Comparative gene co-expression networks show enrichment of brassinosteroid and vitamin B processes in a seagrass under simulated ocean warming and extreme climatic events.

Introduction: Ocean warming combined with extreme climatic events, such as marine heatwaves and flash flooding events, threaten seagrasses globally. How seagrasses cope with these challenges is uncertain, particularly for range-edge populations of species such as Posidonia australis in Shark Bay, Western Australia. Analyzing gene expression while manipulating multiple stressors provides insight into the genetic response and resilience of seagrasses to climate change. We conducted a gene expression study on a polyploid clone of P. australis during an 18-week mesocosm experiment to assess the responses to single and combined future climate change-associated stressors. Methods: Plants were exposed to (1) future ocean warming temperature (baseline +1.5°C) followed by a simulated marine heat wave (baseline +5.5°C), (2) light deprivation simulating observed marine heatwave driven turbidity (95% shade) at baseline temperatures, or (3) both stressors simultaneously. Basal leaf meristems were sampled for gene expression analysis using RNA-seq at four time points during the experiment. Weighted gene co-expression network analysis, GO term enrichment, and KEGG pathway enrichment analyses were used to identify stress responses. Results: Shaded plants showed specific gene enrichment for shade avoidance (programmed cell death) after three weeks of stress, and before any heated tanks showed a specific heat response. Shaded plants were positively correlated with programmed cell death and stress-related processes at the end of the experiment. Once ocean warming temperatures (+1.5°C) were in effect, gene enrichment for heat stress (e.g., ROS scavenging and polyamine metabolism) was present. Vitamin B processes, RNA polymerase II processes. and light-related meristematic phase changes were expressed with the addition of simulated MHW. Heated plants showed meristematic growth signatures as well as trehalose and salicylic acid metabolism. Brassinosteroid-related processes were significantly enriched in all stressor treatments at all time points, except for the isolated heat-stressed plants three weeks after stressor initiation. Discussion: Gene expression responses to the interaction between heat waves and turbidity-induced light reduction support the observed geographical scale mortality in seagrasses observed for P. australis in Shark Bay, suggesting that even this giant polyploid clone will be negatively impacted by more extreme climate change projections.

Extensive polyploid clonality was a successful strategy for seagrass to expand into a newly submerged environment.

Polyploidy has the potential to allow organisms to outcompete their diploid progenitor(s) and occupy new environments. Shark Bay, Western Australia, is a World Heritage Area dominated by temperate seagrass meadows including Poseidon's ribbon weed, Posidonia australis. This seagrass is at the northern extent of its natural geographic range and experiences extremes in temperature and salinity. Our genomic and cytogenetic assessments of 10 meadows identified geographically restricted, diploid clones (2n = 20) in a single location, and a single widespread, high-heterozygosity, polyploid clone (2n = 40) in all other locations. The polyploid clone spanned at least 180 km, making it the largest known example of a clone in any environment on earth. Whole-genome duplication through polyploidy, combined with clonality, may have provided the mechanism for P. australis to expand into new habitats and adapt to new environments that became increasingly stressful for its diploid progenitor(s). The new polyploid clone probably formed in shallow waters after the inundation of Shark Bay less than 8500 years ago and subsequently expanded via vegetative growth into newly submerged habitats.

High-Powered Magnet Exposures in Children: A Multi-Center Cohort Study.

BACKGROUND AND OBJECTIVES: High-powered magnets were effectively removed from the US market by the Consumer Product Safety Commission (CPSC) in 2012 but returned in 2016 after federal court decisions. The United States Court of Appeals for the 10th Circuit cited imprecise data among other reasons as justification for overturning CPSC protections. Since then, incidence of high-powered magnet exposure has increased markedly, but outcome data are limited. In this study, we aim to describe the epidemiology and outcomes in children seeking medical care for high-powered magnets after reintroduction to market. METHODS: This is a multicenter, retrospective cohort study of patients aged 0 to 21 years with a confirmed high-powered magnet exposure (ie, ingestion or insertion) at 25 children's hospitals in the United States between 2017 and 2019. RESULTS: Of 596 patients with high-powered magnet exposures identified, 362 (60.7%) were male and 566 (95%) were <14 years of age. Nearly all sought care for magnet ingestion (n = 574, 96.3%), whereas 17 patients (2.9%) presented for management of nasal or aural magnet foreign bodies, 4 (0.7%) for magnets in their genitourinary tract, and 1 patient (0.2%) had magnets in their respiratory tract. A total of 57 children (9.6%) had a life-threatening morbidity; 276 (46.3%) required an endoscopy, surgery, or both; and 332 (55.7%) required hospitalization. There was no reported mortality. CONCLUSIONS: Despite being intended for use by those >14 years of age, high-powered magnets frequently cause morbidity and lead to high need for invasive intervention and hospitalization in children of all ages.

Profiling the cell walls of seagrasses from A (Amphibolis) to Z (Zostera).

BACKGROUND: The polyphyletic group of seagrasses shows an evolutionary history from early monocotyledonous land plants to the marine environment. Seagrasses form important coastal ecosystems worldwide and large amounts of seagrass detritus washed on beaches might also be valuable bioeconomical resources. Despite this importance and potential, little is known about adaptation of these angiosperms to the marine environment and their cell walls. RESULTS: We investigated polysaccharide composition of nine seagrass species from the Mediterranean, Red Sea and eastern Indian Ocean. Sequential extraction revealed a similar seagrass cell wall polysaccharide composition to terrestrial angiosperms: arabinogalactans, pectins and different hemicelluloses, especially xylans and/or xyloglucans. However, the pectic fractions were characterized by the monosaccharide apiose, suggesting unusual apiogalacturonans are a common feature of seagrass cell walls. Detailed analyses of four representative species identified differences between organs and species in their constituent monosaccharide composition and lignin content and structure. Rhizomes were richer in glucosyl units compared to leaves and roots. Enhalus had high apiosyl and arabinosyl abundance, while two Australian species of Amphibolis and Posidonia, were characterized by high amounts of xylosyl residues. Interestingly, the latter two species contained appreciable amounts of lignin, especially in roots and rhizomes whereas Zostera and Enhalus were lignin-free. Lignin structure in Amphibolis was characterized by a higher syringyl content compared to that of Posidonia. CONCLUSIONS: Our investigations give a first comprehensive overview on cell wall composition across seagrass families, which will help understanding adaptation to a marine environment in the evolutionary context and evaluating the potential of seagrass in biorefinery incentives.

Management of esophageal button battery ingestions: resource utilization and outcomes.

PURPOSE: Institutions are adopting the North American Society for Pediatric Gastroenterology, Hepatology and Nutrition (NASPGHAN) guidelines for pediatric esophageal button battery ingestion (EBBI). Our objective was to evaluate the guidelines' impact on in-hospital resource utilization and short-term clinical outcomes in hemodynamically stable patients after endoscopic battery removal. METHODS: A single-center retrospective review of all EBBI admissions from 2010 to 2020. Patients were divided into two groups based on adoption of national guidelines: pre-guideline (2010-2015) and post-guideline (2016-2020). RESULTS: Sixty-five patients were studied (pre-guideline n = 23; post-guideline n = 42). Compared with pre-guideline, post-guideline use of magnetic resonance imaging (MRI) increased (2/23 [8.7%]; 30/42 [71.4%]; p < 0.001). Post-guideline increases resulted for median days (IQR) receiving antibiotics (0 [0, 4]; 6 [3, 8]; p = 0.01), total pediatric intensive care unit admission (0 [0, 1]; 3 [0, 6]; p < 0.001), and total hospital length of stay (5 [2, 11]; 11.5 [4, 17]; p = 0.02). Two patients in the post-guideline group had delayed presentations despite normal imaging: one with TEF and one with aorto-esophageal fistula. All survived to discharge. CONCLUSION: In EBBI cases managed using the consensus based NASPHAGN guidelines, we report increased resource utilization without improved patient outcomes. Further research should evaluate post-guideline costs and resource utilization.

PRO-C3, a Serological Marker of Fibrosis, During Childhood and Correlations With Fibrosis in Pediatric NAFLD.

Nonalcoholic fatty liver disease (NAFLD) is a common chronic liver disease in children and may lead to cirrhosis requiring liver transplant. Thus, prompt diagnosis of advanced fibrosis is essential. Our objectives were to examine PRO-C3 (a neo-epitope pro-peptide of type III collagen formation) levels across childhood/adolescence and associations with advanced fibrosis in pediatric NAFLD. This cross-sectional study included 88 children and adolescents with biopsy-proven NAFLD (mean age: 13.9 ± 2.9 years, 71% male) and 65 healthy participants (11.8 ± 4.5 years, 38% male). PRO-C3, and the bone remodeling biomarkers C-terminal telopeptide of type I collagen (CTX-I; bone resorption) and osteocalcin (N-MID; bone formation), were measured in serum by enzyme-linked immunosorbent assay. Fibrosis was assessed by liver biopsy in participants with NAFLD, who were categorized as having advanced (Ishak score ≥ 3) or none/mild fibrosis (Ishak score ≤ 2). Overall, PRO-C3 was similar in participants with NAFLD (median [interquartile range]: 20.6 [15.8, 25.9] ng/mL) versus healthy participants (19.0 [13.8, 26.0] ng/mL), but was significantly lower in older adolescents ≥ 15 years old (16.4 [13.0, 21.2] ng/mL) compared with children ≤ 10 years old (22.9 [18.1, 28.4] ng/mL; P < 0.001) or 11-14 years old (22.4 [18.3, 31.2] ng/mL; P < 0.001). PRO-C3 was also directly correlated with levels of CTX-I and N-MID (r = 0.64 and r = 0.62, respectively; both P < 0.001). Among participants with NAFLD, PRO-C3 was higher in those with advanced fibrosis (median [IQR]: 28.5 [21.6, 37.6]) compared with none/mild fibrosis (20.3 [18.2, 22.8]; P = 0.020) in models adjusted for age, sex, and body mass index z-score. However, associations were attenuated after additionally adjusting for bone-remodeling CTX-I (P = 0.09) or N-MID (P = 0.08). Conclusion: Collectively, these findings show that PRO-C3 levels are higher in children with advanced fibrosis in NAFLD, but are also influenced by age and pubertal growth spurt, assessed by bone remodeling biomarkers, and therefore may not be a reliable biomarker for liver fibrosis in pediatric NAFLD until late adolescence.

Development and repair of aorto-esophageal fistula following esophageal button battery impaction: A case report.

BACKGROUND: Complications from esophageal button battery impactions remain a real fear for practicing pediatric gastroenterologists and surgeons. This case describes a child who developed an aorto-esophageal fistula 25 days after initial battery ingestion and survived due to prompt placement of an aortic stent via minimally invasive surgery, avoiding an open procedure. CASE PRESENTATION: A 6-year-old female presented acutely with a mid-esophageal button battery impaction witnessed by her parents. Presenting symptoms included chest pain and emesis. Button battery location and size were confirmed on X-ray. She underwent removal with flexible esophagogastroduodenoscopy (EGD) and rigid esophagoscopy. She was admitted to the hospital and received conservative medical management, with serial cross-sectional imaging via chest MRIs to assess the evolution of her injury according to available national guidelines, and was discharged after 12 days of close inpatient monitoring. Despite these measures the patient represented 25 days post-ingestion with hematemesis from a new aorto-esophageal fistula, requiring emergent cardiac catheterization with successful, life-saving aortic stent placement. She remained admitted for an additional 12 days of monitoring as her diet was advanced slowly post-catheterization. Since this second hospitalization she continues to do well, with outpatient follow-up by multiple subspecialists. CONCLUSIONS: This case highlights the continued uncertainty regarding the risk of developing this complication, as well as gaps in the current literature and guidelines for managing these patients following ingestion and esophageal injury. It also details the unique course following development of this complication and its surgical repair.

Physical activity assessment among men undergoing genetic counseling for inherited prostate cancer: a teachable moment for improved survivorship.

BACKGROUND: Genetic counseling (GC) presents an opportunity to address modifiable cancer risk factors, such as obesity, which is impacted by non-adherence to physical activity (PA) guidelines. Adherence to PA guidelines has not been assessed among men undergoing GC for prostate cancer (PCA). We conducted a targeted analysis of men undergoing PCA GC to assess adherence to PA recommendations. METHODS: Using a cross-sectional design, a total of 158 men from the Genetic Evaluation of Men (GEM) study at two academic cancer centers with a diagnosis or at risk for PCA completed a structured lifestyle survey, including questions about the number of days and intensity of PA over the past year. One-sample t tests assessed adherence of participants to PA recommendations. Chi-square analyses compared differences in PA adherence by PCA status, aggressiveness, family history, and body mass index. Logistic regression analyses identified predictors of PA adherence. RESULTS: High proportions of GEM participants were overweight (44.9%) or obese (38.0%, p = 0.002). Men with PCA engaged in less moderate (p = 0.019) and vigorous (p = 0.005) aerobic activity than men without PCA. Higher education was predictive of adherence to light (p = 0.008), moderate (p = 0.019), and vigorous (p = 0.002) intensity PA. Older age (p = 0.015) and higher education (p = 0.001) were predictive of adherence to strength-based recommendations. CONCLUSIONS: High proportions of men receiving PCA GC were overweight/obese and lacked adherence to PA recommendations. GC represents a teachable moment to address PA to reduce cancer risk and promote cancer survivorship.

Serial MRI Findings After Endoscopic Removal of Button Battery From the Esophagus.

OBJECTIVE. The purpose of this study was to evaluate findings at serial MRI after endoscopic removal of a button battery from the esophagus in a series of pediatric patients. MATERIALS AND METHODS. Serial MRI examinations after removal of a button battery from the esophagus were reviewed retrospectively for the presence of mediastinal edema; imaging characteristics of the aorta and arteries; imaging characteristics of the trachea; and imaging characteristics of the esophageal wall at the level of injury. RESULTS. A total of 48 MRI examinations were performed on 19 patients, 89% (17/19) in the first 48 hours after battery removal. Serial MRI was performed for 84% (16/19) of patients. Initial MRI showed extensive mediastinal edema in all 17 patients who underwent MRI in the first 48 hours. Edema directly abutted major arteries in all 17 patients and abutted the airway in all 10 patients with proximal esophageal injury. Arterial vascular changes were seen in 30% (3/10) of patients with proximal esophageal injury and 57% (4/7) of patients with mid or distalesophageal injury. Airway changes were seen in 80% (8/10) of patients with proximal esophageal injury. Serial MRI showed improvement of airway changes in all patients and improvement in vessel wall changes in all but one (25%, 1/4) of the patients who had mid or distal esophageal injury. Four patients (21% [4/19]) had contained esophageal leak on esophagrams. No patients in our series developed a tracheoesophageal or vascular-enteric fistula. CONCLUSION. Our case series provides important information on natural history of MRI findings in children after endoscopic removal of a button battery from the esophagus. Further studies are needed to determine the imaging findings most sensitive and specific for severe complications, such as tracheoesophageal fistula and vascular-enteric fistula.

Human Immunodeficiency Virus (HIV)-Infected CCR6+ Rectal CD4+ T Cells and HIV Persistence On Antiretroviral Therapy.

BACKGROUND: Identifying where human immunodeficiency virus (HIV) persists in people living with HIV and receiving antiretroviral therapy is critical to develop cure strategies. We assessed the relationship of HIV persistence to expression of chemokine receptors and their chemokines in blood (n = 48) and in rectal (n = 20) and lymph node (LN; n = 8) tissue collected from people living with HIV who were receiving suppressive antiretroviral therapy. METHODS: Cell-associated integrated HIV DNA, unspliced HIV RNA, and chemokine messenger RNA were quantified by quantitative polymerase chain reaction. Chemokine receptor expression on CD4+ T cells was determined using flow cytometry. RESULTS: Integrated HIV DNA levels in CD4+ T cells, CCR6+CXCR3+ memory CD4+ T-cell frequency, and CCL20 expression (ligand for CCR6) were highest in rectal tissue, where HIV-infected CCR6+ T cells accounted for nearly all infected cells (median, 89.7%). Conversely in LN tissue, CCR6+ T cells were infrequent, and there was a statistically significant association of cell-associated HIV DNA and RNA with CCL19, CCL21, and CXCL13 chemokines. CONCLUSIONS: HIV-infected CCR6+ CD4+ T cells accounted for the majority of infected cells in rectal tissue. The different relationships between HIV persistence and T-cell subsets and chemokines in rectal and LN tissue suggest that different tissue-specific strategies may be required to eliminate HIV persistence and that assessment of biomarkers for HIV persistence may not be generalizable between blood and other tissues.

Delayed gastrointestinal-associated lymphoid tissue reconstitution in duodenum compared with rectum in HIV-infected patients initiating antiretroviral therapy.

BACKGROUND: We aimed to characterize the impact of antiretroviral therapy (ART) initiation on gastrointestinal-associated lymphoid tissue at various sites along the gastrointestinal site. METHODOLOGY: Peripheral blood and duodenal and rectal biopsies were obtained from 12 HIV to 33 treatment-naive HIV participants at baseline and after 9 months ART. Tissue was digested for immunophenotyping. Inflammatory, bacterial translocation and intestinal damage markers were measured in plasma. RESULTS: Twenty-six HIV patients completed follow-up. The lowest reconstitution of CD4 T cells and the lowest CD4/CD8 ratio during ART compared with blood were observed in the duodenum with the rectum being either intermediate or approaching blood levels. Regulatory T cells were in higher proportions in the duodenum than the rectum and neither declined significantly during ART. Several correlations with biomarkers of microbial translocation were observed including increases in lipoteichoic acid levels, which reflects Gram-positive bacterial translocation, correlated with increases in %CD4 T cells in the duodenum (Rho 0.773, P = 0.033), and with decreases in duodenal regulatory T-cell populations (Rho -0.40, P = 0.045). CONCLUSION: HIV-mediated immunological disruption is greater in the duodenum than rectum and blood before and during ART. Small intestine damage may represent a unique environment for T-cell depletion, which might be attenuated by interaction with Gram-positive bacteria.

p53 mutant-type in human prostate cancer cells determines the sensitivity to phenethyl isothiocyanate induced growth inhibition.

BACKGROUND: We reported previously that phenethyl isothiocyanate (PEITC), a dietary compound, can reactivate p53R175H mutant in vitro and in SK-BR-3 (p53R175H) breast xenograft model resulting in tumor inhibition. Because of the diversity of human cancers with p53 mutations, these findings raise important questions whether this mechanism operates in different cancer types with same or different p53 mutations. In this study, we investigated whether PEITC recuses mutant p53 in prostate cancer cells harboring different types of p53 mutants, structural and contact, in vitro and in vivo. METHODS: Cell proliferation, cell apoptosis and cell cycle arrest assays were performed to examine the effects of PEITC on prostate cancer cell lines with p53 mutation(s), wild-type p53, p53 null or normal prostate cells in vitro. Western blot analysis was used to monitor the expression levels of p53 protein, activation of ATM and upregulation of canonical p53 targets. Immunoprecipitation, subcellular protein fraction and qRT-PCR was performed to determine change in conformation and restoration of transactivation functions/ inhibition of gain-of-function (GOF) activities to p53 mutant(s). Mice xenograft models were established to evaluate the antitumor efficacy of PEITC and PEITC-induced reactivation of p53 mutant(s) in vivo. Immunohistochemistry of xenograft tumor tissues was performed to determine effects of PEITC on expression of Ki67 and mutant p53 in vivo. RESULTS: We demonstrated that PEITC inhibits the growth of prostate cancer cells with different "hotspot" p53 mutations (structural and contact), however, preferentially towards structural mutants. PEITC inhibits proliferation and induces apoptosis by rescuing mutant p53 in p53R248W contact (VCaP) and p53R175H structural (LAPC-4) mutant cells with differential potency. We further showed that PEITC inhibits the growth of DU145 cells that co-express p53P223L (structural) and p53V274F (contact) mutants by targeting p53P223L mutant selectively, but not p53V274F. The mutant p53 restored by PEITC induces apoptosis in DU145 cells by activating canonical p53 targets, delaying cells in G1 phase and phosphorylating ATM. Importantly, PEITC reactivated p53R175H and p53P223L/V274F mutants in LAPC-4 and DU145 prostate xenograft models, respectively, resulting in significant tumor inhibition. CONCLUSION: Our studies provide the first evidence that PEITC's anti-cancer activity is cancer cell type-independent, but p53 mutant-type dependent.

The effects of IFITM1 and IFITM3 gene deletion on IFNγ stimulated protein synthesis.

Interferon-induced transmembrane proteins IFITM1 and IFITM3 (IFITM1/3) play a role in both RNA viral restriction and in human cancer progression. Using immunohistochemical staining of FFPE tissue, we identified subgroups of cervical cancer patients where IFITM1/3 protein expression is inversely related to metastasis. Guide RNA-CAS9 methods were used to develop an isogenic IFITM1/IFITM3 double null cervical cancer model in order to define dominant pathways triggered by presence or absence of IFITM1/3 signalling. A pulse SILAC methodology identified IRF1, HLA-B, and ISG15 as the most dominating IFNγ inducible proteins whose synthesis was attenuated in the IFITM1/IFITM3 double-null cells. Conversely, SWATH-IP mass spectrometry of ectopically expressed SBP-tagged IFITM1 identified ISG15 and HLA-B as dominant co-associated proteins. ISG15ylation was attenuated in IFNγ treated IFITM1/IFITM3 double-null cells. Proximity ligation assays indicated that HLA-B can interact with IFITM1/3 proteins in parental SiHa cells. Cell surface expression of HLA-B was attenuated in IFNγ treated IFITM1/IFITM3 double-null cells. SWATH-MS proteomic screens in cells treated with IFITM1-targeted siRNA cells resulted in the attenuation of an interferon regulated protein subpopulation including MHC Class I molecules as well as IFITM3, STAT1, B2M, and ISG15. These data have implications for the function of IFITM1/3 in mediating IFNγ stimulated protein synthesis including ISG15ylation and MHC Class I production in cancer cells. The data together suggest that pro-metastatic growth associated with IFITM1/3 negative cervical cancers relates to attenuated expression of MHC Class I molecules that would support tumor immune escape.

Cryopreservation of human mucosal tissues.

BACKGROUND: Cryopreservation of leukocytes isolated from the cervicovaginal and colorectal mucosa is useful for the study of cellular immunity (see Hughes SM et al. PLOS ONE 2016). However, some questions about mucosal biology and sexually transmitted infections are better addressed with intact mucosal tissue, for which there is no standard cryopreservation protocol. METHODS AND FINDINGS: To find an optimal preservation protocol for mucosal tissues, we tested slow cooling (1°C/min) with 10% dimethylsulfoxide (designated "cryopreservation") and fast cooling (plunge in liquid nitrogen) with 20% dimethylsulfoxide and 20% ethylene glycol ("vitrification"). We compared fresh and preserved human cervicovaginal and colorectal tissues in a range of assays, including metabolic activity, human immunodeficiency virus infection, cell phenotype, tissue structure by hematoxylin-and-eosin staining, cell number and viability, production of cytokines, and microbicide drug concentrations. Metabolic activity, HIV infectability, and tissue structure were similar in cryopreserved and vitrified vaginal tissues. However, vitrification led to poor cell recovery from the colorectal mucosa, with 90% fewer cells recovered after isolation from vitrified colorectal tissues than from cryopreserved. HIV infection rates were similar for fresh and cryopreserved ectocervical tissues, whereas cryopreserved colorectal tissues were less easily infected than fresh tissues (hazard ratio 0.7 [95% confidence interval 0.4, 1.2]). Finally, we compared isolation of cells before and after cryopreservation. Cell recoveries were higher when cells were isolated after freezing and thawing (71% [59-84%]) than before (50% [38-62%]). Cellular function was similar to fresh tissue in both cases. Microbicide drug concentrations were lower in cryopreserved explants compared to fresh ones. CONCLUSIONS: Cryopreservation of intact cervicovaginal and colorectal tissues with dimethylsulfoxide works well in a range of assays, while the utility of vitrification is more limited. Cell yields are higher from cryopreserved intact tissue pieces than from thawed cryopreserved single cell suspensions isolated before freezing, but T cell functions are similar.

Hypomethylation of CYP2E1 and DUSP22 Promoters Associated With Disease Activity and Erosive Disease Among Rheumatoid Arthritis Patients.

OBJECTIVE: Epigenetic modifications have previously been associated with rheumatoid arthritis (RA). In this study, we aimed to determine whether differential DNA methylation in peripheral blood cell subpopulations is associated with any of 4 clinical outcomes among RA patients. METHODS: Peripheral blood samples were obtained from 63 patients in the University of California, San Francisco RA cohort (all satisfied the American College of Rheumatology classification criteria; 57 were seropositive for rheumatoid factor and/or anti-cyclic citrullinated protein). Fluorescence-activated cell sorting was used to separate the cells into 4 immune cell subpopulations (CD14+ monocytes, CD19+ B cells, CD4+ naive T cells, and CD4+ memory T cells) per individual, and 229 epigenome-wide DNA methylation profiles were generated using Illumina HumanMethylation450 BeadChips. Differentially methylated positions and regions associated with the Clinical Disease Activity Index score, erosive disease, RA Articular Damage score, Sharp score, medication at time of blood draw, smoking status, and disease duration were identified using robust regression models and empirical Bayes variance estimators. RESULTS: Differential methylation of CpG sites associated with clinical outcomes was observed in all 4 cell types. Hypomethylated regions in the CYP2E1 and DUSP22 gene promoters were associated with active and erosive disease, respectively. Pathway analyses suggested that the biologic mechanisms underlying each clinical outcome are cell type-specific. Evidence of independent effects on DNA methylation from smoking, medication use, and disease duration were also identified. CONCLUSION: Methylation signatures specific to RA clinical outcomes may have utility as biomarkers or predictors of exposure, disease progression, and disease severity.

Chronic stress is associated with reduced circulating hematopoietic progenitor cell number: A maternal caregiving model.

BACKGROUND: Chronic psychological stress is a risk factor for cardiovascular disease and mortality. Circulating hematopoietic progenitor cells (CPCs) maintain vascular homeostasis, correlate with preclinical atherosclerosis, and prospectively predict cardiovascular events. We hypothesize that (1) chronic caregiving stress is related to reduced CPC number, and (2) this may be explained in part by negative interactions within the family. METHODS: We investigated levels of stress and CPCs in 68 healthy mothers - 31 of these had children with an autism spectrum disorder (M-ASD) and 37 had neurotypical children (M-NT). Participants provided fasting blood samples, and CD45+CD34+KDR+ and CD45+CD133+KDR+ CPCs were assayed by flow cytometry. We averaged the blom-transformed scores of both CPCs to create one index. Participants completed the perceived stress scale (PSS), the inventory for depressive symptoms (IDS), and reported on daily interactions with their children and partners, averaged over 7 nights. RESULTS: M-ASD exhibited lower CPCs than M-NT (Cohen's d=0.83; p⩽0.01), controlling for age, BMI, and physical activity. Across the whole sample, positive interactions were related to higher CPCs, and negative interactions to lower CPCs (allp's<0.05). The adverse effects of group on CPCs were significantly mediated through negative interactions with the child (indirect ß=-0.24, p⩽0.01). In the full model, greater age (ß=-0.19, p=0.04), BMI (ß=-0.18, p=0.04), and negative interactions with the child (ß=-0.33, p<0.01) were independently associated with lower CPCs. M-ASD had a less healthy lipid profile (total cholesterol/HDL), which in turn, was associated with lower CPCs. CONCLUSIONS: Chronic stress adversely impacts CPC number, an early-stage biomarker that predicts subclinical atherosclerosis and future CVD events, independent of traditional cardiovascular risk factors and inflammatory factors. Among maternal caregivers, child-related interpersonal stress appears to be a key psychological predictor of stress-related CVD risk.

Nucleotide excision repair deficiency increases levels of acrolein-derived cyclic DNA adduct and sensitizes cells to apoptosis induced by docosahexaenoic acid and acrolein.

The acrolein derived cyclic 1,N(2)-propanodeoxyguanosine adduct (Acr-dG), formed primarily from ω-3 polyunsaturated fatty acids such as docosahexaenoic acid (DHA) under oxidative conditions, while proven to be mutagenic, is potentially involved in DHA-induced apoptosis. The latter may contribute to the chemopreventive effects of DHA. Previous studies have shown that the levels of Acr-dG are correlated with apoptosis induction in HT29 cells treated with DHA. Because Acr-dG is shown to be repaired by the nucleotide excision repair (NER) pathway, to further investigate the role of Acr-dG in apoptosis, in this study, NER-deficient XPA and its isogenic NER-proficient XAN1 cells were treated with DHA. The Acr-dG levels and apoptosis were sharply increased in XPA cells, but not in XAN1 cells when treated with 125µM of DHA. Because DHA can induce formation of various DNA damage, to specifically investigate the role of Acr-dG in apoptosis induction, we treated XPA knockdown HCT116+ch3 cells with acrolein. The levels of both Acr-dG and apoptosis induction increased significantly in the XPA knockdown cells. These results clearly demonstrate that NER deficiency induces higher levels of Acr-dG in cells treated with DHA or acrolein and sensitizes cells to undergo apoptosis in a correlative manner. Collectively, these results support that Acr-dG, a ubiquitously formed mutagenic oxidative DNA adduct, plays a role in DHA-induced apoptosis and suggest that it could serve as a biomarker for the cancer preventive effects of DHA.

Impact of Two Measures of Micrometastatic Disease on Clinical Outcomes in Patients with Newly Diagnosed Ewing Sarcoma: A Report from the Children's Oncology Group.

PURPOSE: Flow cytometry and RT-PCR can detect occult Ewing sarcoma cells in the blood and bone marrow. These techniques were used to evaluate the prognostic significance of micrometastatic disease in Ewing sarcoma. EXPERIMENTAL DESIGN: Newly diagnosed patients with Ewing sarcoma were enrolled on two prospective multicenter studies. In the flow cytometry cohort, patients were defined as "positive" for bone marrow micrometastatic disease if their CD99(+)/CD45(-) values were above the upper limit in 22 control patients. In the PCR cohort, RT-PCR on blood or bone marrow samples classified the patients as "positive" or "negative" for EWSR1/FLI1 translocations. The association between micrometastatic disease burden with clinical features and outcome was assessed. Coexpression of insulin-like growth factor-1 receptor (IGF-1R) on detected tumor cells was performed in a subset of flow cytometry samples. RESULTS: The median total bone marrow CD99(+)CD45(-) percent was 0.0012% (range 0%-1.10%) in the flow cytometry cohort, with 14 of 109 (12.8%) of Ewing sarcoma patients defined as "positive." In the PCR cohort, 19.6% (44/225) patients were "positive" for any EWSR1/FLI1 translocation in blood or bone marrow. There were no differences in baseline clinical features or event-free or overall survival between patients classified as "positive" versus "negative" by either method. CD99(+)CD45(-) cells had significantly higher IGF-1R expression compared with CD45(+) hematopoietic cells (mean geometric mean fluorescence intensity 982.7 vs. 190.9; P < 0.001). CONCLUSIONS: The detection of micrometastatic disease at initial diagnosis by flow cytometry or RT-PCR is not associated with outcome in newly diagnosed patients with Ewing sarcoma. Flow cytometry provides a tool to characterize occult micrometastatic tumor cells for proteins of interest. Clin Cancer Res; 22(14); 3643-50. ©2016 AACR.

Reproduction at the extremes: pseudovivipary, hybridization and genetic mosaicism in Posidonia australis (Posidoniaceae).

BACKGROUND AND AIMS: Organisms occupying the edges of natural geographical ranges usually survive at the extreme limits of their innate physiological tolerances. Extreme and prolonged fluctuations in environmental conditions, often associated with climate change and exacerbated at species' geographical range edges, are known to trigger alternative responses in reproduction. This study reports the first observations of adventitious inflorescence-derived plantlet formation in the marine angiosperm Posidonia australis, growing at the northern range edge (upper thermal and salinity tolerance) in Shark Bay, Western Australia. These novel plantlets are described and a combination of microsatellite DNA markers and flow cytometry is used to determine their origin. METHODS: Polymorphic microsatellite DNA markers were used to generate multilocus genotypes to determine the origin of the adventitious inflorescence-derived plantlets. Ploidy and genome size were estimated using flow cytometry. KEY RESULTS: All adventitious plantlets were genetically identical to the maternal plant and were therefore the product of a novel pseudoviviparous reproductive event. It was found that 87 % of the multilocus genotypes contained three alleles in at least one locus. Ploidy was identical in all sampled plants. The genome size (2 C value) for samples from Shark Bay and from a separate site much further south was not significantly different, implying they are the same ploidy level and ruling out a complete genome duplication (polyploidy). CONCLUSIONS: Survival at range edges often sees the development of novel responses in the struggle for survival and reproduction. This study documents a physiological response at the trailing edge, whereby reproductive strategy can adapt to fluctuating conditions and suggests that the lower-than-usual water temperature triggered unfertilized inflorescences to 'switch' to growing plantlets that were adventitious clones of their maternal parent. This may have important long-term implications as both genetic and ecological constraints may limit the ability to adapt or range-shift; this seagrass meadow in Shark Bay already has low genetic diversity, no sexual reproduction and no seedling recruitment.