Family-Based Telehealth Initiative to Improve Nutrition and Physical Activity for Children With Obesity and Its Utility During COVID-19: A Mixed Methods Evaluation.

Guidelines recommend primary care providers refer children with obesity to behavioral interventions, but given limited program availability, access, and parental engagement, referrals remain rare. We developed telehealth coaching interventions for families whose children received care at a health system in Pennsylvania, United States in 2019-2020. Intervention referrals were facilitated by the pediatrician and/or project team for 6-12-year-old children with obesity following well-child visits. Participants chose one of three 26-week interventions focused on healthy eating, physical activity, or a hybrid clinical/nutrition intervention. Interventions engaged parents as change agents, enhancing self-efficacy to model and reinforce behavior and providing resources to help create a healthy home environment. We enrolled 77 of 183 eligible parent/child dyads. We used mixed methods to evaluate the interventions. Repeated measures models among participants showed significant reductions in obesogenic nutrition behaviors post-intervention and at 1-year follow-up, including a reduction in sugar-sweetened beverage intake of 2.14 servings/week (95% confidence interval: -3.45, -0.82). There were also improvements in obesoprotective nutrition behaviors (e.g., frequency of family meals, parental self-efficacy related to meal management). One year post-baseline, we observed no significant differences in changes in body mass index (BMI) z-scores comparing child participants with matched controls. Given potential impacts of COVID-19 community restrictions on study outcomes, we conducted qualitative interviews with 13 participants during restrictions, which exemplified how disrupted routines constrained children's healthy behaviors but that intervention participation prepared parents by providing cooking and physical activities at home. Findings support the potential of a telehealth-delivered nutrition intervention to support adoption of healthy weight behaviors.

UHRF2 regulates cell cycle, epigenetics and gene expression to control the timing of retinal progenitor and ganglion cell differentiation.

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.

Communicating personalized risk of diabetes and offering weight reduction program choice: Recruitment, participation, and outcomes.

OBJECTIVE: Low patient recruitment into diabetes prevention programs is a challenge. The primary aim of this study was to demonstrate that an increased recruitment rate can be achieved by communicating personalized risk of progression to type 2 diabetes, estimating risk reduction with weight loss, and offering program choice. Secondary aims included program participation rate, weight loss, and short-term decreased diabetes risk. METHODS: In this single-arm study, persons with prediabetes from 3 primary care sites received a letter that communicated their personalized risk of progression to diabetes within 3-years, estimated risk reduction with 5, 10, 15 % weight loss, reported in pounds, and offered a choice of 5 free, 6-month, programs. A one-sided test was used to compare the recruitment rate against the maximum expected rate of (10 %). RESULTS: Recruitment response rate was 25.3 % (81/328, 95 % CI=[20.0 %, 29.4 %]) which was significantly higher than expected (p < 0.0001). Overall, 65 % of participants completed >75 % of contacts. BMI, HbA1c, and diabetes risk (all p < 0.0001) improved at 6 months; BMI (p < 0.0001) and HbA1c (p < 0.05) improved at 12 months. CONCLUSION: Recruitment response rate was better than expected. PRACTICE IMPLICATIONS: Communicating personalized risk and reduction estimates with a choice of programs resulted in favorable outcomes, sustained at 1-year.

Advanced Health Information Technologies to Engage Parents, Clinicians, and Community Nutritionists in Coordinating Responsive Parenting Care: Descriptive Case Series of the Women, Infants, and Children Enhancements to Early Healthy Lifestyles for Baby (WEE Baby) Care Randomized Controlled Trial.

BACKGROUND: Socioeconomically disadvantaged newborns receive care from primary care providers (PCPs) and Women, Infants, and Children (WIC) nutritionists. However, care is not coordinated between these settings, which can result in conflicting messages. Stakeholders support an integrated approach that coordinates services between settings with care tailored to patient-centered needs. OBJECTIVE: This analysis describes the usability of advanced health information technologies aiming to engage parents in self-reporting parenting practices, integrate data into electronic health records to inform and facilitate documentation of provided responsive parenting (RP) care, and share data between settings to create opportunities to coordinate care between PCPs and WIC nutritionists. METHODS: Parents and newborns (dyads) who were eligible for WIC care and received pediatric care in a single health system were recruited and randomized to a RP intervention or control group. For the 6-month intervention, electronic systems were created to facilitate documentation, data sharing, and coordination of provided RP care. Prior to PCP visits, parents were prompted to respond to the Early Healthy Lifestyles (EHL) self-assessment tool to capture current RP practices. Responses were integrated into the electronic health record and shared with WIC. Documentation of RP care and an 80-character, free-text comment were shared between WIC and PCPs. A care coordination opportunity existed when the dyad attended a WIC visit and these data were available from the PCP, and vice versa. Care coordination was demonstrated when WIC or PCPs interacted with data and documented RP care provided at the visit. RESULTS: Dyads (N=131) attended 459 PCP (3.5, SD 1.0 per dyad) and 296 WIC (2.3, SD 1.0 per dyad) visits. Parents completed the EHL tool prior to 53.2% (244/459) of PCP visits (1.9, SD 1.2 per dyad), PCPs documented provided RP care at 35.3% (162/459) of visits, and data were shared with WIC following 100% (459/459) of PCP visits. A WIC visit followed a PCP visit 50.3% (231/459) of the time; thus, there were 1.8 (SD 0.8 per dyad) PCP to WIC care coordination opportunities. WIC coordinated care by documenting RP care at 66.7% (154/231) of opportunities (1.2, SD 0.9 per dyad). WIC visits were followed by a PCP visit 58.9% (116/197) of the time; thus, there were 0.9 (SD 0.8 per dyad) WIC to PCP care coordination opportunities. PCPs coordinated care by documenting RP care at 44.0% (51/116) of opportunities (0.4, SD 0.6 per dyad). CONCLUSIONS: Results support the usability of advanced health information technology strategies to collect patient-reported data and share these data between multiple providers. Although PCPs and WIC shared data, WIC nutritionists were more likely to use data and document RP care to coordinate care than PCPs. Variability in timing, sequence, and frequency of visits underscores the need for flexibility in pragmatic studies. TRIAL REGISTRATION: ClinicalTrials.gov NCT03482908; https://clinicaltrials.gov/ct2/show/NCT03482908. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): RR2-10.1186/s12887-018-1263-z.

Inhalation exposure to cigarette smoke and inflammatory agents induces epigenetic changes in the lung.

Smoking-related lung tumors are characterized by profound epigenetic changes including scrambled patterns of DNA methylation, deregulated histone acetylation, altered gene expression levels, distorted microRNA profiles, and a global loss of cytosine hydroxymethylation marks. Here, we employed an enhanced version of bisulfite sequencing (RRBS/oxRRBS) followed by next generation sequencing to separately map DNA epigenetic marks 5-methyl-dC and 5-hydroxymethyl-dC in genomic DNA isolated from lungs of A/J mice exposed whole-body to environmental cigarette smoke for 10 weeks. Exposure to cigarette smoke significantly affected the patterns of cytosine methylation and hydroxymethylation in the lungs. Differentially hydroxymethylated regions were associated with inflammatory response/disease, organismal injury, and respiratory diseases and were involved in regulation of cellular development, function, growth, and proliferation. To identify epigenetic changes in the lung associated with exposure to tobacco carcinogens and inflammation, A/J mice were intranasally treated with the tobacco carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), the inflammatory agent lipopolysaccharide (LPS), or both. NNK alone caused minimal epigenetic alterations, while exposure either to LPS or NNK/LPS in combination led to increased levels of global cytosine methylation and formylation, reduced cytosine hydroxymethylation, decreased histone acetylation, and altered expression levels of multiple genes. Our results suggest that inflammatory processes are responsible for epigenetic changes contributing to lung cancer development.

Multi-Omics Characterization of Inflammatory Bowel Disease-Induced Hyperplasia/Dysplasia in the Rag2-/-/Il10-/- Mouse Model.

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.

Epigenetic Changes in Alveolar Type II Lung Cells of A/J Mice Following Intranasal Treatment with Lipopolysaccharide.

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.

Maintenance DNA Methyltransferase Activity in the Presence of Oxidized Forms of 5-Methylcytosine: Structural Basis for Ten Eleven Translocation-Mediated DNA Demethylation.

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.

Mapping three guanine oxidation products along DNA following exposure to three types of reactive oxygen species.

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-ß-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 GG and AG 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.

Surgical weight-loss to improve functional status trajectories following total knee arthroplasty: SWIFT trial: Rationale, design, and methods.

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.

Reversible DNA-Protein Cross-Linking at Epigenetic DNA Marks.

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. Herein, 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 exerts potent growth-suppressive activity on leukemia through DNA hypermethylation reversal in leukemia cells.

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.

Synthesis of DNA Oligodeoxynucleotides Containing Site-Specific 1,3-Butadiene-Deoxyadenosine Lesions.

Post-oligomerization synthesis is a useful technique for preparing site-specifically modified DNA oligomers. This approach involves site-specific incorporation of inherently reactive halogenated nucleobases into DNA strands using standard solid-phase synthesis, followed by post-oligomerization nucleophilic aromatic substitution (SNAr) reactions with carcinogen-derived synthons. In these reactions, the inherent reactivities of DNA and carcinogen-derived species are reversed: the modified DNA nucleobase acts as an electrophile, while the carcinogen-derived species acts as a nucleophile. In the present protocol, we describe the use of the post-oligomerization approach to prepare DNA strands containing site- and stereospecific N6-adenine and N1,N6-adenine adducts induced by epoxide metabolites of the known human and animal carcinogen 1,3-butadiene (BD). The resulting oligomers containing site-specific, structurally defined DNA adducts can be used in structural and biological studies to reveal the roles of specific BD adducts in carcinogenesis and mutagenesis.

Bisubstrate Inhibitors of Biotin Protein Ligase in Mycobacterium tuberculosis Resistant to Cyclonucleoside Formation.

Mycobacterium tuberculosis (Mtb), the etiological agent of tuberculosis, is the leading cause bacterial infectious diseases mortality. Biotin protein ligase (BirA) globally regulates lipid metabolism in Mtb through the posttranslational biotinylation of acyl coenzyme A carboxylases (ACCs) involved in lipid biosynthesis and is essential for Mtb survival. We previously developed a rationally designed bisubstrate inhibitor of BirA that displays potent enzyme inhibition and whole-cell activity against multidrug resistant and extensively drug resistant Mtb strains. Here we present the design, synthesis and evaluation of a focused series of inhibitors, which are resistant to cyclonucleoside formation, a key decomposition pathway of our initial analogue. Improved chemical stability is realized through replacement of the adenosyl N-3 nitrogen and C-5' oxygen atom with carbon as well as incorporation of bulky group on the nucleobase to prevent the required syn-conformation necessary for proper alignment of N-3 with C-5'.

Identification of new inhibitors for low molecular weight protein tyrosine phosphatase isoform B.

The National Cancer Institute Diversity Set II (1356 compounds) and Diversity Set III (1597 compounds) were screened via in silico methods as potential inhibitors of low molecular weight protein tyrosine phosphatase (LWM-PTP) isoform B (EC 3.1.3.48). Those candidates that demonstrated comparable or better docking scores than that of pyridoxal 5'-phosphate (PLP), one of the most potent known inhibitors of LMW-PTP with a competitive inhibitor dissociation constant (Kis) of 7.6µM (pH 5.0), were analyzed via in vitro kinetic assays against LMW-PTP isoform B. While none of the compounds tested in vitro was significantly better that PLP, five compounds showed comparable inhibition. These five compounds are very diverse in structure and represent new therapeutic leads for inhibition of this isozyme.

Origin of pancreatic ductal adenocarcinoma from atypical flat lesions: a comparative study in transgenic mice and human tissues.

Pancreatic ductal adenocarcinoma (PDAC) and its precursor lesions, pancreatic intraepithelial neoplasia (PanIN), display a ductal phenotype. However, there is evidence in genetically defined mouse models for PDAC harbouring a mutated kras under the control of a pancreas-specific promoter that ductal cancer might arise in the centroacinar-acinar region, possibly through a process of acinar-ductal metaplasia (ADM). In order to further elucidate this model of PDAC development, an extensive expression analysis and molecular characterization of the putative and already established (PanIN) precursor lesions were performed in the Kras(G12D/+) ; Ptf1a-Cre(ex1/+) mouse model and in human tissues, focusing on lineage markers, developmental pathways, cell cycle regulators, apomucins, and stromal activation markers. The results of this study show that areas of ADM are very frequent in the murine and human pancreas and represent regions of increased proliferation of cells with precursor potential. Moreover, atypical flat lesions originating in areas of ADM are the most probable precursors of PDAC in the Kras(G12D/+); Ptf1a-Cre(ex1/+) mice and similar lesions were also found in the pancreas of three patients with a strong family history of PDAC. In conclusion, PDAC development in Kras(G12D/+); Ptf1a-Cre(ex1/+) mice starts from ADM and a similar process might also take place in patients with a strong family history of PDAC.

Hypothetical progression model of pancreatic cancer with origin in the centroacinar-acinar compartment.

OBJECTIVES: Based mainly on animal models, 2 lesions have been suggested as possible precursors of pancreatic ductal adenocarcinoma (PDAC): pancreatic intraepithelial neoplasia (PanIN) and tubular complexes (TCs). The aim of the study was to find support for either of the 2 models through the analysis of a large panel of human pancreatic tissues. METHODS: Ninety-two PDAC, 45 chronic pancreatitis, and 27 serous cystadenoma cases were investigated using conventional morphology and immunohistochemistry. RESULTS: Most of the cases (78% of PDAC, 93% of chronic pancreatitis, and 67% of serous cystadenoma) exhibited putative precursor lesions, predominantly TC and low-grade PanIN lesions, often present in the same tissue area. High-grade lesions were exclusively observed in PDAC specimens. In 50% to 70% of the cases with TC and associated PanIN, a transitional zone of acinar-ductular transformation with mucinous differentiation of the ductular epithelium was identified. Expression of acinar and centroacinar markers was detected in TC, in the ductular structures of the transitional zones, as well as within the epithelium of mature PanINs. CONCLUSIONS: The results of the present study show that the coexistence of 2 different putative PDAC precursor lesions might not be a contradiction. A progression model that originates in the centroacinar-acinar compartment and ends with the development of PanIN lesions is suggested.