Hypertensive disorders of pregnancy and long-term maternal cardiovascular risk: Bridging epidemiological knowledge into personalized postpartum care and follow-up.

Cardiovascular disease (CVD) is globally the leading cause of death and disability. Sex-specific causes of female CVD are under-investigated. Pregnancy remains an underinvestigated sex-specific stress test for future CVD and a hitherto missed opportunity to initiate prevention of CVD at a young age. Population-based studies show a strong association between female CVD and hypertensive disorders of pregnancy. This association is also present after other pregnancy complications that are associated with placental dysfunction, including fetal growth restriction, preterm delivery and gestational diabetes mellitus. Few women are, however, offered systematic cardio-preventive follow-up after such pregnancy complications. These women typically seek help from the health system at first clinical symptom of CVD, which may be decades later. By this time, morbidity is established and years of preventive opportunities have been missed out. Early identification of modifiable risk factors starting postpartum followed by systematic preventive measures could improve maternal cardiovascular health trajectories, promoting healthier societies. In this non-systematic review we briefly summarize the epidemiological associations and pathophysiological hypotheses for the associations. We summarize current clinical follow-up strategies, including some proposed by international and national guidelines as well as user support groups. We address modifiable factors that may be underexploited in the postpartum period, including breastfeeding and blood pressure management. We suggest a way forward and discuss the remaining knowledge gaps and barriers for securing the best evidence-based follow-up, relative to available resources after a hypertensive pregnancy complication in order to prevent or delay onset of premature CVD.

Correlating transcription and protein expression profiles of immune biomarkers following lipopolysaccharide exposure in lung epithelial cells.

Universal and early recognition of pathogens occurs through recognition of evolutionarily conserved pathogen associated molecular patterns (PAMPs) by innate immune receptors and the consequent secretion of cytokines and chemokines. The intrinsic complexity of innate immune signaling and associated signal transduction challenges our ability to obtain physiologically relevant, reproducible and accurate data from experimental systems. One of the reasons for the discrepancy in observed data is the choice of measurement strategy. Immune signaling is regulated by the interplay between pathogen-derived molecules with host cells resulting in cellular expression changes. However, these cellular processes are often studied by the independent assessment of either the transcriptome or the proteome. Correlation between transcription and protein analysis is lacking in a variety of studies. In order to methodically evaluate the correlation between transcription and protein expression profiles associated with innate immune signaling, we measured cytokine and chemokine levels following exposure of human cells to the PAMP lipopolysaccharide (LPS) from the Gram-negative pathogen Pseudomonas aeruginosa. Expression of 84 messenger RNA (mRNA) transcripts and 69 proteins, including 35 overlapping targets, were measured in human lung epithelial cells. We evaluated 50 biological replicates to determine reproducibility of outcomes. Following pairwise normalization, 16 mRNA transcripts and 6 proteins were significantly upregulated following LPS exposure, while only five (CCL2, CSF3, CXCL5, CXCL8/IL8, and IL6) were upregulated in both transcriptomic and proteomic analysis. This lack of correlation between transcription and protein expression data may contribute to the discrepancy in the immune profiles reported in various studies. The use of multiomic assessments to achieve a systems-level understanding of immune signaling processes can result in the identification of host biomarker profiles for a variety of infectious diseases and facilitate countermeasure design and development.

Fetal-origin cells in maternal circulation correlate with placental dysfunction, fetal sex, and severe hypertension during pregnancy.

Fetal microchimerism (FMc) arises when fetal cells enter maternal circulation, potentially persisting for decades. Increased FMc is associated with fetal growth restriction, preeclampsia, and anti-angiogenic shift in placenta-associated proteins in diabetic and normotensive term pregnancies. The two-stage model of preeclampsia postulates that placental dysfunction causes such shift in placental growth factor (PlGF) and soluble fms-like tyrosine kinase-1 (sFLt-1), triggering maternal vascular inflammation and endothelial dysfunction. We investigated whether anti-angiogenic shift, fetal sex, fetal growth restriction, and severe maternal hypertension correlate with FMc in hypertensive disorders of pregnancy with new-onset features (n = 125). Maternal blood was drawn pre-delivery at > 25 weeks' gestation. FMc was detected by quantitative polymerase chain reaction targeting paternally inherited unique fetal alleles. PlGF and sFlt-1 were measured by immunoassay. We estimated odds ratios (ORs) by logistic regression and detection rate ratios (DRRs) by negative binomial regression. PlGF correlated negatively with FMc quantity (DRR = 0.2, p = 0.005) and female fetal sex correlated positively with FMc prevalence (OR = 5.0, p < 0.001) and quantity (DRR = 4.5, p < 0.001). Fetal growth restriction no longer correlated with increased FMc quantity after adjustment for correlates of placental dysfunction (DRR = 1.5, p = 0.272), whereas severe hypertension remained correlated with both FMc measures (OR = 5.5, p = 0.006; DRR = 6.3, p = 0.001). Our findings suggest that increased FMc is independently associated with both stages of the two-stage preeclampsia model. The association with female fetal sex has implications for microchimerism detection methodology. Future studies should target both male and female-origin FMc and focus on clarifying which placental mechanisms impact fetal cell transfer and how FMc impacts the maternal vasculature.

Fetal microchimerism and the two-stage model of preeclampsia.

Fetal cells cross the placenta during pregnancy and some have the ability to persist in maternal organs and circulation long-term, a phenomenon termed fetal microchimerism. These cells often belong to stem cell or immune cell lineages. The long-term effects of fetal microchimerism are likely mixed, potentially depending on the amount of fetal cells transferred, fetal-maternal histocompatibility and fetal cell-specific properties. Both human and animal data indicate that fetal-origin cells partake in tissue repair and may benefit maternal health overall. On the other hand, these cells have been implicated in inflammatory diseases by studies showing increased fetal microchimerism in women with autoimmune diseases such as systemic lupus erythematosus and rheumatoid arthritis. During pregnancy, preeclampsia is associated with increased cell-transfer between the mother and fetus, and an increase in immune cell subsets. In the current review, we discuss potential mechanisms of transplacental transfer, including passive leakage across the compromised diffusion barrier and active recruitment of cells residing in the placenta or fetal circulation. Within the conceptual framework of the two-stage model of preeclampsia, where syncytiotrophoblast stress is a common pathophysiological pathway to maternal and fetal clinical features of preeclampsia, we argue that microchimerism may represent a mechanistic link between stage 1 placental dysfunction and stage 2 maternal cardiovascular inflammation and endothelial dysfunction. Finally, we postulate that fetal microchimerism may contribute to the known association between placental syndromes and increased long-term maternal cardiovascular disease risk. Fetal microchimerism research represents an exciting opportunity for developing new disease biomarkers and targeted prophylaxis against maternal diseases.

Poor glucose control and markers of placental dysfunction correlate with increased circulating fetal microchimerism in diabetic pregnancies.

Fetal microchimerism (FMc) arises during pregnancy as fetal cells enter maternal circulation and remain decades postpartum. Circulating FMc is increased in preeclampsia, fetal growth restriction, and as we recently showed, is associated with biomarkers of placental dysfunction in normotensive term pregnancies. Diabetes mellitus (DM) also correlates with placental dysfunction. We hypothesize that poor glucose control and markers of placental dysfunction are associated with increased circulating FMc in diabetic pregnancies. We included 122 pregnancies preceding active labor (pregestational DM, n = 77, gestational DM (GDM), n = 45) between 2001 and 2017. Maternal and fetal samples were genotyped for various human leukocyte antigen (HLA) loci, and other polymorphisms to identify fetus-specific alleles. We used validated polymerase chain reaction (PCR) assays to quantify FMc in maternal peripheral blood buffy coat. Negative binomial regression with adjustment for confounders was used to assess FMc quantity. In pregestational DM, increased circulating FMc correlated with elevation of HbA1c (≥ 6.0 %) (detection rate ratio (DRR) = 4.9, p = 0.010) and a 1000 pg/mL rise in the anti-angiogenic biomarker soluble fms-like tyrosine kinase-1 (sFlt-1) (DRR = 1.1, p = 0.011). In GDM, increased FMc correlated with elevated 2-hour oral glucose tolerance test results (DRR = 2.3, p = 0.046) and birthweight < 10th or > 90th percentile (DRR = 4.2, p = 0.049). These findings support our novel hypothesis that FMc correlates with poor glucose control and various aspects of placental dysfunction in DM. Whether increased FMc in pregnancies with poor glucose control and placental dysfunction contributes to the risk of preeclampsia in diabetic pregnancies and to the increased risk of chronic cardiovascular disease later in life remains to be investigated.

Markers of placental function correlate with prevalence and quantity of nucleated fetal cells in maternal circulation in normotensive term pregnancies.

INTRODUCTION: Transplacental fetal cell transfer results in the engraftment of fetal-origin cells in the pregnant woman's body, a phenomenon termed fetal microchimerism. Increased fetal microchimerism measured decades postpartum is implicated in maternal inflammatory disease. Understanding which factors cause increased fetal microchimerism is therefore important. During pregnancy, circulating fetal microchimerism and placental dysfunction increase with increasing gestational age, particularly towards term. Placental dysfunction is reflected by changes in circulating placenta-associated markers, specifically placental growth factor (PlGF), decreased by several 100 pg/mL, soluble fms-like tyrosine kinase-1 (sFlt-1), increased by several 1000 pg/mL, and the sFlt-1/PlGF ratio, increased by several 10 (pg/mL)/(pg/mL). We investigated whether such alterations in placenta-associated markers correlate with an increase in circulating fetal-origin cells. MATERIAL AND METHODS: We included 118 normotensive, clinically uncomplicated pregnancies (gestational age 37+1 up to 42+2 weeks' gestation) pre-delivery. PlGF and sFlt-1 (pg/mL) were measured by Elecsys® Immunoassays. We extracted DNA from maternal and fetal samples and genotyped four human leukocyte antigen loci and 17 other autosomal loci. Paternally inherited, unique fetal alleles served as polymerase chain reaction (PCR) targets for detecting fetal-origin cells in maternal buffy coat. Fetal-origin cell prevalence was assessed by logistic regression, and quantity by negative binomial regression. Statistical exposures included gestational age (weeks), PlGF (100 pg/mL), sFlt-1 (1000 pg/mL) and the sFlt-1/PlGF ratio (10 (pg/mL)/(pg/mL)). Regression models were adjusted for clinical confounders and PCR-related competing exposures. RESULTS: Gestational age was positively correlated with fetal-origin cell quantity (DRR = 2.2, P = 0.003) and PlGF was negatively correlated with fetal-origin cell prevalence (odds ratio [OR]100 = 0.6, P = 0.003) and quantity (DRR100 = 0.7, P = 0.001). The sFlt-1 and the sFlt-1/PlGF ratios were positively correlated with fetal-origin cell prevalence (OR1000 = 1.3, P = 0.014 and OR10 = 1.2, P = 0.038, respectively), but not quantity (DRR1000 = 1.1, P = 0.600; DRR10 = 1.1, P = 0.112, respectively). CONCLUSIONS: Our results suggest that placental dysfunction as evidenced by placenta-associated marker changes, may increase fetal cell transfer. The magnitudes of change tested were based on ranges in PlGF, sFlt-1 and the sFlt-1/PlGF ratio previously demonstrated in pregnancies near and post-term, lending clinical significance to our findings. Our results were statistically significant after adjusting for confounders including gestational age, supporting our novel hypothesis that underlying placental dysfunction potentially is a driver of increased fetal microchimerism.

Serum amyloid A1 and pregnancy zone protein in pregnancy complications and correlation with markers of placental dysfunction.

BACKGROUND: Hypertensive disorders of pregnancy (preeclampsia, gestational hypertension, and chronic hypertension), diabetes mellitus, and placental dysfunction confer an increased risk of long-term maternal cardiovascular disease. Preeclampsia is also associated with acute atherosis that involves lesions of uteroplacental spiral arteries, resembling early stages of atherosclerosis. Serum amyloid A1 is involved in hypercoagulability and atherosclerosis and may aggregate into amyloid-aggregations of misfolded proteins. Pregnancy zone protein may inhibit amyloid aggregation. Amyloid is involved in Alzheimer's disease and cardiovascular disease; it has been identified in preeclampsia, but its role in preeclampsia pathophysiology is unclear. OBJECTIVE: We hypothesized that serum amyloid A1 would be increased and pregnancy zone protein decreased in hypertensive disorders of pregnancy and diabetic pregnancies and that serum amyloid A1 and pregnancy zone protein would correlate with placental dysfunction markers (fetal growth restriction and dysregulated angiogenic biomarkers) and acute atherosis. STUDY DESIGN: Serum amyloid A1 is measurable in both the serum and plasma. In our study, plasma from 549 pregnancies (normotensive, euglycemic controls: 258; early-onset preeclampsia: 71; late-onset preeclampsia: 98; gestational hypertension: 30; chronic hypertension: 9; diabetes mellitus: 83) was assayed for serum amyloid A1 and pregnancy zone protein. The serum levels of angiogenic biomarkers soluble fms-like tyrosine kinase-1 and placental growth factor were available for 547 pregnancies, and the results of acute atherosis evaluation were available for 313 pregnancies. The clinical characteristics and circulating biomarkers were compared between the pregnancy groups using the Mann-Whitney U, chi-squared, or Fisher exact test as appropriate. Spearman's rho was calculated for assessing correlations. RESULTS: In early-onset preeclampsia, serum amyloid A1 was increased compared with controls (17.1 vs 5.1 µg/mL, P<.001), whereas pregnancy zone protein was decreased (590 vs 892 µg/mL, P=.002). Pregnancy zone protein was also decreased in diabetes compared with controls (683 vs 892 µg/mL, P=.01). Serum amyloid A1 was associated with placental dysfunction (fetal growth restriction, elevated soluble fms-like tyrosine kinase-1 to placental growth factor ratio). Pregnancy zone protein correlated negatively with soluble fms-like tyrosine kinase-1 to placental growth factor ratio in all study groups. Acute atherosis was not associated with serum amyloid A1 or pregnancy zone protein. CONCLUSION: Proteins involved in atherosclerosis, hypercoagulability, and protein misfolding are dysregulated in early-onset preeclampsia and placental dysfunction, which links them and potentially contributes to future maternal cardiovascular disease.

Circulating cardiovascular biomarkers during and after preeclampsia: Crosstalk with placental function?

Cardiovascular disease (CVD) is the leading cause of death in women, yet sex-specific risk factors remain understudied. Preeclampsia and other adverse pregnancy outcomes imply an increased maternal cardiovascular risk. We hypothesized that cardiac troponin T (cTnT), N-terminal pro-Brain Natriuretic Peptide (NT-proBNP) and growth differentiation factor 15 (GDF-15) are increased in such pregnancies and correlate with markers of placental dysfunction. We also investigated these cardiovascular biomarkers 1 or 3 years postpartum. Prior to delivery, we included serum from 417 pregnant women: 55 early-onset preeclampsia (EO-PE), 63 late-onset preeclampsia (LO-PE), 30 gestational hypertension (GH) and 269 healthy controls. Postpartum, we included 341 women 1 or 3 years after delivery: 26 EO-PE, 107 LO-PE, 61 GH, and 147 healthy pregnancies. Prior to delivery, median cTnT and NT-proBNP concentrations were higher in women with EO-PE, LO-PE, or GH than in controls. Median GDF-15 was higher in EO-PE and LO-PE compared to controls. Postpartum, GDF-15 was elevated in women with previous EO-PE. Markers of placental dysfunction correlated with CVD biomarkers in pregnancy, but not postpartum. Our findings underscore the cardiovascular burden of hypertensive disorders of pregnancy and the crosstalk with placental function. The upregulation of circulating GDF-15 following early-onset preeclampsia is in line with the epidemiological excessive risk of premature CVD in this group of women. GDF-15 may be explored for targeting postpartum women with most to gain from intensified preventive follow-up for CVD.

Cardiovascular biomarkers in pregnancy with diabetes and associations to glucose control.

AIM: Cardiovascular disease (CVD) is a leading cause of death in both men and women. Type 1 and 2 diabetes mellitus (DM1 and DM2) are well-known risk factors for CVD. In addition, gestational diabetes mellitus (GDM) is a female sex-specific risk factor for CVD. Here, we measure circulating concentrations of cardiac troponin T (cTNT), N-terminal pro-B-type natriuretic peptide (NT-proBNP) and growth differentiation factor 15 (GDF-15) during pregnancy-a window of time often referred to as a cardiovascular stress test for women. METHODS: This study utilized data from 384 pregnant women: 64 with DM1, 16 with DM2, 35 with GDM and 269 euglycemic controls. Blood was predominantly sampled within a week before delivery. Cardiovascular biomarker concentrations were measured in serum using electrochemiluminescence immunoassay. RESULT: Circulating cTnT levels were higher in women with DM1, DM2 and GDM as compared to controls, whereas NT-proBNP and GDF-15 levels were only increased in women with DM1. Glucose dysregulation, assessed by third trimester HbA1c levels, positively correlated with all three CVD biomarker levels, whereas pregestational body mass index correlated negatively with GDF-15. CONCLUSIONS: Our results support the presence of myocardial affection in women with diabetic disorders during pregnancy. Although pregestational DM1 in this study was associated with the most adverse CVD biomarker profile, women with GDM displayed an adverse cTnT profile similar to what we found in women with pregestational DM2. This supports that women with GDM should be offered long-term intensified cardiovascular follow-up and lifestyle advice following delivery, similarly to the well-established CV follow-up of women with pregestational DM.

Acute Atherosis Lesions at the Fetal-Maternal Border: Current Knowledge and Implications for Maternal Cardiovascular Health.

Decidua basalis, the endometrium of pregnancy, is an important interface between maternal and fetal tissues, made up of both maternal and fetal cells. Acute atherosis is a uteroplacental spiral artery lesion. These patchy arterial wall lesions containing foam cells are predominantly found in the decidua basalis, at the tips of the maternal arteries, where they feed into the placental intervillous space. Acute atherosis is prevalent in preeclampsia and other obstetric syndromes such as fetal growth restriction. Causal factors and effects of acute atherosis remain uncertain. This is in part because decidua basalis is challenging to sample systematically and in large amounts following delivery. We summarize our decidua basalis vacuum suction method, which facilitates tissue-based studies of acute atherosis. We also describe our evidence-based research definition of acute atherosis. Here, we comprehensively review the existing literature on acute atherosis, its underlying mechanisms and possible short- and long-term effects. We propose that multiple pathways leading to decidual vascular inflammation may promote acute atherosis formation, with or without poor spiral artery remodeling and/or preeclampsia. These include maternal alloreactivity, ischemia-reperfusion injury, preexisting systemic inflammation, and microbial infection. The concept of acute atherosis as an inflammatory lesion is not novel. The lesions themselves have an inflammatory phenotype and resemble other arterial lesions of more extensively studied etiology. We discuss findings of concurrently dysregulated proteins involved in immune regulation and cardiovascular function in women with acute atherosis. We also propose a novel hypothesis linking cellular fetal microchimerism, which is prevalent in women with preeclampsia, with acute atherosis in pregnancy and future cardiovascular and neurovascular disease. Finally, women with a history of preeclampsia have an increased risk of premature cardiovascular disease. We review whether presence of acute atherosis may identify women at especially high risk for premature cardiovascular disease.

Optical Biosensor Platforms Display Varying Sensitivity for the Direct Detection of Influenza RNA.

Detection methods that do not require nucleic acid amplification are advantageous for viral diagnostics due to their rapid results. These platforms could provide information for both accurate diagnoses and pandemic surveillance. Influenza virus is prone to pandemic-inducing genetic mutations, so there is a need to apply these detection platforms to influenza diagnostics. Here, we analyzed the Fast Evaluation of Viral Emerging Risks (FEVER) pipeline on ultrasensitive detection platforms, including a waveguide-based optical biosensor and a flow cytometry bead-based assay. The pipeline was also evaluated in silico for sequence coverage in comparison to the U.S. Centers for Disease Control and Prevention's (CDC) influenza A and B diagnostic assays. The influenza FEVER probe design had a higher tolerance for mismatched bases than the CDC's probes, and the FEVER probes altogether had a higher detection rate for influenza isolate sequences from GenBank. When formatted for use as molecular beacons, the FEVER probes detected influenza RNA as low as 50 nM on the waveguide-based optical biosensor and 1 nM on the flow cytometer. In addition to molecular beacons, which have an inherently high background signal we also developed an exonuclease selection method that could detect 500 pM of RNA. The combination of high-coverage probes developed using the FEVER pipeline coupled with ultrasensitive optical biosensors is a promising approach for future influenza diagnostic and biosurveillance applications.

Circulating HLA-G and its association with cardiovascular markers in pregnancy.

Human Leukocyte Antigen-G (HLA-G) prevents the activity of immune cells and is decreased in women with preeclampsia. We aimed to investigate the associations between circulating soluble HLA-G (sHLA-G) and 92 cardiovascular disease-related biomarkers from a previously published multiplex study in women with preeclampsia and controls. We found 15 markers significantly associated with circulating sHLA-G in univariate analyses. After multivariable adjusted regression, only proto-oncogene tyrosine-protein kinase Src (SRC) and vascular endothelial growth factor D were significantly associated with sHLA-G. Low SRC, previously observed in the circulation of preeclamptic women, may be regulated by low sHLA-G, and reflect decreased trophoblast differentiation and syncytical formation.

A possible role for HLA-G in development of uteroplacental acute atherosis in preeclampsia.

HLA-G, a non-classical HLA molecule expressed by extravillous trophoblasts, plays a role in the maternal immune tolerance towards fetal cells. HLA-G expression is regulated by genetic polymorphisms in the 3' untranslated region (3'UTR). Low levels of HLA-G in the maternal circulation and placental tissue are linked to preeclampsia. Our objective was to investigate whether variants of the 3'UTR of the HLA-G gene in mother and fetus are associated with acute atherosis, a pregnancy specific arterial lesion of the decidua basalis that is prevalent in preeclampsia. Paired maternal and fetal DNA samples from 83 normotensive and 83 preeclamptic pregnancies were analyzed. We sequenced the part of the HLA-G 3'UTR containing a 14-bp insertion/deletion region and seven single nucleotide polymorphisms (SNPs). Associations with acute atherosis were tested by logistic regression. The frequency of heterozygosity for the 14-bp polymorphism (Ins/Del) and the +3142 SNP (C/G) variant in the fetus are associated with acute atherosis in preeclampsia (66.7 % vs. 39.6 %, p = 0.039, and 69.0 % vs. 43.4 %, p = 0.024). Furthermore, the fetal UTR-3 haplotype, which encompasses the 14-bp deletion and the +3142G variant, is associated with acute atherosis in preeclampsia (15 % vs. 3.8 %, p = 0.016). In conclusion, HLA-G polymorphisms in the fetus are associated with acute atherosis. We hypothesize that these polymorphisms lead to altered HLA-G expression in the decidua basalis, affecting local feto-maternal immune tolerance and development of acute atherosis.

Pregnancy and postpartum levels of circulating maternal sHLA-G in preeclampsia.

Preeclampsia is a leading cause of maternal and offspring mortality and morbidity, and predicts increased future cardiovascular disease risk. Placental dysfunction and immune system dysregulation are likely key pathophysiological factors. Soluble human leukocyte antigen G (sHLA-G) may dampen the specific immune response towards placental trophoblasts. Previous studies have shown low sHLA-G levels in preeclampsia, but postpartum, levels are unknown. Furthermore, the relationship between sHLA-G and sFlt-1 and PlGF, placental function markers, is unknown. We hypothesized that low maternal sHLA-G during pregnancy would be associated with placental dysfunction, including preeclampsia, gestational hypertension, and dysregulated sFlt-1 and PlGF, and that sHLA-G would remain decreased following preeclampsia. We included 316 pregnant women: 58 with early-onset preeclampsia (<34 weeks' gestation), 81 with late-onset preeclampsia (≥34 weeks' gestation), 25 with gestational hypertension, and 152 normotensive controls. Postpartum (1 or 3 years), we included 321 women: 29 with early-onset preeclampsia, 98 with late-onset preeclampsia, 57 with gestational hypertension, and 137 who were normotensive during their index pregnancies. In pregnancy, plasma sHLA-G was significantly lower both in the early- and late-onset preeclampsia groups compared to controls. In women with preeclampsia or gestational hypertension, sHLA-G was inversely correlated with serum sFlt-1. Postpartum, plasma sHLA-G levels were significantly higher in women who had had early-onset preeclampsia compared to controls. Our results support that sHLA-G may be important for placental function. Unexpectedly, sHLA-G was elevated up to 3 years after early-onset preeclampsia, suggesting an excessively activated immune system following this severe preeclampsia form, potentially contributing to future cardiovascular disease risk.

A genome assembly and the somatic genetic and epigenetic mutation rate in a wild long-lived perennial Populus trichocarpa.

BACKGROUND: Plants can transmit somatic mutations and epimutations to offspring, which in turn can affect fitness. Knowledge of the rate at which these variations arise is necessary to understand how plant development contributes to local adaption in an ecoevolutionary context, particularly in long-lived perennials. RESULTS: Here, we generate a new high-quality reference genome from the oldest branch of a wild Populus trichocarpa tree with two dominant stems which have been evolving independently for 330 years. By sampling multiple, age-estimated branches of this tree, we use a multi-omics approach to quantify age-related somatic changes at the genetic, epigenetic, and transcriptional level. We show that the per-year somatic mutation and epimutation rates are lower than in annuals and that transcriptional variation is mainly independent of age divergence and cytosine methylation. Furthermore, a detailed analysis of the somatic epimutation spectrum indicates that transgenerationally heritable epimutations originate mainly from DNA methylation maintenance errors during mitotic rather than during meiotic cell divisions. CONCLUSION: Taken together, our study provides unprecedented insights into the origin of nucleotide and functional variation in a long-lived perennial plant.

Phosphatidylinositol 4-kinase III beta regulates cell shape, migration, and focal adhesion number.

Cell shape is regulated by cell adhesion and cytoskeletal and membrane dynamics. Cell shape, adhesion, and motility have a complex relationship and understanding them is important in understanding developmental patterning and embryogenesis. Here we show that the lipid kinase phosphatidylinositol 4-kinase III beta (PI4KIIIß) regulates cell shape, migration, and focal adhesion (FA) number. PI4KIIIß generates phosphatidylinositol 4-phosphate (PI4P) from phosphatidylinositol and is highly expressed in a subset of human breast cancers. PI4KIIIß and the PI4P it generates regulate a variety of cellular functions, ranging from control of Golgi structure, fly fertility, and Akt signaling. Here, we show that loss of PI4KIIIß expression decreases cell migration and alters cell shape in NIH3T3 fibroblasts. The changes are accompanied by an increase in the number of FA in cells lacking PI4KIIIß. Furthermore, we find that PI4P-containing vesicles move to the migratory leading edge during migration and that some of these vesicles tether to and fuse with FA. Fusion is associated with FA disassembly. This suggests a novel regulatory role for PI4KIIIß and PI4P in cell adhesion and cell shape maintenance.

Repeated social defeat promotes persistent inflammatory changes in splenic myeloid cells; decreased expression of ß-arrestin-2 (ARRB2) and increased expression of interleukin-6 (IL-6).

BACKGROUND: Previous studies suggest that persistent exposure to social stress in mammals may be associated with multiple physiological effects. Here, we examine the effects of social stress in rats, i.e. repeated social defeat, on behavior, hypothalamic-pituitary-adrenal (HPA)-axis and immune system. METHODS: A resident-intruder paradigm, where an intruder rat was exposed to social stress by a dominant resident rat for 1 hour each day for 7 consecutive days was used. The day after the last stress exposure in the paradigm the data were analyzed. Variation in social interaction was observed manually, whereas locomotion was analyzed off-line by a purpose-made software. Gene expression in the pituitary gland, adrenal gland and myeloid cells isolated from the spleen was measured by qPCR. RESULTS: The exposure to social stress induced decreased weight gain and increased locomotion. An increased nuclear receptor subfamily group C number 1 (NR3C1) expression in the pituitary gland was also shown. In myeloid cells harvested from the spleen, we observed decreased expression of the ß2-adrenergic receptor (ADRB2) and ß-arrestin-2 (ARRB2), but increased expression of interleukin-6 (IL-6). Subsequent analyses in the same cells showed that ARRB2 was negatively correlated with IL-6 following the stress exposure. CONCLUSION: Our results show that that the experience of social stress in the form of repeated social defeat in rats is a potent stressor that in myeloid cells in the spleen promotes persistent inflammatory changes. Future research is needed to examine whether similar inflammatory changes also can explain the impact of social stress, such as bullying and harassment, among humans.

Exposure to workplace bullying, microRNAs and pain; evidence of a moderating effect of miR-30c rs928508 and miR-223 rs3848900.

Prolonged exposure to bullying behaviors may give rise to symptoms such as anxiety, depression and chronic pain. Earlier data suggest that these symptoms often are associated with stress-induced low-grade systemic inflammation. Here, using data from both animals and humans, we examined the moderating role of microRNAs (miRNAs, miRs) in this process. In the present study, a resident-intruder paradigm, blood samples, tissue harvesting and subsequent qPCR analyses were used to screen for stress-induced changes in circulating miRNAs in rats. The negative acts questionnaire (NAQ), TaqMan assays and a numeric rating scale (NRS) for pain intensity were then used to examine the associations among bullying behaviors, relevant miRNA polymorphisms and pain in a probability sample of 996 Norwegian employees. In rats, inhibited weight gain, reduced pituitary POMC expression, adrenal Nr3c1 mRNA downregulation, as well as increased miR-146a, miR-30c and miR-223 in plasma were observed following 1 week of repeated exposure to social stress. When following up the miRNA findings from the animal study in the human working population, a stronger relationship between NAQ and NRS scores was observed in subjects with the miR-30c GG genotype (rs928508) compared to other subjects. A stronger relationship between NAQ and NRS scores was also seen in men with the miR-223 G genotype (rs3848900) as compared to other men. Our findings show that social stress may induce many physiological changes including changed expression of miRNAs. We conclude that the miR-30c GG genotype in men and women, and the miR-223 G genotype in men, amplify the association between exposure to bullying behaviors and pain.Lay summaryUsing an animal model of social stress, we identified miR-146a, miR-30c and miR-223 as potentially important gene regulatory molecules that may be involved in the stress response. Interestingly, human genotypes affecting the expression of mature miR-30c and miR-223 had a moderating effect on the association between exposure to bullying and pain. Subjects with the miR-30c rs928508 GG genotype had a significantly stronger association between exposure to bullying behaviors and pain than other subjects. The same was observed in men with the miR-223 rs3848900 G genotype, as compared to other men.

Up-regulation of circulating microRNA-17 is associated with lumbar radicular pain following disc herniation.

BACKGROUND: Previous studies suggest that regulatory microRNAs (miRs) may modulate neuro-inflammatory processes. The purpose of the present study was to examine the role of miR-17 following intervertebral disc herniation. METHODS: In a cohort of 97 patients with leg pain and disc herniation verified on MRI, we investigated the association between circulating miR-17 and leg pain intensity. A rat model was used to examine possible changes in miR-17 expression in nucleus pulposus (NP) associated with leak of NP tissue out of the herniated disc. The functional role of miR-17 was addressed by transfection of miR-17 into THP-1 cells (human monocyte cell line). RESULTS: An association between the level of miR-17 in serum and the intensity of lumbar radicular pain was shown. Up-regulation of miR-17 in the rat NP tissue when applied onto spinal nerve roots and increased release of TNF following transfection of miR-17 into THP-1 cells were also observed. Hence, our data suggest that miR-17 may be involved in the pathophysiology underlying lumbar radicular pain after disc herniation. CONCLUSIONS: We conclude that miR-17 may be associated with the intensity of lumbar radicular pain after disc herniation, possibly through a TNF-driven pro-inflammatory mechanism.

The 5-HTTLPR rs25531 LALA-genotype increases the risk of insomnia symptoms among shift workers.

BACKGROUND: Previous studies indicate that shift work tolerance may be associated with individual factors including genetic variability in the gene encoding the serotonin transporter 5-HTT (SLC6A4). The present study aimed to explore the interaction between work schedule (shift work versus non-shift work), genetic variability in SLC6A4 and insomnia symptoms. METHODS: The study was based on a national probability sample survey of 987 Norwegian employees drawn from The Norwegian Central Employee Register by Statistics Norway. Insomnia symptoms were assessed by three items reflecting problems with sleep onset, sleep maintenance, and early morning awakenings. Genotyping concerning SLC6A4 (the 5-HTTLPR S versus L and the SNP rs25531 A versus G) was carried out using a combination of gel-electrophoresis and TaqMan assay. RESULTS: Using the LALA genotype as a reference a main effect of the SS genotype (B = 0.179; 95% CI = 0.027-0.330) was found. In addition, a main effect of work schedule (0 = non shift, 1 = shift work) was found (B = 0.504; 95% CI = 0.185-0.823). The genotype x work schedule interaction was significant for all genotypes; SLA (B = -0.590; 95% CI = -0.954-0.216), LALG (B = -0.879; 95% CI = -1.342-0.415), SLG (B = -0.705; 95% CI = -1.293-0.117) and SS (B = -0.773; 95% CI = -1.177-0.369) indicating higher insomnia symptom scores among LALA-participants compared to participants with other genotypes when working shifts. CONCLUSIONS: The ability to cope with shift work is associated with the combination of the SLC6A4 variants 5-HTTLPR and SNP rs25531. Our findings demonstrated that the LALA-genotype increases the risk of insomnia symptoms among shift workers.