Liquid chromatography mass spectrometry has become one of the analytical platforms of choice for metabolomics studies. However, LC-MS metabolomics data can suffer from the effects of various ...systematic biases. These include batch effects, day-to-day variations in instrument performance, signal intensity loss due to time-dependent effects of the LC column performance, accumulation of contaminants in the MS ion source and MS sensitivity among others. In this study we aimed to test a singular value decomposition-based method, called EigenMS, for normalization of metabolomics data. We analyzed a clinical human dataset where LC-MS serum metabolomics data and physiological measurements were collected from thirty nine healthy subjects and forty with type 2 diabetes and applied EigenMS to detect and correct for any systematic bias. EigenMS works in several stages. First, EigenMS preserves the treatment group differences in the metabolomics data by estimating treatment effects with an ANOVA model (multiple fixed effects can be estimated). Singular value decomposition of the residuals matrix is then used to determine bias trends in the data. The number of bias trends is then estimated via a permutation test and the effects of the bias trends are eliminated. EigenMS removed bias of unknown complexity from the LC-MS metabolomics data, allowing for increased sensitivity in differential analysis. Moreover, normalized samples better correlated with both other normalized samples and corresponding physiological data, such as blood glucose level, glycated haemoglobin, exercise central augmentation pressure normalized to heart rate of 75, and total cholesterol. We were able to report 2578 discriminatory metabolite peaks in the normalized data (p<0.05) as compared to only 1840 metabolite signals in the raw data. Our results support the use of singular value decomposition-based normalization for metabolomics data.
In the early to mid‐20th century, reductionism as a concept in biology was challenged by key thinkers, including Ludwig von Bertalanffy. He proposed that living organisms were specific examples of ...complex systems and, as such, they should display characteristics including hierarchical organisation and emergent behaviour. Yet the true study of complete biological systems (for example, metabolism) was not possible until technological advances that occurred 60 years later. Technology now exists that permits the measurement of complete levels of the biological hierarchy, for example the genome and transcriptome. The complexity and scale of these data require computational models for their interpretation. The combination of these – systems thinking, high‐dimensional data and computation – defines systems biology, typically accompanied by some notion of iterative model refinement. Only sequencing‐based technologies, however, offer full coverage. Other ‘omics’ platforms trade coverage for sensitivity, although the densely connected nature of biological networks suggests that full coverage may not be necessary. Systems biology models are often characterised as either ‘bottom‐up’ (mechanistic) or ‘top‐down’ (statistical). This distinction can mislead, as all models rely on data and all are, to some degree, ‘middle‐out’. Systems biology has matured as a discipline, and its methods are commonplace in many laboratories. However, many challenges remain, especially those related to large‐scale data integration.
Components of, and challenges in, systems biology.
Abstract The transcription factor STAT3 is overexpressed and hyperactivated in T cells from SLE patients. STAT3 plays a central role in T cell differentiation into Th17 and T follicular helper cells, ...two subsets that orchestrate autoimmune responses in SLE. Moreover, STAT3 is important in chemokine-mediated T cell migration. To better understand its role in SLE, we inhibited STAT3 in lupus-prone mice using the small molecule Stattic. Stattic-treated mice exhibited delayed onset of proteinuria (3 weeks later than controls), and had lower levels of anti-dsDNA antibodies and inflammatory cytokines. Inhibitor treatment reduced lymphadenopathy, resulted in a 3-fold decrease in total T cell number, and a 4-fold decrease in the numbers of T follicular helper cells. In vitro experiments showed that Stattic-treated T cells exhibited decreased proliferation and a decrease in ability to migrate to CXCL12. We propose that STAT3 inhibition represents a therapeutic target in SLE, particularly lupus nephritis.
ObjectivesTo characterise the sketetal muscle metabolic phenotype during early critical illness.MethodsVastus lateralis muscle biopsies and serum samples (days 1 and 7) were obtained from 63 ...intensive care patients (59% male, 54.7±18.0 years, Acute Physiology and Chronic Health Evaluation II score 23.5±6.5).Measurements and main resultsFrom day 1 to 7, there was a reduction in mitochondrial beta-oxidation enzyme concentrations, mitochondrial biogenesis markers (PGC1α messenger mRNA expression (−27.4CN (95% CI −123.9 to 14.3); n=23; p=0.025) and mitochondrial DNA copy number (−1859CN (IQR −5557–1325); n=35; p=0.032). Intramuscular ATP content was reduced compared tocompared with controls on day 1 (17.7mmol/kg /dry weight (dw) (95% CI 15.3 to 20.0) vs. 21.7 mmol/kg /dw (95% CI 20.4 to 22.9); p<0.001) and decreased over 7 days (−4.8 mmol/kg dw (IQR −8.0–1.2); n=33; p=0.001). In addition, the ratio of phosphorylated:total AMP-K (the bioenergetic sensor) increased (0.52 (IQR −0.09–2.6); n=31; p<0.001). There was an increase in intramuscular phosphocholine (847.2AU (IQR 232.5–1672); n=15; p=0.022), intramuscular tumour necrosis factor receptor 1 (0.66 µg (IQR −0.44–3.33); n=29; p=0.041) and IL-10 (13.6 ng (IQR 3.4–39.0); n=29; p=0.004). Serum adiponectin (10.3 µg (95% CI 6.8 to 13.7); p<0.001) and ghrelin (16.0 ng/mL (IQR −7–100); p=0.028) increased. Network analysis revealed a close and direct relationship between bioenergetic impairment and reduction in muscle mass and between intramuscular inflammation and impaired anabolic signaling. ATP content and muscle mass were unrelated to lipids delivered.ConclusionsDecreased mitochondrial biogenesis and dysregulated lipid oxidation contribute to compromised skeletal muscle bioenergetic status. In addition, intramuscular inflammation was associated with impaired anabolic recovery with lipid delivery observed as bioenergetically inert. Future clinical work will focus on these key areas to ameliorate acute skeletal muscle wasting.Trial registration number NCT01106300.
Changes in the respiratory microbiome are associated with disease progression in idiopathic pulmonary fibrosis (IPF). The role of the host response to the respiratory microbiome remains unknown.
To ...explore the host-microbial interactions in IPF.
Sixty patients diagnosed with IPF were prospectively enrolled together with 20 matched control subjects. Subjects underwent bronchoalveolar lavage (BAL), and peripheral whole blood was collected into PAXgene tubes for all subjects at baseline. For subjects with IPF, additional samples were taken at 1, 3, and 6 months and (if alive) 1 year. Gene expression profiles were generated using Affymetrix Human Gene 1.1 ST arrays.
By network analysis of gene expression data, we identified two gene modules that strongly associated with a diagnosis of IPF, BAL bacterial burden (determined by 16S quantitative polymerase chain reaction), and specific microbial operational taxonomic units, as well as with lavage and peripheral blood neutrophilia. Genes within these modules that are involved in the host defense response include NLRC4, PGLYRP1, MMP9, and DEFA4. The modules also contain two genes encoding specific antimicrobial peptides (SLPI and CAMP). Many of these particular transcripts were associated with survival and showed longitudinal overexpression in subjects experiencing disease progression, further strengthening the relationship of the transcripts with disease.
Integrated analysis of the host transcriptome and microbial signatures demonstrated an apparent host response to the presence of an altered or more abundant microbiome. These responses remained elevated in longitudinal follow-up, suggesting that the bacterial communities of the lower airways may act as persistent stimuli for repetitive alveolar injury in IPF.
Acute Skeletal Muscle Wasting in Critical Illness Puthucheary, Zudin A; Rawal, Jaikitry; McPhail, Mark ...
JAMA : the journal of the American Medical Association,
10/2013, Volume:
310, Issue:
15
Journal Article
Peer reviewed
IMPORTANCE Survivors of critical illness demonstrate skeletal muscle wasting with associated functional
impairment. OBJECTIVE To perform a comprehensive prospective characterization of skeletal ...muscle wasting, defining the
pathogenic roles of altered protein synthesis and breakdown. DESIGN, SETTING, AND PARTICIPANTS Sixty-three critically ill patients (59% male; mean age: 54.7 years 95% CI, 50.0-59.6 years)
with an Acute Physiology and Chronic Health Evaluation II score of 23.5 (95% CI, 21.9-25.2) were
prospectively recruited within 24 hours following intensive care unit (ICU) admission from August
2009 to April 2011 at a university teaching and a community hospital in England. Patients were
recruited if older than 18 years and were anticipated to be intubated for longer than 48 hours, to
spend more than 7 days in critical care, and to survive ICU stay. MAIN OUTCOMES AND MEASURES Muscle loss was determined through serial ultrasound measurement of the rectus femoris
cross-sectional area (CSA) on days 1, 3, 7, and 10. In a subset of patients, the fiber CSA area was
quantified along with the ratio of protein to DNA on days 1 and 7. Histopathological analysis was
performed. In addition, muscle protein synthesis, breakdown rates, and respective signaling pathways
were characterized. RESULTS There were significant reductions in the rectus femoris CSA observed at day 10 (−17.7% 95%
CI, −25.9% to 8.1%; P < .001). In the 28 patients assessed by
all 3 measurement methods on days 1 and 7, the rectus femoris CSA decreased by 10.3% (95% CI, 6.1%
to 14.5%), the fiber CSA by 17.5% (95% CI, 5.8% to 29.3%), and the ratio of protein to DNA by 29.5%
(95% CI, 13.4% to 45.6%). Decrease in the rectus femoris CSA was greater in patients who experienced
multiorgan failure by day 7 (−15.7%; 95% CI, −27.7% to 11.4%) compared with single organ
failure (−3.0%; 95% CI, −5.3% to 2.1%) (P < .001), even
by day 3 (−8.7% 95% CI, −59.3% to 50.6% vs −1.8% 95% CI, −12.3% to
10.5%, respectively; P = .03). Myofiber necrosis occurred in 20 of 37
patients (54.1%). Protein synthesis measured by the muscle protein fractional synthetic rate was
depressed in patients on day 1 (0.035%/hour; 95% CI, 0.023% to 0.047%/hour) compared with rates
observed in fasted healthy controls (0.039%/hour; 95% CI, 0.029% to 0.048%/hour)
(P = .57) and increased by day 7 (0.076% 95% CI, 0.032%-0.120%/hour;
P = .03) to rates associated with fed controls (0.065%/hour 95% CI,
0.049% to 0.080%/hour; P = .30), independent of nutritional load. Leg
protein breakdown remained elevated throughout the study (8.5 95% CI, 4.7 to 12.3 to 10.6 95% CI,
6.8 to 14.4 μmol of phenylalanine/min/ideal body weight × 100;
P = .40). The pattern of intracellular signaling supported increased
breakdown (n = 9, r = −0.83,
P = .005) and decreased synthesis (n = 9,
r = −0.69, P = .04). CONCLUSIONS AND RELEVANCE Among these critically ill patients, muscle wasting occurred early and rapidly during the first
week of critical illness and was more severe among those with multiorgan failure compared with
single organ failure. These findings may provide insights into skeletal muscle wasting in critical
illness.
The T-cell receptor (TCR) interacts with peptide-major histocompatibility complexes (pMHC) to discriminate pathogens from self-antigens and trigger adaptive immune responses. Direct physical contact ...is required between the T cell and the antigen-presenting cell for cross-junctional binding where the TCR and pMHC are anchored on two-dimensional (2D) membranes of the apposing cells. Despite their 2D nature, TCR-pMHC binding kinetics have only been analysed three-dimensionally (3D) with a varying degree of correlation with the T-cell responsiveness. Here we use two mechanical assays to show high 2D affinities between a TCR and its antigenic pMHC driven by rapid on-rates. Compared to their 3D counterparts, 2D affinities and on-rates of the TCR for a panel of pMHC ligands possess far broader dynamic ranges that match that of their corresponding T-cell responses. The best 3D predictor of response is the off-rate, with agonist pMHC dissociating the slowest. In contrast, 2D off-rates are up to 8,300-fold faster, with the agonist pMHC dissociating the fastest. Our 2D data suggest rapid antigen sampling by T cells and serial engagement of a few agonist pMHCs by TCRs in a large self pMHC background. Thus, the cellular environment amplifies the intrinsic TCR-pMHC binding to generate broad affinities and rapid kinetics that determine T-cell responsiveness.
BackgroundWe report updated safety, efficacy, and immunogenicity of AZD1222 (ChAdOx1 nCoV-19) from an ongoing phase 3 trial.MethodsAdults at increased risk of SARS-CoV-2 infection were randomized ...(2:1), stratified by age, to receive 2 doses of AZD1222 or placebo. The primary efficacy end point was confirmed SARS-CoV-2 reverse-transcriptase PCR-positive (RT-PCR-positive) symptomatic COVID-19 at 15 or more days after a second dose in baseline SARS-CoV-2-seronegative participants. The 21,634 and 10,816 participants were randomized to AZD1222 and placebo, respectively.FindingsData cutoff for this analysis was July 30, 2021; median follow-up from second dose was 78 and 71 days for the double-blind period (censoring at unblinding or nonstudy COVID-19 vaccination) and 201 and 82 days for the period to nonstudy COVID-19 vaccination (regardless of unblinding) in the AZD1222 and placebo groups, respectively. For the primary efficacy end point in the double-blind period (141 and 184 events; incidence rates: 39.2 and 118.8 per 1,000 person years), vaccine efficacy was 67.0% (P < 0.001). In the period to nonstudy COVID-19 vaccination, incidence of events remained consistently low and stable through 6 months in the AZD1222 group; for the primary efficacy end point (328 and 219 events; incidence rates: 36.4, 108.4) and severe/critical disease (5 and 13 events; incidence rates: 0.6, 6.4), respective vaccine efficacy estimates were 65.1% and 92.1%. AZD1222 elicited humoral immune responses over time, with waning at day 180. No emergent safety issues were seen.ConclusionAZD1222 is safe and well tolerated, demonstrating durable protection and immunogenicity with median follow-up (AZD1222 group) of 6 months.Trial registrationClinicalTrials.gov NCT04516746.FundingAstraZeneca; US government.
Despite significant advancements in the care of patients with hypoplastic left heart syndrome (HLHS) morbidity and mortality remain high. Postnatal right ventricular dysfunction and tricuspid ...regurgitation (TR) are associated with worse outcomes in HLHS. We aim to determine if right ventricle functional parameters and TR on fetal echocardiogram are associated with postnatal outcomes in HLHS patients. Retrospective review was performed on all fetuses with HLHS from 2014 to 2022 at our institution. Initial and follow up fetal echocardiogram measurements of right ventricular myocardial performance index (MPI), fractional area change (FAC) and global longitudinal strain (GLS) were retrospectively measured. The presence and severity of TR was recorded from the fetal echocardiogram reports. Postnatal outcomes including transplant-free survival, hospital length of stay > 30 days after initial palliation and need for bidirectional Glenn at < 4 months were reviewed. Forty-three subjects met inclusion criteria. Mean gestational age at presentation was 26.1 ± 5.9 weeks. Nine subjects died and 3 required heart transplantation. Initial fetal echocardiogram MPI was significantly lower (better) (0.36 ± 0.06 vs 0.44 ± 0.11; p = < 0.001) and FAC was significantly higher (better) (45 ± 6% vs 40 ± 8%; p = 0.035) in transplant-free survivors. Fetal right ventricular GLS and presence of mild TR were not associated with postnatal outcome. In fetuses with HLHS, abnormal MPI and right ventricular FAC are associated with decreased transplant-free survival. There was no observed association between GLS and postnatal outcomes. To our knowledge this is the first study examining fetal right ventricular function and GLS in HLHS patients and its link to postnatal outcomes.
Dietary nitrate supplementation can enhance exercise performance in healthy people, but it is not clear if it is beneficial in COPD. We investigated the hypotheses that acute nitrate dosing would ...improve exercise performance and reduce the oxygen cost of submaximal exercise in people with COPD.
We performed a double-blind, placebo-controlled, cross-over single dose study. Subjects were randomised to consume either nitrate-rich beetroot juice (containing 12.9 mmoles nitrate) or placebo (nitrate-depleted beetroot juice) 3 hours prior to endurance cycle ergometry, performed at 70% of maximal workload assessed by a prior incremental exercise test. After a minimum washout period of 7 days the protocol was repeated with the crossover beverage.
21 subjects successfully completed the study (age 68 ± 7 years; BMI 25.2 ± 5.5 kg/m2; FEV1 percentage predicted 50.1 ± 21.6%; peak VO2 18.0 ± 5.9 ml/min/kg). Resting diastolic blood pressure fell significantly with nitrate supplementation compared to placebo (-7 ± 8 mmHg nitrate vs. -1 ± 8 mmHg placebo; p = 0.008). Median endurance time did not differ significantly; nitrate 5.65 (3.90-10.40) minutes vs. placebo 6.40 (4.01-9.67) minutes (p = 0.50). However, isotime oxygen consumption (VO2) was lower following nitrate supplementation (16.6 ± 6.0 ml/min/kg nitrate vs. 17.2 ± 6.0 ml/min/kg placebo; p = 0.043), and consequently nitrate supplementation caused a significant lowering of the amplitude of the VO2-percentage isotime curve.
Acute administration of oral nitrate did not enhance endurance exercise performance; however the observation that beetroot juice caused reduced oxygen consumption at isotime suggests that further investigation of this treatment approach is warranted, perhaps targeting a more hypoxic phenotype.
ISRCTN Registry ISRCTN66099139.
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