Atrial fibrillation (AF) affects over 30 million individuals worldwide and is associated with an increased risk of stroke, heart failure, and death. AF is highly heritable, yet the genetic basis for the arrhythmia remains incompletely understood.
Methods & Results
To identify new AF-related genes, we utilized a multifaceted approach, combining large-scale genotyping in two ethnically distinct populations, cis-eQTL mapping, and functional validation. Four novel loci were identified in individuals of European descent near the genes NEURL (rs12415501, RR=1.18, 95%CI 1.13 – 1.23, p=6.5×10−16), GJA1 (rs13216675, RR=1.10, 95%CI 1.06 – 1.14, p=2.2×10−8), TBX5 (rs10507248, RR=1.12, 95%CI 1.08 – 1.16, p=5.7×10−11), and CAND2 (rs4642101, RR=1.10, 95%CI 1.06 – 1.14, p=9.8×10−9). In Japanese, novel loci were identified near NEURL (rs6584555, RR=1.32, 95%CI 1.26–1.39, p=2.0×10−25) and CUX2 (rs6490029, RR=1.12, 95%CI 1.08–1.16, p=3.9×10−9). The top SNPs or their proxies were identified as cis-eQTLs for the genes CAND2 (p=2.6×10−19), GJA1 (p=2.66×10−6), and TBX5 (p=1.36×10−05). Knockdown of the zebrafish orthologs of NEURL and CAND2 resulted in prolongation of the atrial action potential duration (17% and 45%, respectively).
We have identified five novel loci for AF. Our results further expand the diversity of genetic pathways implicated in AF and provide novel molecular targets for future biological and pharmacological investigation.
atrial fibrillation; genetics; epidemiology; expression; functional analysis; zebrafish
Spiritual distress is common in the ICU, and spiritual care providers are often called upon to provide care for patients and their families. Our goal was to evaluate the activities spiritual care providers’ conduct to support patients and families, and whether those activities are associated with family satisfaction with ICU care.
Prospective cohort study.
350-bed, 65-ICU bed tertiary care teaching hospital.
Spiritual care providers and family members of patients who died in the ICU or within 30 hours of transfer from the ICU.
Spiritual care providers completed surveys reporting their activities. Family members completed validated measures of satisfaction with care and satisfaction with spiritual care. Clustered regression was used to assess the association between activities completed by spiritual care providers and family ratings of care.
Of 494 eligible patients, 275 family members completed surveys (response rate, 56%). Fifty-seven spiritual care providers received surveys relating to 268 patients, completing 285 surveys for 244 patients (response rate, 91%). Spiritual care providers commonly reported activities related to supporting religious and spiritual needs (>=90%) and providing support for family feelings (90%). Discussions about the patient’s wishes for end-of-life care and a greater number of spiritual care activities performed were both associated with increased overall family satisfaction with ICU care (p<0.05). Discussions about a patient’s end-of-life wishes, preparation for a family conference, and total number of activities performed were associated with improved family satisfaction with decision-making in the ICU (p<0.05).
Spiritual care providers engage in a variety of activities with families of ICU patients; several are associated with increased family satisfaction with ICU care in general and decision-making in the ICU specifically. These findings provide insight into spiritual care provider activities and provide guidance for interventions to improve spiritual care delivered to families of critically ill patients.
intensive care; critical care; spiritual care; end-of-life care; dying; death; palliative care
This study examined children’s ability to follow audio-visual instructions presented in noise and reverberation.
Children (8–12 years) with normal hearing followed instructions in noise or noise plus reverberation. Performance was compared for a single talker (ST), multiple talkers (MT) and multiple talkers with competing comments (MTC). Working memory was assessed using measures of digit span.
Performance was better for children in noise than for those in noise plus reverberation. In noise, performance for ST was better than for either MT or MTC and performance for MT was better than for MTC. In noise plus reverberation, performance for ST and MT were better than for MTC but there were no differences between ST and MT. Digit span did not account for significant variance in the task.
Overall, children performed better in noise than in noise plus reverberation. However, differing patterns across conditions for the two environments suggested that the addition of reverberation may have affected performance in a way that was not apparent in noise alone. Continued research is needed to examine the differing effects of noise and reverberation on children’s speech understanding.
Noise; speech perception; children
Epstein-Barr virus (EBV) is a well-established B-cell-tropic virus associated with various lymphoproliferative diseases of both B-cell and non-B-cell origin. EBV is associated with a number of T-cell lymphomas; however, in vitro studies utilizing prototypical EBV type 1 (EBV-1) laboratory strains have generally failed to readily infect mature T cells in culture. The difficulties in performing in vitro T-cell experiments have left questions regarding the role of EBV in the pathogenesis of EBV-positive T-cell lymphoproliferative diseases largely unresolved. We report here that the EBV type 2 (EBV-2) strain displays a unique cell tropism for T cells. In remarkable contrast to EBV-1, EBV-2 readily infects primary T cells in vitro, demonstrating a propensity for CD8+ T cells. EBV-2 infection of purified T cells results in expression of latency genes and ultimately leads to T-cell activation, substantial proliferation, and profound alteration of cytokine expression. The pattern of cytokine production is strikingly skewed toward chemokines with roles in lymphocyte migration, demonstrating that EBV-2 has the ability to modulate normal T-cell processes. Collectively, these novel findings identify a previously unknown cell population potentially utilized by EBV-2 to establish latency and lay the foundation for further studies to elucidate the role of EBV in the pathogenesis of T-cell lymphoproliferative diseases.
IMPORTANCE The ability of EBV to infect T cells is made apparent by its association with a variety of T-cell lymphoproliferative disorders. However, studies to elucidate the pathogenic role of EBV in these diseases have been limited by the inability to conduct in vitro T-cell infection experiments. Here, we report that EBV-2 isolates, compromised in the capacity to immortalize B cells, infect CD3+ T cells ex vivo and propose a working model of EBV-2 persistence where alteration of T-cell functions resulting from EBV-2 infection enhances the establishment of latency in B cells. If indeed EBV-2 utilizes T cells to establish a persistent infection, this could provide one mechanism for the association of EBV with T-cell lymphomas. The novel finding that EBV-2 infects T cells in culture will provide a model to understand the role EBV plays in the development of T-cell lymphomas.
Tight junctions are multicomponent structures, with claudin proteins defining paracellular permeability. Claudin 3 is a candidate for the exceptional “tightness” of human urothelium, being localised to the terminal tight junction (TJ) of superficial cells. Our aim was to determine whether claudin 3 plays an instigating and/or a functional role in the urothelial TJ.
Materials and Methods
Normal human urothelial (NHU) cells maintained as non-immortalised cell lines were retrovirally-transduced to over-express or silence claudin 3 expression. Stable sublines induced to stratify or differentiate were assessed for TJ formation by immunocytochemistry and transepithelial electrical resistance (TER). Expression of claudin 3, ZO-1 and ZO-1α+ was examined in native urothelium by immunohistochemistry.
Claudin 3 expression was associated with differentiation and development of a tight barrier and along with ZO-1 and ZO-1α+ was localised to the apical tight junction in native urothelium. Knockdown of claudin 3 inhibited formation of a tight barrier in three independent cell lines, however, overexpression of claudin 3 was not sufficient to induce tight barrier development in the absence of differentiation. A differentiation-dependent induction of the ZO-1α+ isoform was found to coincide with barrier formation. Whereas claudin 3 overexpression did not induce the switch to co-expression of ZO-1α−/ZO-1α+, claudin 3 knockdown decreased localisation of ZO-1 to the TJ and resulted in compromised barrier function.
Urothelial cytodifferentiation is accompanied by induction of claudin 3 which is essential for the development of a terminal TJ. A coordinated switch to the ZO-1α+ isotype was also observed and for the first time may indicate that ZO-1α+ is involved in the structural assembly and function of the urothelial terminal TJ.
claudin; differentiation; tight junction; urothelium; zonula occludens
D-dimer, a fibrin degradation product, is related to risk of cardiovascular disease and venous thromboembolism. Genetic determinants of D-dimer are not well characterized; notably, few data have been reported for African American (AA), Asian, and Hispanic populations.
Materials and Methods
We conducted a large-scale candidate gene association study to identify variants in genes associated with D-dimer levels in multi-ethnic populations. Four cohorts, comprising 6,848 European Americans (EAs), 2,192 AAs, 670 Asians, and 1,286 Hispanics in the NHLBI Candidate Gene Association Resource (CARe) consortium, were assembled. Approximately 50,000 genotyped SNPs in 2,000 cardiovascular disease gene loci were analyzed by linear regression, adjusting for age, sex, study site, and principal components in each cohort and ethnic group. Results across studies were combined within each ethnic group by meta-analysis.
Twelve SNPs in coagulation factor V (F5) and 3 SNPs in the fibrinogen alpha chain (FGA) were significantly associated with D-dimer level in EAs with p < 2.0×10−6. The signal for the most associated SNP in F5 (rs6025, F5 Leiden) was replicated in Hispanics (p = 0.023), while that for the top functional SNP in FGA (rs6050) was replicated in AAs (p = 0.006). No additional SNPs were significantly associated with D-dimer.
Our study replicated previously reported associations of D-dimer with SNPs in F5 (F5-Leiden) and FGA in EAs; we demonstrated replication of the association of D-dimer with FGA rs6050 in AAs and the F5-Leiden variant in Hispanics.
D-dimer; genetic association study; CARe consortium; single nucleotide polymorphisms
We sought to examine the influence of social and clinical factors on risk of progression of chronic kidney disease (CKD) to end-stage renal disease (ESRD) in the urban poor. We studied 15,353 individuals with moderate-to-advanced CKD who received ambulatory care within a large public health system during 1996–2005. The primary outcome was progression to ESRD. Overall, 559 cases of ESRD occurred over a median follow-up of 2.8 years. Among traditional predictors of ESRD, younger age, male sex, non-White race/ ethnicity, public health insurance coverage, diabetes, lower kidney function, higher proteinuria, lower hemoglobin level, and lower serum albumin concentration were significantly associated with a higher adjusted ESRD risk (p < .001 for all). There was no significant association between HIV/AIDS (p=.07), viral hepatitis (p=.11), or non-English language (p=.27) and ESRD risk. Our results highlight the importance of addressing traditional risk factors for progressive CKD to reduce the disproportionate burden of ESRD among disadvantaged populations.
End-stage renal disease; chronic kidney disease; urban poor; race or ethnicity; public health care; disparities
Oral infection of C57BL/6J mice with the protozoan parasite Toxoplasma gondii leads to a lethal inflammatory ileitis.
Mice lacking the purinergic receptor P2X7R are acutely susceptible to toxoplasmic ileitis, losing significantly more weight than C57BL/6J mice and exhibiting much greater intestinal inflammatory pathology in response to infection with only 10 cysts of T. gondii. This susceptibility is not dependent on the ability of P2X7R-deficient mice to control the parasite, which they accomplish just as efficiently as C57BL/6J mice. Rather, susceptibility is associated with elevated ileal concentrations of pro-inflammatory cytokines, reactive nitrogen intermediates and altered regulation of elements of NFκB activation in P2X7R-deficient mice.
Our data support the thesis that P2X7R, a well-documented activator of pro-inflammatory cytokine production, also plays an important role in the regulation of intestinal inflammation.
Tissue plasminogen activator (tPA), a serine protease, catalyzes the conversion of plasminogen to plasmin, the major enzyme responsible for endogenous fibrinolysis. In some populations, elevated plasma levels of tPA have been associated with myocardial infarction and other cardiovascular diseases (CVD). We conducted a meta-analysis of genome-wide association studies (GWAS) to identify novel correlates of circulating levels of tPA.
Approach and Results
Fourteen cohort studies with tPA measures (N=26,929) contributed to the meta-analysis. Three loci were significantly associated with circulating tPA levels (P <5.0×10−8). The first locus is on 6q24.3, with the lead SNP (rs9399599, P=2.9×10−14) within STXBP5. The second locus is on 8p11.21. The lead SNP (rs3136739, P=1.3×10−9) is intronic to POLB and less than 200kb away from the tPA encoding gene PLAT. We identified a non-synonymous SNP (rs2020921) in modest LD with rs3136739 (r2 = 0.50) within exon 5 of PLAT (P=2.0×10−8). The third locus is on 12q24.33, with the lead SNP (rs7301826, P=1.0×10−9) within intron 7 of STX2. We further found evidence for association of lead SNPs in STXBP5 and STX2 with expression levels of the respective transcripts. In in vitro cell studies, silencing STXBP5 decreased release of tPA from vascular endothelial cells, while silencing of STX2 increased tPA release. Through an in-silico lookup, we found no associations of the three lead SNPs with coronary artery disease or stroke.
We identified three loci associated with circulating tPA levels, the PLAT region, STXBP5 and STX2. Our functional studies implicate a novel role for STXBP5 and STX2 in regulating tPA release.
tissue plasminogen activator; genome-wide association study; meta-analysis; cardiovascular disease risk; fibrinolysis; hemostasis
Epstein-Barr virus (EBV) is associated with Burkitt’s lymphoma (BL), and in regions of sub-Saharan Africa where endemic BL is common, both the EBV Type 1 (EBV-1) and EBV Type 2 strains (EBV-2) are found. Little is known about genetic variation of EBV strains in areas of sub-Saharan Africa. In the present study, spontaneous lymphoblastoid cell lines (LCLs) were generated from samples obtained from Kenya. Polymerase chain reaction (PCR) amplification of the EBV genome was done using multiple primers and sequenced by next-generation sequencing (NGS). Phylogenetic analyses against the published EBV-1 and EBV-2 strains indicated that one sample, LCL10 was closely related to EBV-2, while the remaining 3 LCL samples were more closely related to EBV-1. Moreover, single nucleotide polymorphism (SNP) analyses showed clustering of LCL variants. We further show by analysis of EBNA-1, BLLF1, BPLF1, and BRRF2 that latent genes are less conserved than lytic genes in these LCLs from a single geographic region. In this study we have shown that NGS is highly useful for deciphering detailed inter and intra-variations in EBV genomes and that within a geographic region different EBV genetic variations can co-exist, the implications of which warrant further investigation. The findings will enhance our understanding of potential pathogenic variants critical to the development and maintenance of EBV-associated malignancies.
Etoposide is a topoisomerase II poison
that is utilized to treat
a broad spectrum of human cancers. Despite its wide clinical use,
2–3% of patients treated with etoposide eventually develop
treatment-related acute myeloid leukemias (t-AMLs) characterized by
rearrangements of the MLL gene. The molecular basis
underlying the development of these t-AMLs is not well understood;
however, previous studies have implicated etoposide metabolites (i.e.,
etoposide quinone) and topoisomerase IIβ in the leukemogenic
process. Although interactions between etoposide quinone and topoisomerase
IIα have been characterized, the effects of the drug metabolite
on the activity of human topoisomerase IIβ have not been reported.
Thus, we examined the ability of etoposide quinone to poison human
topoisomerase IIβ. The quinone induced ∼4 times more
enzyme-mediated DNA cleavage than did the parent drug. Furthermore,
the potency of etoposide quinone was ∼2 times greater against
topoisomerase IIβ than it was against topoisomerase IIα,
and the drug reacted ∼2–4 times faster with the β
isoform. Etoposide quinone induced a higher ratio of double- to single-stranded
breaks than etoposide, and its activity was less dependent on ATP.
Whereas etoposide acts as an interfacial topoisomerase II poison,
etoposide quinone displayed all of the hallmarks of a covalent poison:
the activity of the metabolite was abolished by reducing agents, and
the compound inactivated topoisomerase IIβ when it was incubated
with the enzyme prior to the addition of DNA. These results are consistent
with the hypothesis that etoposide quinone contributes to etoposide-related
leukemogenesis through an interaction with topoisomerase IIβ.
venous thrombosis; smoking; hormone therapy; interaction; menopause
The protozoan Eimeria tenella is a common parasite of chickens, causing avian coccidiosis, a disease of on-going concern to agricultural industries. The high prevalence of E. tenella can be attributed to the resilient oocyst stage, which is transmitted between hosts in the environment. As in related Coccidia, development of the eimerian oocyst appears to be dependent on completion of the parasite’s sexual cycle. RNA Seq transcriptome profiling offers insights into the mechanisms governing the biology of E. tenella sexual stages (gametocytes) and the potential to identify targets for blocking parasite transmission.
Comparisons between the sequenced transcriptomes of E. tenella gametocytes and two asexual developmental stages, merozoites and sporozoites, revealed upregulated gametocyte transcription of 863 genes. Many of these genes code for proteins involved in coccidian sexual biology, such as oocyst wall biosynthesis and fertilisation, and some of these were characterised in more depth. Thus, macrogametocyte-specific expression and localisation was confirmed for two proteins destined for incorporation into the oocyst wall, as well as for a subtilisin protease and an oxidoreductase. Homologues of an oocyst wall protein and oxidoreductase were found in the related coccidian, Toxoplasma gondii, and shown to be macrogametocyte-specific. In addition, a microgametocyte gamete fusion protein, EtHAP2, was discovered.
The need for novel vaccine candidates capable of controlling coccidiosis is rising and this panel of gametocyte targets represents an invaluable resource for development of future strategies to interrupt parasite transmission, not just in Eimeria but in other Coccidia, including Toxoplasma, where transmission blocking is a relatively unexplored strategy.
Electronic supplementary material
The online version of this article (doi:10.1186/s12864-015-1298-6) contains supplementary material, which is available to authorized users.
Eimeria tenella; RNA Seq; Microgametocyte; Macrogametocyte; Oocyst; Fertilisation; Transmission
The fluorescent lifetimes of the BODIPY–BODIPY dyad appear to correlate with the viscosity of the media, thus making this rotor a suitable small molecule viscometer.
A symmetrical BODIPY–BODIPY dyad with a diyne linker was prepared in two steps; the lifetime decay of this rotor appeared to correlate with the viscosity of the media, thus making this dyad a suitable small molecule viscometer for molecular solvents. The potential of using the rotor to probe the viscosity of ionic liquids was also investigated.
The apicomplexan parasite Toxoplasma gondii is cosmopolitan in nature, largely as a result of its highly flexible life cycle. Felids are its only definitive hosts and a wide range of mammals and birds serve as intermediate hosts. The latent bradyzoite stage is orally infectious in all warm-blooded vertebrates and establishes chronic, transmissible infections. When bradyzoites are ingested by felids, they transform into merozoites in enterocytes and expand asexually as part of their coccidian life cycle. In all other intermediate hosts, however, bradyzoites differentiate exclusively to tachyzoites, and disseminate extraintestinally to many cell types. Both merozoites and tachyzoites undergo rapid asexual population expansion, yet possess different effector fates with respect to the cells and tissues they develop in and the subsequent stages they differentiate into.
To determine whether merozoites utilize distinct suites of genes to attach, invade, and replicate within feline enterocytes, we performed comparative transcriptional profiling on purified tachyzoites and merozoites. We used high-throughput RNA-Seq to compare the merozoite and tachyzoite transcriptomes. 8323 genes were annotated with sequence reads across the two asexually replicating stages of the parasite life cycle. Metabolism was similar between the two replicating stages. However, significant stage-specific expression differences were measured, with 312 transcripts exclusive to merozoites versus 453 exclusive to tachyzoites. Genes coding for 177 predicted secreted proteins and 64 membrane- associated proteins were annotated as merozoite-specific. The vast majority of known dense-granule (GRA), microneme (MIC), and rhoptry (ROP) genes were not expressed in merozoites. In contrast, a large set of surface proteins (SRS) was expressed exclusively in merozoites.
The distinct expression profiles of merozoites and tachyzoites reveal significant additional complexity within the T. gondii life cycle, demonstrating that merozoites are distinct asexual dividing stages which are uniquely adapted to their niche and biological purpose.
Electronic supplementary material
The online version of this article (doi:10.1186/s12864-015-1225-x) contains supplementary material, which is available to authorized users.
Toxoplasma gondii; Apicomplexa; Coccidia; Cat; Enteroepithelial development; Merozoite; Schizont; Comparative transcriptomics; Surface antigen; Stage-specific gene expression
Recently, many statistical methods have been proposed to test for associations between rare genetic variants and complex traits. Most of these methods test for association by aggregating genetic variations within a predefined region, such as a gene. Although there is evidence that “aggregate” tests are more powerful than the single marker test, these tests generally ignore neutral variants and therefore are unable to identify specific variants driving the association with phenotype. We propose a novel aggregate rare-variant test that explicitly models a fraction of variants as neutral, tests associations at the gene-level, and infers the rare-variants driving the association. Simulations show that in the practical scenario where there are many variants within a given region of the genome with only a fraction causal our approach has greater power compared to other popular tests such as the Sequence Kernel Association Test (SKAT), the Weighted Sum Statistic (WSS), and the collapsing method of Morris and Zeggini (MZ). Our algorithm leverages a fast variational Bayes approximate inference methodology to scale to exome-wide analyses, a significant computational advantage over exact inference model selection methodologies. To demonstrate the efficacy of our methodology we test for associations between von Willebrand Factor (VWF) levels and VWF missense rare-variants imputed from the National Heart, Lung, and Blood Institute’s Exome Sequencing project into 2,487 African Americans within the VWF gene. Our method suggests that a relatively small fraction (~10%) of the imputed rare missense variants within VWF are strongly associated with lower VWF levels in African Americans.
Exome sequencing study; approximate inference; von Willebrand Factor genetics
Acoustical changes in the prosody of mothers’ speech to infants are distinct and near universal. However, less is known about the visible properties mothers’ infant-directed (ID) speech, and their relation to speech acoustics. Mothers’ head movements were tracked as they interacted with their infants using ID speech, and compared to movements accompanying their adult-directed (AD) speech. Movement measures along three dimensions of head translation, and three axes of head rotation were calculated. Overall, more head movement was found for ID than AD speech, suggesting that mothers exaggerate their visual prosody in a manner analogous to the acoustical exaggerations in their speech. Regression analyses examined the relation between changing head position and changing acoustical pitch (F0) over time. Head movements and voice pitch were more strongly related in ID speech than in AD speech. When these relations were examined across time windows of different durations, stronger relations were observed for shorter time windows (< 5 sec). However, the particular form of these more local relations did not extend or generalize to longer time windows. This suggests that the multimodal correspondences in speech prosody are variable in form, and occur within limited time spans.
Plasma fibrinogen is an acute phase protein playing an important role in the blood coagulation cascade having strong associations with smoking, alcohol consumption and body mass index (BMI). Genome-wide association studies (GWAS) have identified a variety of gene regions associated with elevated plasma fibrinogen concentrations. However, little is yet known about how associations between environmental factors and fibrinogen might be modified by genetic variation. Therefore, we conducted large-scale meta-analyses of genome-wide interaction studies to identify possible interactions of genetic variants and smoking status, alcohol consumption or BMI on fibrinogen concentration. The present study included 80,607 subjects of European ancestry from 22 studies. Genome-wide interaction analyses were performed separately in each study for about 2.6 million single nucleotide polymorphisms (SNPs) across the 22 autosomal chromosomes. For each SNP and risk factor, we performed a linear regression under an additive genetic model including an interaction term between SNP and risk factor. Interaction estimates were meta-analysed using a fixed-effects model. No genome-wide significant interaction with smoking status, alcohol consumption or BMI was observed in the meta-analyses. The most suggestive interaction was found for smoking and rs10519203, located in the LOC123688 region on chromosome 15, with a p value of 6.2×10−8. This large genome-wide interaction study including 80,607 participants found no strong evidence of interaction between genetic variants and smoking status, alcohol consumption or BMI on fibrinogen concentrations. Further studies are needed to yield deeper insight in the interplay between environmental factors and gene variants on the regulation of fibrinogen concentrations.
Statins effectively lower LDL cholesterol levels in large studies and the observed interindividual response variability may be partially explained by genetic variation. Here we perform a pharmacogenetic meta-analysis of genome-wide association studies (GWAS) in studies addressing the LDL cholesterol response to statins, including up to 18,596 statin-treated subjects. We validate the most promising signals in a further 22,318 statin recipients and identify two loci, SORT1/CELSR2/PSRC1 and SLCO1B1, not previously identified in GWAS. Moreover, we confirm the previously described associations with APOE and LPA. Our findings advance the understanding of the pharmacogenetic architecture of statin response.
Statins are effectively used to prevent and manage cardiovascular disease, but patient response to these drugs is highly variable. Here, the authors identify two new genes associated with the response of LDL cholesterol to statins and advance our understanding of the genetic basis of drug response.
We report, for the first time, the saturated excitation (SAX) of fluorescent proteins for subdiffraction-limited imaging of living cells in three-dimensions. To achieve saturation, a bright yellow and green fluorescent protein (Venus and EGFP) that exhibits a strong nonlinear fluorescence response to the high excitation intensity at the laser focus is used. Harmonic demodulation of the fluorescence signal produced by a modulated excitation light extracts the nonlinear fluorescence signals. After constructing the image from the nonlinear components, we obtain fluorescence images of living cells with spatial resolution beyond the diffraction limit. We also applied linear deconvolution to SAX microscopy and found it effective in further enhancing the contrast of small intracellular structures in the SAX image, confirming the expansion of the optical transfer function in SAX microscopy.
high resolution; live-cell imaging; fluorescence microscopy; saturated excitation; confocal microscopy
Estimates of the heritability of plasma fibrinogen concentration, an established predictor of cardiovascular disease (CVD), range from 34 to 50%. Genetic variants so far identified by genome-wide association (GWA) studies only explain a small proportion (< 2%) of its variation.
Methods and Results
We conducted a meta-analysis of 28 GWA studies, including more than 90,000 subjects of European ancestry, the first GWA meta-analysis of fibrinogen levels in 7 African Americans studies totaling 8,289 samples, and a GWA study in Hispanic-Americans totaling 1,366 samples. Evaluation for association of SNPs with clinical outcomes included a total of 40,695 cases and 85,582 controls for coronary artery disease (CAD), 4,752 cases and 24,030 controls for stroke, and 3,208 cases and 46,167 controls for venous thromboembolism (VTE). Overall, we identified 24 genome-wide significant (P<5×10−8) independent signals in 23 loci, including 15 novel associations, together accounting for 3.7% of plasma fibrinogen variation. Gene-set enrichment analysis highlighted key roles in fibrinogen regulation for the three structural fibrinogen genes and pathways related to inflammation, adipocytokines and thyrotrophin-releasing hormone signaling. Whereas lead SNPs in a few loci were significantly associated with CAD, the combined effect of all 24 fibrinogen-associated lead SNPs was not significant for CAD, stroke or VTE.
We identify 23 robustly associated fibrinogen loci, 15 of which are new. Clinical outcome analysis of these loci does not support a causal relationship between circulating levels of fibrinogen and CAD, stroke or VTE.
Fibrinogen; cardiovascular disease; genome-wide association study
Ceruloplasmin (Cp) decreases nitric oxide bioavailability in blood and has been associated with cardiovascular disease (CVD) in clinical studies. We assessed the association between Cp and incident heart failure (HF), death and CVD in the Atherosclerosis Risk in Communities (ARIC) Study.
Methods and Results
Cp was measured at ARIC visit 4 (1996–1998). We studied 9,240 individuals without HF or CVD at ARIC visit 4, and followed them for a mean of 10.5 years. Genome-wide association study was performed to identify genetic determinants of Cp levels and evaluate their association with incident HF. Cp levels (mean±standard deviation) were higher in women vs men (335±79 vs 258±44 mg/L, p<0.0001), women on vs not on hormone-replacement therapy (398±89 vs 291±60 mg/L, p<0.0001) and African Americans vs Caucasians (299±63 vs 293±74 mg/L, p=0.0005). After adjusting for traditional risk factors, high-sensitivity C-reactive protein, N-terminal pro–B-type natriuretic peptide, and high-sensitivity cardiac troponin T, higher levels of Cp were associated with HF (hazard ratio [HR] 1.44, 95% confidence interval [CI] 1.13–1.83) and mortality (HR 1.38, 95% CI 1.11–1.63). A locus on the ceruloplasmin gene on chromosome 3 was significantly associated with Cp levels (normal 295.56±77.60mg/L, heterozygote 316.72±88.02mg/L; homozygote 331.04±85.40mg/L, p=8.3×10−) but not with incident HF. After adjustment for traditional risk factors Cp levels were also weekly associated with CVD.
Cp was associated with incident, HF mortality and CVD in the ARIC population. A single locus on chromosome 3 was associated with Cp levels but not with HF.
ceruloplasmin; heart failure; cardiovascular disease; single nucleotide polymorphism
Transforming growth factor-beta1 (TGF-B1) is a highly pleiotropic cytokine whose functions include a central role in the induction of fibrosis.
To investigate the hypothesis that elevated plasma levels of TGF-B1 are positively associated with incident heart failure (HF).
Participants and Methods
The hypotheses were tested using a two-phase case-control study design, ancillary to the Cardiovascular Health Study – a longitudinal, population-based cohort study. Cases were defined as having an incident HF event after their 1992-93 exam and controls were free of HF at follow-up. TGF-B1 was measured using plasma collected in 1992-93 and data from 89 cases and 128 controls were used for analysis. The association between TGF-B1 and risk of HF was evaluated using the weighted likelihood method, and odds ratios (OR) for risk of HF were calculated for TGF-B1 as a continuous linear variable and across quartiles of TGF-B1.
The OR for HF was 1.88 (95% confidence intervals [CI] 1.26 to 2.81) for each nanogram increase in TGF-B1, and the OR for the highest quartile (compared to the lowest) of TGF-B1 was 5.79 (95% CI 1.65 – 20.34), after adjustment for age, sex, C-reactive protein, platelet count and digoxin use. Further adjustment with other covariates did not change the results.
Higher levels of plasma TGF-B1 were associated with an increased risk of incident heart failure among older adults. However, further study is needed in larger samples to confirm these findings.
transforming growth factor-beta; heart failure; fibrosis; growth factors; cardiac remodeling
In patients with heart failure (HF), mortality is lower in women versus men. However, it is unknown whether the survival advantage in women compared with men is present in both whites and African Americans with HF. The inception cohort consisted of adults ≥65 years with incident HF after enrollment in the CHS, a prospective population-based study of cardiovascular disease. Of 5,888 CHS subjects, 1,264 developed new HF and were followed up for 3 years. Subjects were categorized into 4 race-gender groups, and Cox proportional hazard regression models were used to examine whether 3-year total and cardiovascular mortality differed among the 4 groups after adjusting for sociodemographic factors, co-morbidities, and treatment. A gender-race interaction was also tested for each outcome. In subjects with incident HF, African Americans had more hypertension and diabetes than whites, and white men had more coronary heart disease than other gender-race groups. Receipt of cardiovascular treatments among the 4 groups was similar. Mortality rates after HF were lower in women compared with men (for white women, African-American women, African-American men, and white men, total mortality was 35.5, 33.6, 44.4, and 40.5/100 person-years, and cardiovascular mortality was 18.4, 19.5, 20.2, and 22.7/100 person-years, respectively). After adjusting for covariates, women had a 15% to 20% lower risk of total and cardiovascular mortality compared with men, but there was no significant difference in outcome by race. The gender-race interaction for either outcome was not significant. In conclusion, in older adults with HF, women had significantly better survival than men irrespective of race, suggesting that gender-based survival differences may be more important than race-based differences.
BACKGROUND & AIMS
Identification of intestinal stem cells (ISCs) has relied heavily on the use of transgenic reporters in mice, but this approach is limited by mosaic expression patterns and difficult to directly apply to human tissues. We sought to identify reliable surface markers of ISCs and establish a robust functional assay to characterize ISCs from mouse and human tissues.
We used immunohistochemistry, real-time reverse-transcription polymerase chain reaction, and fluorescence-activated cell sorting (FACS) to analyze intestinal epithelial cells isolated from mouse and human intestinal tissues. We compared different combinations of surface markers among ISCs isolated based on expression of Lgr5–green fluorescent protein. We developed a culture protocol to facilitate the identification of functional ISCs from mice and then tested the assay with human intestinal crypts and putative ISCs.
CD44+CD24loCD166+ cells, isolated by FACS from mouse small intestine and colon, expressed high levels of stem cell–associated genes. Transit-amplifying cells and progenitor cells were then excluded based on expression of GRP78 or c-Kit. CD44+CD24loCD166+ GRP78lo/− putative stem cells from mouse small intestine included Lgr5-GFPhi and Lgr5-GFPmed/lo cells. Incubation of these cells with the GSK inhibitor CHIR99021 and the E-cadherin stabilizer Thiazovivin resulted in colony formation by 25% to 30% of single-sorted ISCs.
We developed a culture protocol to identify putative ISCs from mouse and human tissues based on cell surface markers. CD44+CD24loCD166+, GRP78lo/−, and c-Kit− facilitated identification of putative stem cells from the mouse small intestine and colon, respectively. CD44+CD24−/loCD166+ also identified putative human ISCs. These findings will facilitate functional studies of mouse and human ISCs.
Stemness; Differentiation; Single-Cell Sorting; Flow Cytometry Analysis