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IntroductionThe function of the second receptor for the complement cleavage product C5a, C5aR2, is poorly understood and often neglected in the immunological context. Using mice with a global deficiency of C5aR2, we have previously reported an important role of this receptor in the pathogenesis of the neutrophil-driven autoimmune disease epidermolysis bullosa acquisita (EBA). Based on in vitro analyses, we hypothesized that the absence of C5aR2 specifically on neutrophils is the cause of the observed differences. Here, we report the generation of a new mouse line with a LysM-specific deficiency of C5aR2.MethodsLysM-specific deletion of C5aR2 was achieved by crossing LysMcre mice with tdTomato-C5ar2fl/fl mice in which the tdTomato-C5ar2 gene is flanked by loxP sites. Passive EBA was induced by subcutaneous injection of rabbit anti-mouse collagen type VII IgG. The effects of targeted deletion of C5ar2 on C5a-induced effector functions of neutrophils were examined in in vitro assays.ResultsWe confirm the successful deletion of C5aR2 at both the genetic and protein levels in neutrophils. The mice appeared healthy and the expression of C5aR1 in bone marrow and blood neutrophils was not negatively affected by LysM-specific deletion of C5aR2. Using the antibody transfer mouse model of EBA, we found that the absence of C5aR2 in LysM-positive cells resulted in an overall amelioration of disease progression, similar to what we had previously found in mice with global deficiency of C5aR2. Neutrophils lacking C5aR2 showed decreased activation after C5a stimulation and increased expression of the inhibitory Fcγ receptor FcγRIIb.DiscussionOverall, with the data presented here, we confirm and extend our previous findings and show that C5aR2 in neutrophils regulates their activation and function in response to C5a by potentially affecting the expression of Fcγ receptors and CD11b. Thus, C5aR2 regulates the finely tuned interaction network between immune complexes, Fcγ receptors, CD11b, and C5aR1 that is important for neutrophil recruitment and sustained activation. This underscores the importance of C5aR2 in the pathogenesis of neutrophil-mediated autoimmune diseases.

XLS file - 14K, Transcripts corresponding to 40 probe sets were differentially expressed between mammary glands of the E2 treated ACI and ACI.BN-Emca8 rats

XLS file - 12K, Markers Used in Generating ACI.BN-RNO5 Congenic Rat Strains

PDF file - 56K, Rat strain specific expression of Wnt4, Cd52 and Tspan1

The ACI rat model of 17β-estradiol (E2)-induced mammary cancer has gained wide use in the study of breast cancer etiology, prevention, and genetics. Emca8, a QTL that determines susceptibility to E2-induced mammary cancer, was previously mapped to rat chromosome 5 (RNO5) in an intercross between resistant Brown Norway (BN) and susceptible ACI rats. In this study, a panel of congenic rat strains, each of which carries BN alleles across a defined segment of RNO5 on the ACI genetic background, was generated and used to map more precisely the Emca8 determinants of mammary cancer susceptibility. Three distinct genetic determinants were localized within Emca8, and two of these were mapped to intervals of less than 15 megabases. Emca8.1 harbors Cdkn2a, Cdkn2b, and other genes and is orthologous to the 9p21 breast cancer locus identified in genome-wide and candidate gene association studies. Emca8.2 harbors Cdkn2c and other genes and is orthologous to the 1p32 locus in humans that is frequently deleted in breast cancers. Both Emca8.1 and Emca8.2 harbor copy number variants that are orthologous to copy number variant regions in humans. Gene expression profiles were defined for mammary tissues from E2-treated ACI and ACI.BN-Emca8 rats to define the impact of Emca8 on gene expression and identify differentially expressed genes residing within Emca8.1 and Emca8.2. This study further illustrates the relevance of the ACI rat model of E2-induced mammary cancer for identifying novel genetic determinants of breast cancer susceptibility and defining the mechanisms through which estrogens contribute to breast cancer development. Cancer Prev Res; 6(1); 59–69. ©2012 AACR.

Fiber photometry (FP) is an adaptable method for recording in vivo neural activity in freely behaving animals. It has become a popular tool in neuroscience due to its ease of use, low cost, the ability to combine FP with freely moving behavior, among other advantages. However, analysis of FP data can be a challenge for new users, especially those with a limited programming background. Here, we present Guided Photometry Analysis in Python (GuPPy), a free and open-source FP analysis tool. GuPPy is provided as a Jupyter notebook, a well-commented interactive development environment (IDE) designed to operate across platforms. GuPPy presents the user with a set of graphic user interfaces (GUIs) to load data and provide input parameters. Graphs produced by GuPPy can be exported into various image formats for integration into scientific figures. As an open-source tool, GuPPy can be modified by users with knowledge of Python to fit their specific needs.

Lipopolysaccharide (LPS) is a bacterial endotoxin present in cigarette smoke. LPS is known to induce inflammation and to increase the size and the multiplicity of lung tumors induced by tobacco-specific nitrosamines. However, the means by which LPS contributes to pulmonary carcinogenesis are not known. One possible mechanism includes LPS-mediated epigenetic deregulation, which leads to aberrant expression of genes involved in DNA repair, tumor suppression, cell cycle progression, and cell growth. In the present work, epigenetic effects of LPS were examined in alveolar type II lung cells of A/J mice. Type II cells were selected because they serve as progenitors of lung adenocarcinomas in smoking induced lung cancer. A/J mice were intranasally treated with LPS, followed by isolation of alveolar type II cells from the lung using cell panning. Global levels of DNA methylation and histone acetylation were quantified by mass spectrometry, while genome-wide transcriptomic changes were characterized by RNA-Seq. LPS treatment was associated with epigenetic changes including decreased cytosine formylation and reduced histone H3K14 and H3K23 acetylation, as well as altered expression levels of genes involved in cell adhesion, inflammation, immune response, and epigenetic regulation. These results suggest that exposure to inflammatory agents in cigarette smoke leads to early epigenetic changes in the lung, which may collaborate with genetic changes to drive the development of lung cancer.

MINI: We compared administrative data and National Surgical Quality Improvement Program data regarding the presence of 12 patient-level comorbidities. The level of disagreement between these data sources raises concerns regarding their ability to generate an accurate/unbiased estimate of patient-level risk. Objective To characterize agreement between administrative and registry data in the determination of patient-level comorbidities. Background Previous research finds poor agreement between these 2 types of data in the determination of outcomes. We hypothesized that concordance between administrative and registry data would also be poor. Methods A cohort of inpatient operations (length of stay 1 day or greater) was obtained from a consortium of 8 hospitals. Within each hospital, National Surgical Quality Improvement Program (NSQIP) data were merged with intra-institutional inpatient administrative data. Twelve different comorbidities (diabetes, hypertension, congestive heart failure, hemodialysis-dependence, cancer diagnosis, chronic obstructive pulmonary disease, ascites, sepsis, smoking, steroid, congestive heart failure, acute renal failure, and dyspnea) were analyzed in terms of agreement between administrative and NSQIP data. Results Forty-one thousand four hundred thirty-two inpatient surgical hospitalizations were analyzed in this study. Concordance (Cohen Kappa value) between the 2 data sources varied from 0.79 (diabetes) to 0.02 (dyspnea). Hospital variation in concordance (intersite variation) was quantified using a test of homogeneity. This test found significant intersite variation at a level of P Conclusion This study finds significant differences in how administrative versus registry data assess patient-level comorbidity. These differences are of concern to patients, payers, and providers, each of which had a stake in the integrity of these data. Standardized definitions of comorbidity and periodic audits are necessary to ensure data accuracy and minimize bias.

BACKGROUND We recently demonstrated that the odds of contracting coronavirus disease 2019 (COVID-19) in patients with celiac disease (CeD) is similar to that of the general population. However, how patients with CeD perceive their COVID-19 risk may differ from their actual risk. AIM To investigate risk perceptions of contracting COVID-19 in patients with CeD and determine the factors that may influence their perception. METHODS We distributed a survey throughout 10 countries between March and June 2020 and collected data on demographics, diet, COVID-19 testing, and risk perceptions of COVID-19 in patients with CeD. Participants were recruited through various celiac associations, clinic visits, and social media. Risk perception was assessed by asking individuals whether they believe patients with CeD are at an increased risk of contracting COVID-19 when compared to the general population. Logistic regression was used to determine the influencing factors associated with COVID-19 risk perception, such as age, sex, adherence to a gluten-free diet (GFD), and comorbidities such as cardiac conditions, respiratory conditions, and diabetes. Data was presented as adjusted odds ratios (aORs) RESULTS A total of 10737 participants with CeD completed the survey. From them, 6019 (56.1%) patients with CeD perceived they were at a higher risk or were unsure if they were at a higher risk of contracting COVID-19 compared to the non-CeD population. A greater proportion of patients with CeD perceived an increased risk of contracting COVID-19 when compared to infections in general due to their CeD (56.1% vs 26.7%, P < 0.0001). Consequently, 34.8% reported taking extra COVID-19 precautions as a result of their CeD. Members of celiac associations were less likely to perceive an increased risk of COVID-19 when compared to non-members (49.5% vs 57.4%, P < 0.0001). Older age (aOR: 0.99; 95%CI: 0.99 to 0.99, P < 0.001), male sex (aOR: 0.84; 95%CI: 0.76 to 0.93, P = 0.001), and strict adherence to a GFD (aOR: 0.89; 95%CI: 0.82 to 0.96, P = 0.007) were associated with a lower perception of COVID-19 risk and the presence of comorbidities was associated with a higher perception of COVID-19 risk (aOR: 1.38; 95%CI: 1.22 to 1.54, P < 0.001). CONCLUSION Overall, high levels of risk perceptions, such as those found in patients with CeD, may increase an individual's pandemic-related stress and contribute to negative mental health consequences. Therefore, it is encouraged that public health officials maintain consistent communication with the public and healthcare providers with the celiac community. Future studies specifically evaluating mental health in CeD could help determine the consequences of increased risk perceptions in this population.

The World Health Organization declared coronavirus disease-2019 (COVID-19) a global pandemic in March 2020. Since then, there are more than 34 million cases of COVID-19 leading to more than 1 million deaths worldwide. Numerous studies suggest that celiac disease (CeD), a chronic immune-mediated gastrointestinal condition triggered by gluten, is associated with an increased risk of respiratory infections.(1-3) However, how it relates to the risk of COVID-19 is unknown. To address this gap, we conducted a cross-sectional study to evaluate whether patients with self-reported CeD are at an increased risk of contracting COVID-19.

BackgroundResidents of plain communities constitute an underserved minority population that is not reliably captured in contemporary surgical outcomes research. We hypothesized that plain communities (PC) patients would have higher postoperative complication rates than a general surgical population.MethodsA retrospective review of 30-day postoperative outcomes for PC patients compared to a majority (non-PC) matched patient population from September 2014 to March 2020 was performed. The primary outcome measure was any complication within 30 days of surgery.Results270 PC patients were matched with 493 non-PC patients. The 30-day complication rate was higher for the PC group (6.3% vs 3.7%, P = .09), though not statistically significant. There was significantly lower utilization of preventive care services, and postoperative follow-up among PC patients.DiscussionAlthough our regional PC surgical patient population utilized preventive and postoperative health care services less than the non-PC population, there was no statistically significant difference in overall 30-day postoperative morbidity or mortality.

Epigenetic dysregulation is hypothesized to play a role in the observed association between inflammatory bowel disease (IBD) and colon tumor development. In the present work, DNA methylome, hydroxymethylome, and transcriptome analyses were conducted in proximal colon tissues harvested from the Helicobacter hepaticus (H. hepaticus)-infected murine model of IBD. Reduced representation bisulfite sequencing (RRBS) and oxidative RRBS (oxRRBS) analyses identified 1606 differentially methylated regions (DMR) and 3011 differentially hydroxymethylated regions (DhMR). These DMR/DhMR overlapped with genes that are associated with gastrointestinal disease, inflammatory disease, and cancer. RNA-seq revealed pronounced expression changes of a number of genes associated with inflammation and cancer. Several genes including Duox2, Tgm2, Cdhr5, and Hk2 exhibited changes in both DNA methylation/hydroxymethylation and gene expression levels. Overall, our results suggest that chronic inflammation triggers changes in methylation and hydroxymethylation patterns in the genome, altering the expression of key tumorigenesis genes and potentially contributing to the initiation of colorectal cancer.

Introduction Many patients with celiac disease (CD) experience persistent symptoms despite adhering to the gluten-free diet. Different studies have assessed the use of probiotics as an adjuvant treatment for CD. We performed a systematic review and meta-analysis to evaluate the efficacy of probiotics in improving gastrointestinal (GI) symptoms and quality of life (QOL) in patients with CD. Methods We searched EMBASE, MEDLINE, CINAHL, Web of Science, CENTRAL, and DARE databases up to February 2019 for randomized controlled trials (RCTs) evaluating probiotics compared with placebo for treating CD. We collected data on GI symptoms, QOL, adverse events, serum tumor necrosis factor-α, intestinal permeability, and microbiota composition. Results We screened 2,831 records and found that 7 articles describing 6 RCTs (n = 279 participants) were eligible for quantitative analysis. Probiotics improved GI symptoms when assessed by the GI Symptoms Rating Scale (mean difference symptom reduction: -28.7%; 95% confidence interval [CI] -43.96 to -13.52; P = 0.0002). There was no difference in GI symptoms after probiotics when different questionnaires were pooled. The levels of Bifidobacteria increased after probiotics (mean difference: 0.85 log colony-forming units (CFU) per gram; 95% CI 0.38-1.32 log CFU per gram; P = 0.0003). There were insufficient data on tumor necrosis factor-a levels or QOL for probiotics compared with placebo. No difference in adverse events was observed between probiotics and placebo. The overall certainty of the evidence ranged from very low to low. Discussion Probiotics may improve GI symptoms in patients with CD. High-quality clinical trials are needed to improve the certainty in the evidence (see Visual abstract, Supplementary Digital Content 2, http://links.lww.com/AJG/B595).

Ubiquitin-like, containing PHD and RING finger domains 2 (UHRF2) regulates cell cycle and binds 5-hydroxymethylcytosine (5hmC) to promote completion of DNA demethylation. Uhrf2−/− mice are without gross phenotypic defects; however, the cell cycle and epigenetic regulatory functions of Uhrf2 during retinal tissue development are unclear. Retinal progenitor cells (RPCs) produce all retinal neurons and Müller glia in a predictable sequence controlled by the complex interplay between extrinsic signaling, cell cycle, epigenetic changes and cell-specific transcription factor activation. In this study, we find that UHRF2 accumulates in RPCs, and its conditional deletion from mouse RPCs reduced 5hmC, altered gene expressions and disrupted retinal cell proliferation and differentiation. Retinal ganglion cells were overproduced in Uhrf2-deficient retinae at the expense of VSX2+ RPCs. Most other cell types were transiently delayed in differentiation. Expression of each member of the Tet3/Uhrf2/Tdg active demethylation pathway was reduced in Uhrf2-deficient retinae, consistent with locally reduced 5hmC in their gene bodies. This study highlights a novel role of UHRF2 in controlling the transition from RPCs to differentiated cell by regulating cell cycle, epigenetic and gene expression decisions.

SUMMARYCompulsive behavior is a defining feature of disorders such as substance use disorder and obsessive-compulsive disorder. Current evidence suggests that corticostriatal circuits control the expression of established compulsions, but little is known about the mechanisms regulating the development of compulsions. We hypothesized that dopamine, a critical modulator of striatal synaptic plasticity, could control alterations in corticostriatal circuits leading to the development of compulsions (defined as continued reward-seeking in the face of punishment). We used dual-site fiber photometry to measure dopamine axon activity in the dorsomedial striatum (DMS) and the dorsolateral striatum (DLS) as compulsions emerged. Individual variability in the speed with which compulsions emerged was predicted by DMS dopamine axon activity. Amplifying this dopamine signal accelerated animals’ transitions to compulsion, whereas inhibition led to learning delays. In contrast, amplifying DLS dopamine signaling had no effect on the emergence of compulsions. These results establish DMS dopamine signaling as a key controller of the development of compulsive reward-seeking.

Background Celiac disease is a chronic autoimmune disorder triggered by dietary gluten. The only available treatment for celiac disease is strict compliance to the gluten-free diet. Despite adherence to a gluten-free diet, many celiac patients still experience symptoms which may be mediated by the gut microbiota. Different studies have assessed the use of probiotics as an adjuvant treatment for celiac disease. Aims We performed a systematic review and meta-analysis to evaluate the efficacy of probiotics in improving gastrointestinal symptoms and quality of life in patients with celiac disease. Methods We searched MEDLINE (1966 to February 2019), EMBASE (1974 to February 2019), CINAHL, Web of Science, CENTRAL, DARE, and Sigle up to February 2019 for randomized controlled trials (RCTs) assessing the effects of probiotics on celiac disease. We collected data on gastrointestinal symptoms, quality of life, adverse events, serum tumor necrosis factor α (TNF-α), intestinal permeability, and microbiota composition. Results Out of 2831 records screened, 7 articles from 6 RCTs with a total of 279 participants were eligible for quantitative analysis. Probiotic supplementation for a minimum of 2 weeks improved gastrointestinal symptoms when assessed by the Gastrointestinal Symptoms Rating Scale (GSRS) (2 studies; 131 participants; mean difference (MD) symptom reduction: -28.7%; 95% CI -43.96 to -13.52; P=0.0002). No differences in gastrointestinal symptoms after probiotics were observed when pooling data from the GSRS, Celiac Symptoms Index (CSI), and the Celiac Disease Questionnaire (CDQ) (4 studies; 197 participants; standardized mean difference (SMD) symptom reduction: -0.48; 95% CI -1.21 to +0.25; P=0.20). TNF-α levels did not change after probiotics (SMD: -0.52; 95% CI -1.08 to +0.04; P=0.07). Levels of Bifidobacteria species increased (MD: increase by 0.85 log CFU/g; 95% CI +0.38 to +1.32 log CFU/g; P=0.0003) but there was no difference in Lactobacilli species (MD: change of 1.13 log CFU/g; 95% CI -0.67 to +2.93 log CFU/g; P=0.22) after probiotic supplementation. No difference in quality of life or adverse events was observed after probiotics. There was insufficient evidence to assess the effects of probiotics on intestinal permeability. The overall quality of the evidence ranged from very low to low. Conclusions Probiotics may improve gastrointestinal symptoms in patients with celiac disease; however, our confidence in these results is limited by the low quality of the evidence. Further large clinical trials with a more rigorous design are needed to improve the quality of the evidence. Funding Agencies None

Background & Aims There is controversy over the association between celiac disease (CeD) and inflammatory bowel diseases (IBD). We performed a systematic review and meta-analysis to assess evidence for an association between CeD and IBD. Methods We searched databases including MEDLINE, EMBASE, CENTRAL, Web of Science, CINAHL, DARE, and SIGLE through June 25, 2019 for studies assessing the risk of CeD in patients with IBD, and IBD in patients with CeD, compared with controls of any type. We used the Newcastle-Ottawa Scale to evaluate the risk of bias and GRADE to assess the certainty of the evidence. Results We identified 9791 studies and included 65 studies in our analysis. Moderate certainty evidence found an increased risk of CeD in patients with IBD vs controls (risk ratio [RR] 3.96; 95% confidence interval [CI] 2.23–7.02) and increased risk of IBD in patients with CeD vs controls (RR 9.88; 95% CI 4.03–24.21). There was low-certainty evidence for the risk of anti-Saccharomyces antibodies, a serologic marker of IBD, in patients with CeD vs controls (RR 6.22; 95% CI 2.44–15.84). There was low-certainty evidence for no difference in risk of HLA-DQ2 or DQ8 in patients with IBD vs controls (RR 1.04; 95% CI 0.42–2.56), and very low-certainty evidence for an increased risk of anti-tissue transglutaminase in patients with IBD vs controls (RR 1.52; 95% CI 0.52–4.40). Patients with IBD had a slight decrease in risk of anti-endomysial antibodies vs controls (RR 0.70; 95% CI 0.18–2.74), but these results are uncertain. Conclusions In a systematic review and meta-analysis, we found an increased risk of IBD in patients with CeD and increased risk of CeD in patients with IBD, compared with other patient populations. High-quality prospective cohort studies are needed to assess the risk of CeD-specific and IBD-specific biomarkers in patients with IBD and CeD.

Compulsive behavior is a defining feature of disorders such as substance use disorders. Current evidence suggests that corticostriatal circuits control the expression of established compulsions, but little is known about the mechanisms regulating the development of compulsions. We hypothesized that dopamine, a critical modulator of striatal synaptic plasticity, could control alterations in corticostriatal circuits leading to the development of compulsions (defined here as continued reward seeking in the face of punishment). We used dual-site fiber photometry to measure dopamine axon activity in the dorsomedial striatum (DMS) and the dorsolateral striatum (DLS) as compulsions emerged. Individual variability in the speed with which compulsions emerged was predicted by DMS dopamine axon activity. Amplifying this dopamine signal accelerated animals' transitions to compulsion, whereas inhibition delayed it. In contrast, amplifying DLS dopamine signaling had no effect on the emergence of compulsions. These results establish DMS dopamine signaling as a key controller of the development of compulsive reward seeking.

Total Knee Arthroplasty (TKA, also known as total knee replacement) is a highly effective surgical intervention for the restoration of physical function and improving quality of life in patients with disabling knee osteoarthritis. Recent data suggest that obesity is a major risk factor in the pathogenesis and progression of knee osteoarthritis, with increases in body mass index (BMI, kg/m2) directly correlating with the prevalence of knee osteoarthritis. However, recent data also suggest that there are increased risks associated with TKAs when performed in patients with morbid obesity (BMI > 40 kg/m2). Patients with morbid obesity are routinely referred for weight management prior to surgery. Many of these patients fail to meet the recommended weight loss goals prior to TKA, potentially making them ineligible for surgery or placing them at increased risk for sub-optimal outcomes. Thus, the purpose of this study is to examine the potential therapeutic impact and long-term outcomes of surgically induced weight loss on TKA outcomes. Specifically, these outcomes will include measures of physical function, mobility, and indices of joint function at 1 and 2 years post-TKA compared between extremely obese patients who undergo TKA (Control group, n = 150) and those with TKA performed ~1 year after bariatric surgery (Test group, n = 150). An additional primary endpoint will be the percent of bariatric patients that negate or delay the need for TKA. Secondary endpoints include perioperative outcomes after TKA.

Background Previous studies have documented significant differences between administrative data and registry data in the determination of postoperative venous thromboembolism (VTE). The goal of this study was to characterize the discordance between administrative and registry data in the determination of postoperative VTE. Study Design This study was performed using data from the American College of Surgeons NSQIP merged with administrative data from 8 different hospitals (5 different medical centers) between 2013 and 2015. Occurrences of postoperative vein thrombosis (VT) and pulmonary embolism (PE) as ascertained by administrative data and NSQIP data were compared. In each situation where the 2 sources disagreed (discordance), a 2-clinician chart review was performed to characterize the reasons for discordance. Results The cohort used for analysis included 43,336 patients, of which 53.3% were female and the mean age was 59.5 years. Concordance between administrative and NSQIP data was worse for VT (κ 0.57; 95% CI 0.51 to 0.62) than for PE (κ 0.83; 95% CI 0.78 to 0.89). A total of 136 cases of discordance were noted in the assessment of VT; of these, 50 (37%) were explained by differences in the criteria used by administrative vs NSQIP systems. In the assessment of postoperative PE, administrative data had a higher accuracy than NSQIP data (odds ratio for accuracy 2.86; 95% CI 1.11 to 7.14) when compared with the 2-clinician chart review. Conclusions This study identifies significant problems in ability of both NSQIP and administrative data to assess postoperative VT/PE. Administrative data functioned more accurately than NSQIP data in the identification of postoperative PE. The mechanisms used to translate VTE measurement into quality improvement should be standardized and improved.

5-Methylcytosine (MeC) is an endogenous modification of DNA that plays a crucial role in DNA-protein interactions, chromatin structure, epigenetic regulation, and DNA repair. MeC is produced via enzymatic methylation of the C-5 position of cytosine by DNA-methyltransferases (DNMT) which use S-adenosylmethionine (SAM) as a cofactor. Hemimethylated CG dinucleotides generated as a result of DNA replication are specifically recognized and methylated by maintenance DNA methyltransferase 1 (DNMT1). The accuracy of DNMT1-mediated methylation is essential for preserving tissue-specific DNA methylation and thus gene expression patterns. In the present study, we synthesized DNA duplexes containing MeC analogues with modified C-5 side chains and examined their ability to guide cytosine methylation by the human DNMT1 protein. We found that the ability of 5-alkylcytosines to direct cytosine methylation decreased with increased alkyl chain length and rigidity (methyl > ethyl > propyl ∼ vinyl). Molecular modeling studies indicated that this loss of activity may be caused by the distorted geometry of the DNA-protein complex in the presence of unnatural alkylcytosines.

5-Formylcytosine (5fC) is an endogenous DNA modification frequently found within regulatory elements of mammalian genes. Although 5fC is an oxidation product of 5-methylcytosine (5mC), the two epigenetic marks show distinct genome-wide distributions and protein affinities, suggesting that they perform different functions in epigenetic signaling. A unique feature of 5fC is the presence of a potentially reactive aldehyde group in its structure. Here, we show that 5fC bases in DNA readily form Schiff base conjugates with Lys side chains of nuclear proteins in vitro and in vivo. These covalent protein-DNA complexes are reversible (t1/2, 1.8 h), suggesting that they contribute to transcriptional regulation and chromatin remodeling. On the other hand, 5fC mediated DNA-protein cross-links, if present at replication forks or actively transcribed regions, may interfere with DNA replication and transcription.

Thymoquinone (TQ), a bioactive constituent of the volatile oil of Monarda fistulosa and Nigella sativa, possesses cancer-specific growth inhibitory effects, but the underlying molecular mechanisms remain largely elusive. We propose that TQ curbs cancer cell growth through dysfunction of DNA methyltransferase 1 (DNMT1). Molecular docking analysis revealed that TQ might interact with the catalytic pocket of DNMT1 and compete with co-factor SAM/SAH for DNMT1 inhibition. In vitro inhibitory assays showed that TQ decreases DNMT1 methylation activity in a dose-dependent manner with an apparent IC50 of 30 nM. Further, exposure of leukemia cell lines and patient primary cells to TQ resulted in DNMT1 downregulation, mechanistically, through dissociation of Sp1/NFkB complex from DNMT1 promoter. This led to a reduction of DNA methylation, a decrease of colony formation and an increase of cell apoptosis via the activation of caspases. In addition, we developed and validated a sensitive and specific LC-MS/MS method and successfully detected a dynamic change of TQ in mouse plasma after administration of TQ through the tail vein, and determined a tolerable dose of TQ to be 15 mg/kg in mouse. TQ administration into leukemia-bearing mice induced leukemia regression, as indicated by the reversed splenomegaly and the inhibited leukemia cell growth in lungs and livers. Our study for the first time demonstrates that DNMT1-dependent DNA methylation mediates the anticancer actions of TQ, opening a window to develop TQ as a novel DNA hypomethylating agent for leukemia therapy.

We investigate whether customers' overall impression of online property listings can be influenced by the real estate agent, and whether this influence depends on the customer's demographic characteristics. A sample of 1,594 potential homebuyers took an online audio/visual tour of a typically priced home in their area. Subjects were shown one of eight conditions in which we varied agent gender (male/female), agent attractiveness (attractive / less attractive), and pathos (used/not used). The results show that segments of customers are drawn to different real estate agents, but contrary to our expectations, customers were not necessarily drawn to similar agents or more attractive ones.

Background Patients with type 1 Diabetes Mellitus (T1DM) often suffer with gastrointestinal (GI) symptoms, such as abdominal pain, bloating, early satiety, nausea and vomiting. T1DM patients are at a higher risk to develop celiac disease, and those patient with both disorders benefit from a gluten-free diet (GFD). However, it is unknown whether GFD has any benefit in patients with T1DM without celiac disease who present with upper GI symptoms. Aims To investigate the role of GFD in the management of moderate to severe dyspeptic symptoms in non-celiac patients with T1DM. Methods We enrolled adult T1DM patients, in whom celiac disease was ruled out by serology and/or endoscopy, suffering with two or more of upper GI symptoms. The patients were instructed to undergo a strict GFD for a period of 1 month, under supervision of a dietitian. Glycemic levels were monitored by a continuous glucose monitoring device (CGM) for 2 weeks before, and for 2 weeks at the end of the GFD period. Upper GI symptoms, general quality of life, anxiety and depression were assessed using standardized questionnaires. Blood samples were collected to assess glycaemia (Hb1Ac) and lipid profiles. Scintigraphy and videofluoroscopy were used to assess gastric emptying. Results Seven patients finished the study so far. They reported a significant improvement in nausea (p Conclusions One month of GFD improved dyspepsia-like symptoms, general quality of life and anxiety levels in T1D patients without concomitant celiac disease. GFD also improved the blood glucose management of patients with frequent hypoglycemia. Thus, this dietary intervention appears to improve upper GI symptoms in T1D patients but the results need to be replicated in a larger patient cohort. Funding Agencies CIHR

Epigenetic marks of DNA such as 5-methylcytosine control the levels of gene expression in cells and tissues by altering DNA structure, inhibiting transcription factor binding, and recruiting chromatin-modifying enzymes. DNA methylation marks are introduced in both strands of DNA by DNA methyltransferases, allowing for heritable gene silencing. In addition to 5-methylcytosine, cells contain its oxidized forms 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxylcytosine, as well as recently discovered DNA marks, 4-methylcytosine and 6-methyladenosine. Structural identification, quantitation, and mapping of DNA epigenetic marks are critical for our understanding of epigenetic control in healthy cells and to allow for insight into epigenetic deregulation in human disease and the efficacy of epigenetic therapies. This review focuses on experimental techniques and methodologies that can be used to study epigenetic modifications of DNA, including methods for quantifying global levels of epigenetically modified nucleosides and sequencing techniques for mapping the locations of various epigenetic marks along the genome, leading to better understanding of their biological functions and dynamics.

Poly(ethylene glycol)–poly(vinylphosphonic acid) block copolymers (PEG-b-PVPA) of low average molar mass were synthesized by RAFT/MADIX polymerization. Their intrinsic properties (i.e. anionic-neutral character which brings both ability to strongly interact with cations and steric stabilization) allow the one-step in situ synthesis of water-dispersible iron oxide nanoparticles with controlled size and high colloidal stability. The effect of structural properties of the polymer (chain length of PEG and PVPA blocks) on the formation and/or stabilization of nanoparticles has been evaluated. Additionally, PVPA and PAA block complexing abilities were compared and the advantage of phosphonic acid groups over carboxylic acid groups was proven. Hemocompatibility tests carried out with the DHBCs alone and DHBC/iron oxide hybrid nanoparticles revealed that the hemolysis rates observed with phosphonated copolymers were much lower than for their carboxylated analogs.

A precise balance of DNA methylation and demethylation is required for epigenetic control of cell identity, development, and growth. DNA methylation marks are introduced by de novo DNA methyltransferases DNMT3a/b and are maintained throughout cell divisions by DNA methyltransferase 1 (DNMT1), which adds methyl groups to hemimethylated CpG dinucleotides generated during DNA replication. Ten eleven translocation (TET) dioxygenases oxidize 5-methylcytosine (mC) to 5-hydroxymethylcytosine (hmC), 5-formylcytosine (fC), and 5-carboxylcytosine (caC), a process known to induce DNA demethylation and gene reactivation. In this study, we investigated the catalytic activity of human DNMT1 in the presence of oxidized forms of mC. A mass spectrometry-based assay was employed to study the kinetics of DNMT1-mediated cytosine methylation in CG dinucleotides containing C, mC, hmC, fC, or caC across from the target cytosine. Homology modeling, coupled with molecular dynamics simulations, was used to explore the structural consequences of mC oxidation with regard to the geometry of protein-DNA complexes. The DNMT1 enzymatic activity was strongly affected by the oxidation status of mC, with the catalytic efficiency decreasing in the following order: mC > hmC > fC > caC. Molecular dynamics simulations revealed that DNMT1 forms an unproductive complex with DNA duplexes containing oxidized forms of mC as a consequence of altered interactions of the target recognition domain of the protein with the C-5 substituent on cytosine. Our results provide new structural and mechanistic insight into TET-mediated DNA demethylation.

Reactive oxygen and nitrogen species generated during respiration, inflammation, and immune response can damage cellular DNA, contributing to aging, cancer, and neurodegeneration. The ability of oxidized DNA bases to interfere with DNA replication and transcription is strongly influenced by their chemical structures and locations within the genome. In the present work, we examined the influence of local DNA sequence context, DNA secondary structure, and oxidant identity on the efficiency and the chemistry of guanine oxidation in the context of the Kras protooncogene. A novel isotope labeling strategy developed in our laboratory was used to accurately map the formation of 2,2-diamino-4-[(2-deoxy-β-D-erythropentofuranosyl)amino]− 5(2 H)-oxazolone (Z), 8-oxo-7,8-dihydro-2'-deoxyguanosine (OG), and 8-nitroguanine (8-NO2-G) lesions along DNA duplexes following photooxidation in the presence of riboflavin, treatment with nitrosoperoxycarbonate, and oxidation in the presence of hydroxyl radicals. Riboflavin-mediated photooxidation preferentially induced OG lesions at 5' guanines within GG repeats, while treatment with nitrosoperoxycarbonate targeted 3'-guanines within G G and A G dinucleotides. Little sequence selectivity was observed following hydroxyl radical-mediated oxidation. However, Z and 8-NO2-G adducts were overproduced at duplex ends, irrespective of oxidant identity. Overall, our results indicate that the patterns of Z, OG, and 8-NO2-G adduct formation in the genome are distinct and are influenced by oxidant identity and the secondary structure of DNA.

BACKGROUND: Patients with inflammatory bowel disease (IBD) frequently suffer from psychiatric comorbidities, mainly anxiety and depression. Infliximab, an anti-TNFα antibody, is commonly used for treatment of colitis. There are anecdotal reports of IBD patients having improvement in mood only hours after infliximab infusion, well before intestinal mucosal healing could occur. AIMS: To evaluate the immediate effects of infliximab infusion on intestinal and extra-intestinal symptoms and immune markers in IBD patients. METHODS: IBD patients attending the McMaster University and Charlton infusion clinics were enrolled in the study. Patients were examined at baseline (day 0), 1 day, and 7 days after the infliximab infusion. We assessed anxiety and depression (HAD and STAI-Y scales), disease specific quality of life (SIDBQ), as well as disease activity scores using the Harvey Bradshaw Index (HBI) or the Mayo Score. Serum C-reactive protein (CRP), kynurenine, tryptophan, and serotonin levels were measured at each visit. RESULTS: We enrolled 41 IBD patients (Crohn’s disease = 35, Ulcerative Colitis = 5, indeterminate colitis = 1; median age 33 yrs, median disease duration 9 yrs). Disease activity decreased at day 1 compared to baseline (p=0.01), but this was not sustained at day 7. Anxiety scores (HADS-A) decreased significantly at day 1 (p=0.006), and remained lower at day 7 (p=0.009). An improvement in quality of life (SIBDQ) was seen only at day 7 (p=0.017). The decrease in anxiety scores strongly correlated with the improvement in quality of life (SIBDQ r=-0.81; p

Background: Traumatic brain injury (TBI) commonly presents with a constellation of findings, including physical, cognitive, and psychological disturbances. Traditionally, few options have been available for long-term management of mood symptoms for post-TBI patients who are refractory to medications. Repetitive transcranial magnetic stimulation (rTMS) is a promising alternative treatment.Case Report: A 37-year-old male with history of anxiety and bipolar depression incurred a TBI after a 60-foot fall. After his physical recovery, the patient had refractory depression. Eight months after the TBI event, he underwent a 6-week course of rTMS treatment and had a 70.8% improvement in mood symptoms by the end of the therapy as indicated by the Patient Health Questionnaire-9, transitioning from severe to mild depression according to the scoring system. Clinical correlation during the months following conclusion of rTMS therapy showed no signs of remission or adverse side effects. The patient remains stable and lives independently 1 year after treatment with mood-stabilizing medications.Conclusion: This case provides evidence for successful treatment of refractory depressive symptoms after severe TBI with the addition of rTMS to psychotherapy and mood-stabilizing medications, supporting the safety and tolerability of this novel therapeutic approach. Further studies are needed to validate the contribution of rTMS for management of mood symptoms in patients with TBI.

This study is the first to examine the widely debated merits of staging a home for sale. We find that both homeowners and real estate agents believe staging conditions (furnishings and wall color) will significantly impact homeowners’ willingness to pay for a property. Our results show that homeowners rationally do not significantly differ in their valuations based on staging conditions. However, staging conditions do influence the process, as we find a neutral wall color and good furnishings do significantly influence a buyer’s perceived livability and overall opinion of the home. While these are a necessary condition for purchase, staging is not enough to result in a higher selling price.

Background Previous studies have documented significant differences between administrative data and registry data in the determination of postoperative MI. The goal of this study was to characterize discordance between administrative and registry data in the determination of postoperative myocardial infarction (MI). Study Design This study was performed using data from the American College of Surgeons NSQIP merged with administrative data from 8 different hospitals, between 2013 and 2015. From each of these sources, the occurrence of a postoperative MI, as ascertained by administrative data and NSQIP data, were compared. In each situation in which the 2 sources disagreed (discordance), a 2-clinician chart review was performed to generate a "gold standard" determination as to the occurrence of postoperative MI. Results A total of 43,289 operations met our inclusion criteria for analysis. Within this cohort a total of 230 cases of MI were identified by administrative data and/or NSQIP data (administrative rate 0.41%, NSQIP rate 0.42%). A total of 89 discordant ascertainments were identified, of which 42 were admin+/NSQIP- and 47 were admin-/NSQIP+. Accuracy (99.9% for both) and concordance (kappa = 0.89 [95% CI 0.86 to 0.92] for administrative data, kappa = 0.87 [95% CI 0.84 to 0.91] for NSQIP data) of the 2 systems were similar when compared against our gold standard (chart review). The majority of errors were related to false negatives, with sensitivity rates of 81% in both data sources. Conclusions In this multi-institutional study, administrative data and NSQIP demonstrated a similar ability to determine the occurrence of postoperative MI. These findings do not demonstrate an advantage of registry data over administrative data in the determination of postoperative MI.

In contrast with northern Australia and Thailand, in Singapore the incidence of melioidosis and co-incidence of melioidosis and pneumonia have declined. Burkholderia pseudomallei deep abscesses increased 20.4% during 2003–2014. These trends could not be explained by the environmental and climatic factors conventionally ascribed to melioidosis.