Large-Scale Proteomics in Early Pregnancy and Hypertensive Disorders of Pregnancy.

Importance: There is no consensus regarding the best method for prediction of hypertensive disorders of pregnancy (HDP), including gestational hypertension and preeclampsia. Objective: To determine predictive ability in early pregnancy of large-scale proteomics for prediction of HDP. Design, Setting, and Participants: This was a nested case-control study, conducted in 2022 to 2023, using clinical data and plasma samples collected between 2010 and 2013 during the first trimester, with follow-up until pregnancy outcome. This multicenter observational study took place at 8 academic medical centers in the US. Nulliparous individuals during first-trimester clinical visits were included. Participants with HDP were selected as cases; controls were selected from those who delivered at or after 37 weeks without any HDP, preterm birth, or small-for-gestational-age infant. Age, self-reported race and ethnicity, body mass index, diabetes, health insurance, and fetal sex were available covariates. Exposures: Proteomics using an aptamer-based assay that included 6481 unique human proteins was performed on stored plasma. Covariates were used in predictive models. Main Outcomes and Measures: Prediction models were developed using the elastic net, and analyses were performed on a randomly partitioned training dataset comprising 80% of study participants, with the remaining 20% used as an independent testing dataset. Primary measure of predictive performance was area under the receiver operating characteristic curve (AUC). Results: This study included 753 HDP cases and 1097 controls with a mean (SD) age of 26.9 (5.5) years. Maternal race and ethnicity were 51 Asian (2.8%), 275 non-Hispanic Black (14.9%), 275 Hispanic (14.9%), 1161 non-Hispanic White (62.8% ), and 88 recorded as other (4.8%), which included those who did not identify according to these designations. The elastic net model, allowing for forced inclusion of prespecified covariates, was used to adjust protein-based models for clinical and demographic variables. Under this approach, no proteins were selected to augment the clinical and demographic covariates. The predictive performance of the resulting model was modest, with a training set AUC of 0.64 (95% CI, 0.61-0.67) and a test set AUC of 0.62 (95% CI, 0.56-0.68). Further adjustment for study site yielded only minimal changes in AUCs. Conclusions and Relevance: In this case-control study with detailed clinical data and stored plasma samples available in the first trimester, an aptamer-based proteomics panel did not meaningfully add to predictive utility over and above clinical and demographic factors that are routinely available.

Incident heart failure in chronic kidney disease: proteomics informs biology and risk stratification.

BACKGROUND AND AIMS: Incident heart failure (HF) among individuals with chronic kidney disease (CKD) incurs hospitalizations that burden patients and health care systems. There are few preventative therapies, and the Pooled Cohort equations to Prevent Heart Failure (PCP-HF) perform poorly in the setting of CKD. New drug targets and better risk stratification are urgently needed. METHODS: In this analysis of incident HF, SomaScan V4.0 (4638 proteins) was analysed in 2906 participants of the Chronic Renal Insufficiency Cohort (CRIC) with validation in the Atherosclerosis Risk in Communities (ARIC) study. The primary outcome was 14-year incident HF (390 events); secondary outcomes included 4-year HF (183 events), HF with reduced ejection fraction (137 events), and HF with preserved ejection fraction (165 events). Mendelian randomization and Gene Ontology were applied to examine causality and pathways. The performance of novel multi-protein risk models was compared to the PCP-HF risk score. RESULTS: Over 200 proteins were associated with incident HF after adjustment for estimated glomerular filtration rate at P < 1 × 10-5. After adjustment for covariates including N-terminal pro-B-type natriuretic peptide, 17 proteins remained associated at P < 1 × 10-5. Mendelian randomization associations were found for six proteins, of which four are druggable targets: FCG2B, IGFBP3, CAH6, and ASGR1. For the primary outcome, the C-statistic (95% confidence interval [CI]) for the 48-protein model in CRIC was 0.790 (0.735, 0.844) vs. 0.703 (0.644, 0.762) for the PCP-HF model (P = .001). C-statistic (95% CI) for the protein model in ARIC was 0.747 (0.707, 0.787). CONCLUSIONS: Large-scale proteomics reveal novel circulating protein biomarkers and potential mediators of HF in CKD. Proteomic risk models improve upon the PCP-HF risk score in this population.

Proteomics of CKD progression in the chronic renal insufficiency cohort.

Progression of chronic kidney disease (CKD) portends myriad complications, including kidney failure. In this study, we analyze associations of 4638 plasma proteins among 3235 participants of the Chronic Renal Insufficiency Cohort Study with the primary outcome of 50% decline in estimated glomerular filtration rate or kidney failure over 10 years. We validate key findings in the Atherosclerosis Risk in the Communities study. We identify 100 circulating proteins that are associated with the primary outcome after multivariable adjustment, using a Bonferroni statistical threshold of significance. Individual protein associations and biological pathway analyses highlight the roles of bone morphogenetic proteins, ephrin signaling, and prothrombin activation. A 65-protein risk model for the primary outcome has excellent discrimination (C-statistic[95%CI] 0.862 [0.835, 0.889]), and 14/65 proteins are druggable targets. Potentially causal associations for five proteins, to our knowledge not previously reported, are supported by Mendelian randomization: EGFL9, LRP-11, MXRA7, IL-1 sRII and ILT-2. Modifiable protein risk markers can guide therapeutic drug development aimed at slowing CKD progression.

Proteomic cardiovascular risk assessment in chronic kidney disease.

AIMS: Chronic kidney disease (CKD) is widely prevalent and independently increases cardiovascular risk. Cardiovascular risk prediction tools derived in the general population perform poorly in CKD. Through large-scale proteomics discovery, this study aimed to create more accurate cardiovascular risk models. METHODS AND RESULTS: Elastic net regression was used to derive a proteomic risk model for incident cardiovascular risk in 2182 participants from the Chronic Renal Insufficiency Cohort. The model was then validated in 485 participants from the Atherosclerosis Risk in Communities cohort. All participants had CKD and no history of cardiovascular disease at study baseline when ∼5000 proteins were measured. The proteomic risk model, which consisted of 32 proteins, was superior to both the 2013 ACC/AHA Pooled Cohort Equation and a modified Pooled Cohort Equation that included estimated glomerular filtrate rate. The Chronic Renal Insufficiency Cohort internal validation set demonstrated annualized receiver operating characteristic area under the curve values from 1 to 10 years ranging between 0.84 and 0.89 for the protein and 0.70 and 0.73 for the clinical models. Similar findings were observed in the Atherosclerosis Risk in Communities validation cohort. For nearly half of the individual proteins independently associated with cardiovascular risk, Mendelian randomization suggested a causal link to cardiovascular events or risk factors. Pathway analyses revealed enrichment of proteins involved in immunologic function, vascular and neuronal development, and hepatic fibrosis. CONCLUSION: In two sizeable populations with CKD, a proteomic risk model for incident cardiovascular disease surpassed clinical risk models recommended in clinical practice, even after including estimated glomerular filtration rate. New biological insights may prioritize the development of therapeutic strategies for cardiovascular risk reduction in the CKD population.

Does multi-way, long-range chromatin contact data advance 3D genome reconstruction?

BACKGROUND: Methods for inferring the three-dimensional (3D) configuration of chromatin from conformation capture assays that provide strictly pairwise interactions, notably Hi-C, utilize the attendant contact matrix as input. More recent assays, in particular split-pool recognition of interactions by tag extension (SPRITE), capture multi-way interactions instead of solely pairwise contacts. These assays yield contacts that straddle appreciably greater genomic distances than Hi-C, in addition to instances of exceptionally high-order chromatin interaction. Such attributes are anticipated to be consequential with respect to 3D genome reconstruction, a task yet to be undertaken with multi-way contact data. However, performing such 3D reconstruction using distance-based reconstruction techniques requires framing multi-way contacts as (pairwise) distances. Comparing approaches for so doing, and assessing the resultant impact of long-range and multi-way contacts, are the objectives of this study. RESULTS: We obtained 3D reconstructions via multi-dimensional scaling under a variety of weighting schemes for mapping SPRITE multi-way contacts to pairwise distances. Resultant configurations were compared following Procrustes alignment and relationships were assessed between associated Procrustes root mean square errors and key features such as the extent of multi-way and/or long-range contacts. We found that these features had surprisingly limited influence on 3D reconstruction, a finding we attribute to their influence being diminished by the preponderance of pairwise contacts. CONCLUSION: Distance-based 3D genome reconstruction using SPRITE multi-way contact data is not appreciably affected by the weighting scheme used to convert multi-way interactions to pairwise distances.

Analytical and Biological Variability of a Commercial Modified Aptamer Assay in Plasma Samples of Patients with Chronic Kidney Disease.

BACKGROUND: We carried out a study of the aptamer proteomic assay, SomaScan V4, to evaluate the analytical and biological variability of the assay in plasma samples of patients with moderate to severe chronic kidney disease (CKD). METHODS: Plasma samples were selected from 2 sources: (a) 24 participants from the Chronic Renal Insufficiency Cohort (CRIC) and (b) 49 patients from the Brigham and Women's Hospital-Kidney/Renal Clinic. We calculated intra-assay variability from both sources and examined short-term biological variability in samples from the Brigham clinic. We also measured correlations of aptamer measurements with traditional biomarker assays. RESULTS: A total of 4656 unique proteins (4849 total aptamer measures) were analyzed in all samples. Median (interquartile range [IQR] intra-assay CV) was 3.7% (2.8-5.3) in CRIC and 5.0% (3.8-7.0) in Brigham samples. Median (IQR) biological CV among Brigham samples drawn from one individual on 2 occasions separated by median (IQR) 7 (4-14) days was 8.7% (6.2-14). CVs were independent of CKD stage, diabetes, or albuminuria but were higher in patients with systemic lupus erythematosus. Rho correlations between aptamer and traditional assays for biomarkers of interest were cystatin C = 0.942, kidney injury model-1 = 0.905, fibroblast growth factor-23 = 0.541, tumor necrosis factor receptors 1 = 0.781 and 2 = 0.843, P < 10-100 for all. CONCLUSIONS: Intra-assay and within-subject variability for SomaScan in the CKD setting was low and similar to assay variability reported from individuals without CKD. Intra-assay precision was excellent whether samples were collected in an optimal research protocol, as were CRIC samples, or in the clinical setting, as were the Brigham samples.

Assessing chromatin relocalization in 3D using the patient rule induction method.

Three-dimensional (3D) genome architecture is critical for numerous cellular processes, including transcription, while certain conformation-driven structural alterations are frequently oncogenic. Inferring 3D chromatin configurations has been advanced by the emergence of chromatin conformation capture assays, notably Hi-C, and attendant 3D reconstruction algorithms. These have enhanced understanding of chromatin spatial organization and afforded numerous downstream biological insights. Until recently, comparisons of 3D reconstructions between conditions and/or cell types were limited to prescribed structural features. However, multiMDS, a pioneering approach developed by Rieber and Mahony (2019). that performs joint reconstruction and alignment, enables quantification of all locus-specific differences between paired Hi-C data sets. By subsequently mapping these differences to the linear (1D) genome the identification of relocalization regions is facilitated through the use of peak calling in conjunction with continuous wavelet transformation. Here, we seek to refine this approach by performing the search for significant relocalization regions in terms of the 3D structures themselves, thereby retaining the benefits of 3D reconstruction and avoiding limitations associated with the 1D perspective. The search for (extreme) relocalization regions is conducted using the patient rule induction method (PRIM). Considerations surrounding orienting structures with respect to compartmental and principal component axes are discussed, as are approaches to inference and reconstruction accuracy assessment. The illustration makes recourse to comparisons between four different cell types.

Can 3D diploid genome reconstruction from unphased Hi-C data be salvaged?

The three-dimensional (3D) configuration of chromatin impacts numerous cellular processes. However, directly observing chromatin architecture at high resolution is challenging. Accordingly, inferring 3D structure utilizing chromatin conformation capture assays, notably Hi-C, has received considerable attention, with a multitude of reconstruction algorithms advanced. While these have enhanced appreciation of chromatin organization, most suffer from a serious shortcoming when faced with diploid genomes: inability to disambiguate contacts between corresponding loci on homologous chromosomes, making attendant reconstructions potentially meaningless. Three recent proposals offer a computational way forward at the expense of strong assumptions. Here, we show that making plausible assumptions about the components of homologous chromosome contacts provides a basis for rescuing conventional consensus-based, unphased reconstruction. This would be consequential since not only are assumptions needed for diploid reconstruction considerable, but the sophistication of select unphased algorithms affords substantive advantages with regard resolution and folding complexity. Rather than presuming that the requisite salvaging assumptions are met, we exploit a recent imaging technology, in situ genome sequencing (IGS), to comprehensively evaluate their reasonableness. We analogously use IGS to assess assumptions underpinning diploid reconstruction algorithms. Results convincingly demonstrate that, in all instances, assumptions are not met, making further algorithm development, potentially informed by IGS data, essential.

Principal curve approaches for inferring 3D chromatin architecture.

Three-dimensional (3D) genome spatial organization is critical for numerous cellular processes, including transcription, while certain conformation-driven structural alterations are frequently oncogenic. Genome architecture had been notoriously difficult to elucidate, but the advent of the suite of chromatin conformation capture assays, notably Hi-C, has transformed understanding of chromatin structure and provided downstream biological insights. Although many findings have flowed from direct analysis of the pairwise proximity data produced by these assays, there is added value in generating corresponding 3D reconstructions deriving from superposing genomic features on the reconstruction. Accordingly, many methods for inferring 3D architecture from proximity data have been advanced. However, none of these approaches exploit the fact that single chromosome solutions constitute a one-dimensional (1D) curve in 3D. Rather, this aspect has either been addressed by imposition of constraints, which is both computationally burdensome and cell type specific, or ignored with contiguity imposed after the fact. Here, we target finding a 1D curve by extending principal curve methodology to the metric scaling problem. We illustrate how this approach yields a sequence of candidate solutions, indexed by an underlying smoothness or degrees-of-freedom parameter, and propose methods for selection from this sequence. We apply the methodology to Hi-C data obtained on IMR90 cells and so are positioned to evaluate reconstruction accuracy by referencing orthogonal imaging data. The results indicate the utility and reproducibility of our principal curve approach in the face of underlying structural variation.

Increased rates of secondary bacterial infections, including Enterococcus bacteremia, in patients hospitalized with coronavirus disease 2019 (COVID-19).

OBJECTIVE: We compared the rates of hospital-onset secondary bacterial infections in patients with coronavirus disease 2019 (COVID-19) with rates in patients with influenza and controls, and we investigated reports of increased incidence of Enterococcus infections in patients with COVID-19. DESIGN: Retrospective cohort study. SETTING: An academic quaternary-care hospital in San Francisco, California. PATIENTS: Patients admitted between October 1, 2019, and October 1, 2020, with a positive SARS-CoV-2 PCR (N = 314) or influenza PCR (N = 82) within 2 weeks of admission were compared with inpatients without positive SARS-CoV-2 or influenza tests during the study period (N = 14,332). METHODS: National Healthcare Safety Network definitions were used to identify infection-related ventilator-associated complications (IVACs), probable ventilator-associated pneumonia (PVAP), bloodstream infections (BSIs), and catheter-associated urinary tract infections (CAUTIs). A multiple logistic regression model was used to control for likely confounders. RESULTS: COVID-19 patients had significantly higher rates of IVAC and PVAP compared to controls, with adjusted odds ratios of 4.7 (95% confidence interval [CI], 1.7-13.9) and 10.4 (95 % CI, 2.1-52.1), respectively. COVID-19 patients had higher incidence of BSI due to Enterococcus but not BSI generally, and whole-genome sequencing of Enterococcus isolates demonstrated that nosocomial transmission did not explain the increased rate. Subanalyses of patients admitted to the intensive care unit and patients who required mechanical ventilation revealed similar findings. CONCLUSIONS: COVID-19 is associated with an increased risk of IVAC, PVAP, and Enterococcus BSI compared with hospitalized controls, which is not fully explained by factors such as immunosuppressive treatments and duration of mechanical ventilation. The mechanism underlying increased rates of Enterococcus BSI in COVID-19 patients requires further investigation.

Indoor Air Pollution and Susceptibility to Tuberculosis Infection in Urban Vietnamese Children.

Rationale: The Southeast Asian tuberculosis burden is high, and it remains unclear if urban indoor air pollution in this setting is exacerbating the epidemic. Objectives: To determine the associations of latent tuberculosis with common urban indoor air pollution sources (secondhand smoke, indoor motorcycle emissions, and cooking) in Southeast Asia. Methods: We enrolled child household contacts of patients with microbiologically confirmed active tuberculosis in Vietnam, from July 2017 to December 2019. We tested children for latent tuberculosis and evaluated air pollution exposures with questionnaires and personal aerosol sampling. We tested hypotheses using generalized estimating equations. Measurements and Main Results: We enrolled 72 patients with tuberculosis (27% with cavitary disease) and 109 of their child household contacts. Latent tuberculosis was diagnosed in 58 (53%) household contacts at baseline visit. Children experienced a 2.56-fold increased odds of latent tuberculosis for each additional household member who smoked (95% confidence interval, 1.27-5.16). Odds were highest among children exposed to indoor smokers and children <5 years old exposed to household smokers. Each residential floor above street-level pollution decreased the odds of latent tuberculosis by 36% (adjusted odds ratio, 0.64; 95% confidence interval, 0.42-0.96). Motorcycles parked inside children's homes and cooking with liquid petroleum gas compared with electricity increased the odds of latent tuberculosis, whereas kitchen ventilation decreased the effect, but these findings were not statistically significant. Conclusions: Common urban indoor air pollution sources were associated with increased odds of latent tuberculosis infection in child household contacts of patients with active tuberculosis.

Correlates and Consequences of High Serum Irisin Concentration in Patients on Hemodialysis: A Longitudinal Analysis.

OBJECTIVE: Irisin is a hormone released by muscle in response to exercise that acts on white adipose cells to stimulate browning of adipose tissue. We aimed to examine irisin correlates and consequences of irisin in patients receiving hemodialysis. DESIGN AND METHODS: A prospective cohort study was conducted using data from 749 prevalent patients receiving hemodialysis. Multivariable linear regression and multivariable generalized estimating equations were used to determine correlates of baseline and change in serum irisin concentration. Proportional hazards (Cox) regression was used to assess the association between serum irisin concentration and time to death. RESULTS: Age and body mass index were inversely associated with baseline and change in serum irisin concentration. Lower muscle mass as estimated by serum creatinine concentration was associated with lower irisin concentration (-1.38% per mg/dL (95% confidence interval [CI]: -2.45, -0.21) and with a 0.72% decrease in irisin concentration (95% CI: -1.48, -0.04) from baseline to 12 months. Each 50% higher serum interleukin-6 (IL-6) concentration was associated with 1.52% higher serum irisin concentration (95% CI: 0.38, 2.66) at baseline and an increase of 1.04% in irisin concentration over 1 year (95% CI: 0.47, 1.61). Irisin concentration at baseline was associated with higher hazard of death (hazards ratio: 1.45, 95% CI: 1.05 2.00); an increase in irisin concentration over 1 year was associated with a higher hazard of death (hazards ratio: 1.34, 95% CI: 1.01, 1.79). In formal mediation analysis, serum IL-6 was a mediator in the association between serum irisin and mortality. CONCLUSIONS: Lower serum creatinine (reflecting lower muscle mass) and higher serum IL-6 were associated with higher serum irisin concentrations. Higher serum irisin concentrations were associated with higher mortality, which may be mediated by inflammation.

Composition and origin of lung fluid proteome in premature infants and relationship to respiratory outcome.

BACKGROUND: Infants born at extremely low gestational age are at high risk for bronchopulmonary dysplasia and continuing lung disease. There are no early clinical biomarkers for pulmonary outcome and limited therapeutic interventions. OBJECTIVES: We performed global proteomics of premature infant tracheal aspirate (TA) and plasma to determine the composition and source of lung fluid proteins and to identify potential biomarkers of respiratory outcome. METHODS: TA samples were collected from intubated infants in the TOLSURF cohort before and after nitric oxide treatment, and plasma was collected from NO CLD infants. Protein abundance was assayed by HPLC/tandem mass spectrometry and Protein Prospector software. mRNA abundance in mid-gestation fetal lung was assessed by RNA sequencing. Pulmonary morbidity was defined as a need for ventilatory support at term and during the first year. RESULTS: Abundant TA proteins included albumin, hemoglobin, and actin-related proteins. 96 of 137 detected plasma proteins were present in TA (r = 0.69, p<0.00001). Based on lung RNAseq data, ~88% of detected TA proteins in injured infant lung are derived at least in part from lung epithelium with overrepresentation in categories of cell membrane/secretion and stress/inflammation. Comparing 37 infants at study enrollment (7-14 days) who did or did not develop persistent pulmonary morbidity, candidate biomarkers of both lung (eg., annexin A5) and plasma (eg., vitamin D-binding protein) origin were identified. Notably, levels of free hemoglobin were 2.9-fold (p = 0.03) higher in infants with pulmonary morbidity. In time course studies, hemoglobin decreased markedly in most infants after enrollment coincident with initiation of inhaled nitric oxide treatment. CONCLUSIONS: We conclude that both lung epithelium and plasma contribute to the lung fluid proteome in premature infants with lung injury. Early postnatal elevation of free hemoglobin and heme, which are both pro-oxidants, may contribute to persistent lung disease by depleting nitric oxide and increasing oxidative/nitrative stress.

Assessing stationary distributions derived from chromatin contact maps.

BACKGROUND: The spatial configuration of chromosomes is essential to various cellular processes, notably gene regulation, while architecture related alterations, such as translocations and gene fusions, are often cancer drivers. Thus, eliciting chromatin conformation is important, yet challenging due to compaction, dynamics and scale. However, a variety of recent assays, in particular Hi-C, have generated new details of chromatin structure, spawning a number of novel biological findings. Many findings have resulted from analyses on the level of native contact data as generated by the assays. Alternatively, reconstruction based approaches often proceed by first converting contact frequencies into distances, then generating a three dimensional (3D) chromatin configuration that best recapitulates these distances. Subsequent analyses can enrich contact level analyses via superposition of genomic attributes on the reconstruction. But, such advantages depend on the accuracy of the reconstruction which, absent gold standards, is inherently difficult to assess. Attempts at accuracy evaluation have relied on simulation and/or FISH imaging that typically features a handful of low resolution probes. While newly advanced multiplexed FISH imaging offers possibilities for refined 3D reconstruction accuracy evaluation, availability of such data is limited due to assay complexity and the resolution thereof is appreciably lower than the reconstructions being assessed. Accordingly, there is demand for new methods of reconstruction accuracy appraisal. RESULTS: Here we explore the potential of recently proposed stationary distributions, hereafter StatDns, derived from Hi-C contact matrices, to serve as a basis for reconstruction accuracy assessment. Current usage of such StatDns has focussed on the identification of highly interactive regions (HIRs): computationally defined regions of the genome purportedly involved in numerous long-range intra-chromosomal contacts. Consistent identification of HIRs would be informative with respect to inferred 3D architecture since the corresponding regions of the reconstruction would have an elevated number of k nearest neighbors (kNNs). More generally, we anticipate a monotone decreasing relationship between StatDn values and kNN distances. After initially evaluating the reproducibility of StatDns across replicate Hi-C data sets, we use this implied StatDn - kNN relationship to gauge the utility of StatDns for reconstruction validation, making recourse to both real and simulated examples. CONCLUSIONS: Our analyses demonstrate that, as constructed, StatDns do not provide a suitable measure for assessing the accuracy of 3D genome reconstructions. Whether this is attributable to specific choices surrounding normalization in defining StatDns or to the logic underlying their very formulation remains to be determined.

The Rabl configuration limits topological entanglement of chromosomes in budding yeast.

The three dimensional organization of genomes remains mostly unknown due to their high degree of condensation. Biophysical studies predict that condensation promotes the topological entanglement of chromatin fibers and the inhibition of function. How organisms balance between functionally active genomes and a high degree of condensation remains to be determined. Here we hypothesize that the Rabl configuration, characterized by the attachment of centromeres and telomeres to the nuclear envelope, helps to reduce the topological entanglement of chromosomes. To test this hypothesis we developed a novel method to quantify chromosome entanglement complexity in 3D reconstructions obtained from Chromosome Conformation Capture (CCC) data. Applying this method to published data of the yeast genome, we show that computational models implementing the attachment of telomeres or centromeres alone are not sufficient to obtain the reduced entanglement complexity observed in 3D reconstructions. It is only when the centromeres and telomeres are attached to the nuclear envelope (i.e. the Rabl configuration) that the complexity of entanglement of the genome is comparable to that of the 3D reconstructions. We therefore suggest that the Rabl configuration is an essential player in the simplification of the entanglement of chromatin fibers.

Epigenomic profiling of newborns with isolated orofacial clefts reveals widespread DNA methylation changes and implicates metastable epiallele regions in disease risk.

Cleft lip with or without cleft palate (CL/P) is a common human birth defect whose etiologies remain largely unknown. Several studies have demonstrated that periconceptional supplementation of folic acid can reduce risk of CL/P in offspring. In this study, we tested the hypothesis that the preventive effect of folic acid is manifested through epigenetic modifications by determining whether DNA methylation changes are associated with CL/P. To more readily observe the potential effects of maternal folate on the offspring epigenome, we focused on births prior to mandatory dietary folate fortification in the United States (i.e. birth year 1997 or earlier). Genomic DNA methylation levels were assessed from archived newborn bloodspots in a 182-member case-control study using the Illumina® Human Beadchip 450K array. CL/P cases displayed striking epigenome-wide hypomethylation relative to controls: 63% of CpGs interrogated had lower methylation levels in case newborns, a trend which held up in racially stratified sub-groups. 28 CpG sites reached epigenome-wide significance and all were case-hypomethylated. The most significant CL/P-associated differentially methylated region encompassed the VTRNA2-1 gene, which was also hypomethylated in cases (FWER p = 0.014). This region has been previously characterized as a nutritionally-responsive, metastable epiallele and CL/P-associated methylation changes, in general, were greater at or near putative metastable epiallelic regions. Gene Set Enrichment Analysis of CL/P-associated DMRs showed an over-representation of genes involved in palate development such as WNT9B, MIR140 and LHX8. CL/P-associated DNA methylation changes may partly explain the mechanism by which orofacial clefts are responsive to maternal folate levels.

Surfactant status and respiratory outcome in premature infants receiving late surfactant treatment.

BACKGROUND: Many premature infants with respiratory failure are deficient in surfactant, but the relationship to occurrence of bronchopulmonary dysplasia (BPD) is uncertain. METHODS: Tracheal aspirates were collected from 209 treated and control infants enrolled at 7-14 days in the Trial of Late Surfactant. The content of phospholipid, surfactant protein B, and total protein were determined in large aggregate (active) surfactant. RESULTS: At 24 h, surfactant treatment transiently increased surfactant protein B content (70%, p < 0.01), but did not affect recovered airway surfactant or total protein/phospholipid. The level of recovered surfactant during dosing was directly associated with content of surfactant protein B (r = 0.50, p < 0.00001) and inversely related to total protein (r = 0.39, p < 0.0001). For all infants, occurrence of BPD was associated with lower levels of recovered large aggregate surfactant, higher protein content, and lower SP-B levels. Tracheal aspirates with lower amounts of recovered surfactant had an increased proportion of small vesicle (inactive) surfactant. CONCLUSIONS: We conclude that many intubated premature infants are deficient in active surfactant, in part due to increased intra-alveolar metabolism, low SP-B content, and protein inhibition, and that the severity of this deficit is predictive of BPD. Late surfactant treatment at the frequency used did not provide a sustained increase in airway surfactant.

Frailty Among Patients Receiving Hemodialysis: Evolution of Components and Associations With Mortality.

BACKGROUND: Understanding how components of frailty change over time and how they can be modeled as time-dependent predictors of mortality could lead to better risk prediction in the dialysis population. METHODS: We measured frailty at baseline, 12 months, and 24 months among 727 patients receiving hemodialysis in Northern California and Atlanta. We examined the likelihood of meeting frailty components (weight loss, exhaustion, low physical activity, weak grip strength, and slow gait speed) as a function of time in logistic regression analysis and association of frailty components with mortality in time-updated multivariable Cox models. RESULTS: Physical activity and gait speed declined, exhaustion and grip strength did not change, and the odds of meeting the weight loss criterion declined with time. All five components were associated with higher mortality in multivariable analyses, but gait speed was the strongest individual predictor. All frailty components except physical inactivity were independently associated with mortality when all five components were included in the same model. The number of frailty components met was associated with mortality in a gradient that ranged from a hazard ratio of 2.73 for one component to 10.07 for five components met; the model including all five components was the best model based on Akaike information criterion. CONCLUSIONS: Measurement of all frailty components was necessary for optimal mortality prediction, and the number of components met was strongly associated with mortality in this cohort.

Improved accuracy assessment for 3D genome reconstructions.

BACKGROUND: Three dimensional (3D) genome spatial organization is critical for numerous cellular functions, including transcription, while certain conformation-driven structural alterations are frequently oncogenic. Genome conformation had been difficult to elucidate but the advent chromatin conformation capture assays, notably Hi-C, has transformed understanding of chromatin architecture and yielded numerous biological insights. Although most of these findings have flowed from analysis of proximity data produced by these assays, added value in generating 3D reconstructions has been demonstrated, deriving, in part, from superposing genomic features on the reconstruction. However, advantages of 3D structure-based analyses are clearly conditional on the accuracy of the attendant reconstructions, which is difficult to assess. Proponents of competing reconstruction algorithms have evaluated their accuracy by recourse to simulation of toy structures and/or limited fluorescence in situ hybridization (FISH) imaging that features a handful of low resolution probes. Accordingly, new methods of reconstruction accuracy assessment are needed. RESULTS: Here we utilize two recently devised assays to develop methodology for assessing 3D reconstruction accuracy. Multiplex FISH increases the number of probes by an order of magnitude and hence the number of inter-probe distances by two orders, providing sufficient information for structure-level evaluation via mean-squared deviations (MSD). Crucially, underscoring multiplex FISH applications are large numbers of coordinate-system aligned replicates that provide the basis for a referent distribution for MSD statistics. Using this system we show that reconstructions based on Hi-C data for IMR90 cells are accurate for some chromosomes but not others. The second new assay, genome architecture mapping, utilizes large numbers of thin cryosections to obtain a measure of proximity. We exploit the planarity of the cryosections - not used in inferring proximity - to obtain measures of reconstruction accuracy, with referents provided via resampling. Application to mouse embryonic stem cells shows reconstruction accuracies that vary by chromosome. CONCLUSIONS: We have developed methods for assessing the accuracy of 3D genome reconstructions that exploit features of recently advanced multiplex FISH and genome architecture mapping assays. These approaches can help overcome the absence of gold standards for making such assessments which are important in view of the considerable uncertainties surrounding 3D genome reconstruction.

Memory T Cell Proliferation before Hepatitis C Virus Therapy Predicts Antiviral Immune Responses and Treatment Success.

The contribution of the host immune system to the efficacy of new anti-hepatitis C virus (HCV) drugs is unclear. We undertook a longitudinal prospective study of 33 individuals with chronic HCV treated with combination pegylated IFN-α, ribavirin, and telaprevir/boceprevir. We characterized innate and adaptive immune cells to determine whether kinetics of the host response could predict sustained virologic response (SVR). We show that characteristics of the host immune system present before treatment were correlated with successful therapy. Augmentation of adaptive immune responses during therapy was more impressive among those achieving SVR. Most importantly, active memory T cell proliferation before therapy predicted SVR and was associated with the magnitude of the HCV-specific responses at week 12 after treatment start. After therapy initiation, the most important correlate of success was minimal monocyte activation, as predicted by previous in vitro work. In addition, subjects achieving SVR had increasing expression of the transcription factor T-bet, a driver of Th1 differentiation and cytotoxic effector cell maturation. These results show that host immune features present before treatment initiation predict SVR and eventual development of a higher frequency of functional virus-specific cells in blood. Such host characteristics may also be required for successful vaccine-mediated protection.