Gb 3 in urine. Conclusion: Gb 3 is not an ideal marker of Fabry disease or the response to treatment in all patients. Plasma and urine levels of Gb 3 cannot be used as a marker of Fabry disease in patients with the N215S mutation and many heterozygotes do not have elevated Gb 3 levels in plasma. The urine concentration is more informative in heterozygotes and can be used as a measure of the response to therapy. The fall in Gb 3 levels in patients receiving ERT was not sustained in some patients, despite a clinical improvement. Additionally, Gb 3 cannot be used to monitor the response to treatment in patients who initially have normal plasma and urine concentrations of this glycolipid.

[H]e saw Paul coming, a man small in size, bald-headed, bandy-legged, well-built, with eyebrows meeting, rather long-nosed, full of grace. For sometimes he looked like a man, and sometimes he had the countenance of an angel.--The Acts of Paul and Thecla [W]e cannot even regard ourselves as a constant; in this flux of things, our identity itself seems in a perpetual variation; and not infrequently we find our own disguise the strangest in the masquerade.--Robert Louis Stevenson, Virginibus Puerisque The Strangest Disguise Modern thinkers, from Rene Descartes onward, have often had what can seem like a clear-eyed view of themselves. Viewing the human mind as the "mirror of nature," as Richard Rorty puts it, they have included in its reflective field their own pristine images. Descartes thought his self-reflection clear enough to serve as the foundation of an entire philosophical system: self was the one thing that could not be doubted, and thus was launched modern philosophy. Immanuel Kant, too, found in the perusal of his own mind sufficient clarity to limn out the entire architecture of reason, drawing on no other source. In the preface to the first edition of Critique of Pure Reason, he wrote that "I have to deal with nothing save reason and its pure thinking; and to obtain complete knowledge of these, there is no need to go far afield, since I come upon them in my own self" (10). Subsequently, of course, Friedrich Nietzsche took a Darwinian hammer to the looking glass, arguing that the Cartesian "I" was "only a supposition, an assertion" (171). Karl Marx went on to breathe the powers of nature and culture onto the mirror of self-representation, pointing to the real-world conditions under which human consciousness is produced: "Consciousness can never be anything else than conscious existence, and the existence of men is their actual life-process" (169). The story continues with Sigmund Freud's insistence that self-representation is superficial, Jacques Lacan's claim that it is an effect of language, and Luce Irigaray's perception that it is distorted by gendered conceptions. Given their barrage of damaging blows aimed at the mirror of self-consciousness, such post-Kantian thinkers have shown us an image of ourselves that is unlike both the visual awareness of our appearance and the imagination's projection of our identity. However, the chastening of Descartes' and Kant's self-images, and the breaking of their vainglorious mirrors, did not require much besides a careful reading of St. Paul. Robert Louis Stevenson was, perhaps, among the first to recognize this, or at least the first to encode that recognition in fictional form. In that most Pauline of tales, The Strange Case of Dr. Jekyll and Mr. Hyde, he explores subjectivity in a way that both echoes Paul's text and brings to light the puzzling fecundity of the self. This essay attempts to set up a series of reflections between the texts of Stevenson and St. Paul in order to create an image of the Christian subject. In the process of discovering this image, another reflection is produced--the inversion of William James's account of conversion in The Varieties of Religious Experience. He describes the process as one of the reunification of a divided subject: To be converted, to be regenerated, to receive grace, to experience religion, to gain an assurance [...] are so many phrases which denote the process, gradual or sudden, by which a self hitherto divided, and consciously wrong[,] inferior[,] and unhappy, becomes unified and consciously right[,] superior[,] and happy, in consequence of its firmer hold upon religious realities. (189) The subject of Christian conversion that emerges from my exploration moves in the opposite direction--toward the splitting, or disaggregation, of the self. It is precisely this fracturing within the self, and the echoes of St. Paul that can be detected in its delineation, that make Jekyll and Hyde an instructive narrative in the consideration of Christian subjectivity. …

Introduction The tertiary structure of normal podocytes prevents protein from leaking into urine. Patients with lupus nephritis (LN) develop proteinuria, and kidney biopsies from these patients display a number of podocyte abnormalities including retraction of podocyte processes. Autoantibodies have been shown to deposit in the kidneys of patients and mice with LN and are believed to play a key role in causing renal inflammation and dysfunction. The objective of this research was to study the effects of IgG antibodies from patients with LN on cultured human podocytes. Methods We exposed a human podocyte cell line to heat-inactivated (HI) plasma and purified polyclonal IgG from the following groups of subjects; patients with LN, patients with lupus without nephritis, patients with rheumatoid arthritis and healthy controls. We measured expression and intracellular distribution of podocyte-specific proteins and global phosphorylation of tyrosine. We then used mass spectrometry to identify the major protein targets of this phosphorylation. Results HI LN plasma did not alter expression or cellular distribution of podocyte-specific proteins but caused a significant reduction in podocyte protein tyrosine phosphorylation compared with plasma from healthy controls (p=0.0008). This result was replicated using purified IgG but was not seen with plasma from rheumatoid arthritis or non-renal lupus patients. The dominant tyrosine phosphorylated protein in podocytes was 55 kDa in size and was identified as tubulin. Conclusions Since tubulin is an important component of podocyte major processes, these results suggest that autoantibodies from LN patients may exert an important pathogenic effect by dephosphorylation of this protein in podocytes.

Protein glycosylation is an important post-translational modification. It enhances the functional diversity of proteins, half-life and influences their biological activity. Defective glycosylation often leads to multisystem disease and adds itself to the expanding group of ‘Congenital disorders or glycosylation’ which are predominantly disorders of N-linked glycosylation. Another rapidly growing group of disorders are defects in O-linked glycosylation, including a subset of dystroglycanopathies. Current diagnostic strategies for glycosylation disorders are compounded by the multivariate clinical phenotype of many of the diseases. Biochemical tests such as the isoelectric focusing of transferrin and apolipoprotein CIII are used to assess a patient’s glycoform profile before in depth enzyme and genetic analysis is initiated. Whilst the glycoform profiling has been instrumental in screening for many glycosylation disorders, there is a need for a more sensitive and informative test. This short review gives an overview of the recent methods used in glycobiology research that could be used to devise such a test, which alongside currently used diagnostic tests should further facilitate the delineation of CDG subtypes. It provides a view to a potential strategy using marker glycopeptides to develop a mass spectrometry based assay that could be implemented into clinical diagnostic laboratories.

In a country where many view bicycles as mere toys and walking as a relic of a bygone era, why would anyone posit that walking and bicycling (or “active transportation”) are essential to a serious strategy to rein in America’s oil dependence? The simple answer is that overreliance on motor vehicles is at the center of our oil dependence, and walking and bicycling are the most cost-effective ways to curb a substantial portion of projected growth in vehicle-miles traveled. A convergence of popular demand for safe and convenient places to walk or bicycle and fiscal constraints that compel policy makers to make the most of every tax dollar add up to a compelling case for increased investment in active transportation as an integral piece of our strategy to manage our nation’s oil dependence.

We present a 4-generation family in which affected individuals have cataracts, a motor neuronopathy with upper motor neuron signs, short stature, developmental delay, and skeletal abnormalities. An additional symptom is weakness during pregnancy which resolves after delivery. The condition is inherited in an autosomal dominant manner. The manifestations and inheritance are not found in any previously described conditions. We consider that this is a new syndrome.

Alzheimer’s disease (AD) is the most common cause of dementia. Biomarkers are required to identify individuals in the preclinical phase, explain phenotypic diversity, measure progression and estimate prognosis. The development of assays to validate candidate biomarkers is costly and time-consuming. Targeted proteomics is an attractive means of quantifying novel proteins in cerebrospinal and other fluids, and has potential to help overcome this bottleneck in biomarker development. We used a previously validated multiplexed 10-min, targeted proteomic assay to assess 54 candidate cerebrospinal fluid (CSF) biomarkers in two independent cohorts comprising individuals with neurodegenerative dementias and healthy controls. Individuals were classified as ‘AD’ or ‘non-AD’ on the basis of their CSF T-tau and amyloid Aβ1–42 profile measured using enzyme-linked immunosorbent assay; biomarkers of interest were compared using univariate and multivariate analyses. In all, 35/31 individuals in Cohort 1 and 46/36 in Cohort 2 fulfilled criteria for AD/non-AD profile CSF, respectively. After adjustment for multiple comparisons, five proteins were elevated significantly in AD CSF compared with non-AD CSF in both cohorts: malate dehydrogenase; total APOE; chitinase-3-like protein 1 (YKL-40); osteopontin and cystatin C. In an independent multivariate orthogonal projection to latent structures discriminant analysis (OPLS-DA), these proteins were also identified as major contributors to the separation between AD and non-AD in both cohorts. Independent of CSF Aβ1–42 and tau, a combination of these biomarkers differentiated AD and non-AD with an area under curve (AUC)=0.88. This targeted proteomic multiple reaction monitoring (MRM)-based assay can simultaneously and rapidly measure multiple candidate CSF biomarkers. Applying this technique to AD we demonstrate differences in proteins involved in glucose metabolism and neuroinflammation that collectively have potential clinical diagnostic utility.