Arthur Berg

Electronic cigarettes (e-cigs): views of aficionados and clinical/public health perspectives

Background:  Electronic cigarettes (e‐cigs) have experienced a rapid growth in popularity but little is known about how they are used.

Social disadvantage, genetic sensitivity, and children's telomere length

Significance This paper makes two contributions to research on the link between the social environment and health. Using data from a birth cohort study, we show that, among African American boys, those who grow up in highly disadvantaged environments have shorter telomeres (at age 9) than boys who grow up in highly advantaged environments. We also find that the association between the social environment and telomere length (TL) is moderated by genetic variation within the serotonin and dopamine pathways. Boys with the highest genetic sensitivity scores had the shortest TL when exposed to disadvantaged environments and the longest TL when exposed to advantaged environments. To our knowledge, this report is the first to document a gene–social environment interaction for TL, a biomarker of stress exposure. Disadvantaged social environments are associated with adverse health outcomes. This has been attributed, in part, to chronic stress. Telomere length (TL) has been used as a biomarker of chronic stress: TL is shorter in adults in a variety of contexts, including disadvantaged social standing and depression. We use data from 40, 9-y-old boys participating in the Fragile Families and Child Wellbeing Study to extend this observation to African American children. We report that exposure to disadvantaged environments is associated with reduced TL by age 9 y. We document significant associations between low income, low maternal education, unstable family structure, and harsh parenting and TL. These effects were moderated by genetic variants in serotonergic and dopaminergic pathways. Consistent with the differential susceptibility hypothesis, subjects with the highest genetic sensitivity scores had the shortest TL when exposed to disadvantaged social environments and the longest TL when exposed to advantaged environments.

REDD1 enhances protein phosphatase 2A-mediated dephosphorylation of Akt to repress mTORC1 signaling.

REDD1 targets a phosphatase to the kinase Akt, thereby changing the substrate specificity of Akt and inhibiting mTORC1 activity. Altering Kinase Specificity to Limit Cell Growth Metabolic signals are coupled to pathways that mediate cellular growth and proliferation through mTORC1, a complex consisting of the kinase mechanistic target of rapamycin (mTOR) and the regulatory component raptor. Insulin and other growth factors activate the kinase Akt, which then phosphorylates and suppresses the activity of a complex that inhibits mTORC1. Several mechanisms have been proposed to explain how mTORC1 signaling is inhibited by REDD1 (regulated in DNA damage and development 1), a stress-inducible protein. Dennis et al. found that REDD1 acted as a targeting unit for a phosphatase that dephosphorylated Akt at a specific site. Once dephosphorylated by this REDD1-phosphatase complex, Akt had different substrate specificity and did not phosphorylate and inactivate the mTORC1 inhibitor. By changing the substrate specificity of Akt, REDD1 could potentially affect the activity of other signaling pathways in which Akt participates. The protein kinase mTOR (mechanistic target of rapamycin) in complex 1 (mTORC1) promotes cell growth and proliferation in response to anabolic stimuli, including growth factors and nutrients. Growth factors activate mTORC1 by stimulating the kinase Akt, which phosphorylates and inhibits the tuberous sclerosis complex [TSC; which is composed of TSC1, TSC2, and TBC1D7 (Tre2-Bub2-Cdc16 domain family member 7)], thereby stimulating the mTORC1 activator Rheb (Ras homolog enriched in brain). We identified the mechanism through which REDD1 (regulated in DNA damage and development 1) represses the mTORC1 signaling pathway. We found that REDD1 promoted the protein phosphatase 2A (PP2A)–dependent dephosphorylation of Akt on Thr308 but not on Ser473. Consistent with previous studies showing that phosphorylation of Akt on Thr308, but not on Ser473, is necessary for phosphorylation of TSC2, we observed a REDD1-dependent reduction in the phosphorylation of TSC2 and subsequently in the activation state of Rheb. REDD1 and PP2A coimmunoprecipitated with Akt from wild-type but not REDD1 knockout mouse embryonic fibroblasts, suggesting that REDD1 may act as a targeting protein for the catalytic subunit of PP2A. Furthermore, binding to both Akt and PP2A was essential for REDD1 to repress signaling to mTORC1. Overall, the results demonstrate that REDD1 acts not only as a repressor of mTORC1 but also as a constant modulator of the phosphorylation of Akt in response to growth factors and nutrients.

Risk Factors for Surgical Site Infections after Colorectal Resection in Diabetic Patients

BACKGROUND Surgical site infections (SSIs) are a well known complication of gastrointestinal surgery and associated with an increased morbidity, mortality and overall cost. Diabetes mellitus (DM) is a risk factor for SSI. However, there is no clear consensus as to which other risk factors play a significant role. The goal of this study was to identify risk factors associated with SSI in patients with DM undergoing colorectal resection. STUDY DESIGN A retrospective review was conducted of DM patients who underwent colorectal resection from June 2000 to June 2009 at Milton S Hershey Medical Center, Division of Colorectal Surgery. Individual measures were analyzed using chi-square, t-test, and Mann-Whitney U tests, and statistical significance was confirmed using a multiple logistical regression model. RESULTS There were 183 DM patients included in the study, 28 (15%) of whom developed SSI. Glucose levels were significantly higher in the SSI group for each time interval, 0 to 6 hours (211 mg/dL, p = 0.03), 0 to 48 hours (176 mg/dL, p = 0.001), and 48 to 96 hours (167 mg/dL, p = 0.012) postoperatively. Other measures significantly associated with SSI included the use of drains (p = 0.05) and the use of prophylactic antibiotics for more than 24 hours (p = 0.02). Body mass index and stoma creation approached statistical significance (p = 0.08, 0.07, respectively). The type of hypoglycemic regimen, immunosuppression, and emergency surgery were not associated with an increased rate of SSI. CONCLUSIONS Higher than normal glucose control at all postoperative time intervals was associated with SSI. The majority of glucose levels were below the American Diabetes Association recommended level of 200 mg/dL, but patients still developed SSI. Type of perioperative glucose control did not affect the incidence of SSI. These data suggest that DM patients undergoing colectomy should have glucose tightly controlled, avoid placement of drains, and receive antibiotics for less than 24 hours.

Diversification in the genetic architecture of gene expression and transcriptional networks in organ differentiation of Populus

A fundamental goal of systems biology is to identify genetic elements that contribute to complex phenotypes and to understand how they interact in networks predictive of system response to genetic variation. Few studies in plants have developed such networks, and none have examined their conservation among functionally specialized organs. Here we used genetical genomics in an interspecific hybrid population of the model hardwood plant Populus to uncover transcriptional networks in xylem, leaves, and roots. Pleiotropic eQTL hotspots were detected and used to construct coexpression networks a posteriori, for which regulators were predicted based on cis-acting expression regulation. Networks were shown to be enriched for groups of genes that function in biologically coherent processes and for cis-acting promoter motifs with known roles in regulating common groups of genes. When contrasted among xylem, leaves, and roots, transcriptional networks were frequently conserved in composition, but almost invariably regulated by different loci. Similarly, the genetic architecture of gene expression regulation is highly diversified among plant organs, with less than one-third of genes with eQTL detected in two organs being regulated by the same locus. However, colocalization in eQTL position increases to 50% when they are detected in all three organs, suggesting conservation in the genetic regulation is a function of ubiquitous expression. Genes conserved in their genetic regulation among all organs are primarily cis regulated (~92%), whereas genes with eQTL in only one organ are largely trans regulated. Trans-acting regulation may therefore be the primary driver of differentiation in function between plant organs.

Focused, high accuracy 5-methylcytosine quantitation with base resolution by benchtop next-generation sequencing

BackgroundThe growing interest in the role of epigenetic modifications in human health and disease has led to the development of next-generation sequencing methods for whole genome analysis of DNA methylation patterns. However, many projects require targeted methylation analysis of specific genes or genomic regions. We have developed an approach, termed BiSulfite Amplicon Sequencing (BSAS), for hypothesis driven and focused absolute DNA methylation analysis. This approach is applicable both to targeted DNA methylation studies as well as to confirmation of genome-wide studies.ResultsBSAS uses PCR enrichment of targeted regions from bisulfite-converted DNA and transposome-mediated library construction for rapid generation of sequencing libraries from low (1 ng) sample input. Libraries are sequenced using the Illumina MiSeq benchtop sequencer. Generating high levels of sequencing depth (>1,000 ×) provides for quantitatively precise and accurate assessment of DNA methylation levels with base specificity. Dual indexing of sequencing libraries allows for simultaneous analysis of up to 96 samples. We demonstrate the superior quantitative accuracy of this approach as compared to existing Sanger sequencing methods.ConclusionsBSAS can be applied to any genomic region from any DNA source, including tissue and cell culture. Thus, BSAS provides a new validation approach for rapid and highly quantitative absolute CpG methylation analysis of any targeted genomic regions in a high throughput manner.

NOD2/CARD15 Mutations Correlate With Severe Pouchitis After Ileal Pouch-Anal Anastomosis

PURPOSE: Pouchitis and Crohns-like complications can plague patients after IPAA. NOD2 is an intracellular sensor for bacterial cell wall peptidoglycan. NOD2 mutations compromise host response to enteric bacteria and are increased in Crohns disease. We hypothesize that IPAA patients with complications (Crohns disease-like/pouchitis) have a higher rate of NOD2 mutations compared with asymptomatic IPAA patients. METHODS: Patients were retrospectively subclassified into the following groups: 1) IPAA with Crohns-like complications (n = 28, perianal fistula, pouch inlet stricture/upstream small-bowel disease, or biopsies showing granulomata) occurring at least 6 months after ileostomy closure; 2) IPAA with mild pouchitis (n = 33, ≤3 episodes/y for 2 consecutive years); 3) IPAA with severe pouchitis (n = 9, ≥4 episodes/y for 2 consecutive years or need for continuous antibiotics); 4) IPAA without complications or pouchitis (n = 37); 5) patients with Crohns disease with colitis undergoing total proctocolectomy/ileostomy (n = 11); and 6) healthy controls (n = 269). The 3 NOD2 single-nucleotide polymorphism mutations (rs2066844, rs2066845, and rs2066847) previously identified as associated with Crohns disease were genotyped using polymerase chain reaction. Groups were compared by use of &khgr;2 with Yates continuity correction. RESULTS: NOD2 mutations were found in 8.5% of healthy controls. NOD2 mutations were significantly higher in the severe pouchitis group (67%) compared with both asymptomatic IPAA (5.4%, P < .001) and IPAA with Crohns disease-like complications (14.3%, P = .008) groups. CONCLUSIONS: 1) Asymptomatic IPAA patients have a low incidence of NOD2 mutations not significantly different from patients with mild pouchitis or healthy controls. 2) Patients with severe pouchitis had the highest incidence of NOD2 mutations, suggesting that this group may have a compromised host defense mechanism to enteric bacteria. 3) Patients with Crohns-like complications after IPAA have a significantly lower incidence of NOD2 mutations than patients with severe pouchitis, suggesting a different genetic makeup in these 2 patient groups. Preoperative assessment of NOD2 in the equivocal IPAA candidate may predict severe pouchitis and might assist in preoperative surgical decision making.

Circulating Tumor Cells in Melanoma Patients

Circulating tumor cells (CTCs) are of recognized importance for diagnosis and prognosis of cancer patients. With melanoma, most studies do not show any clear relationship between CTC levels and stage of disease. Here, CTCs were enriched (∼400X) from blood of melanoma patients using a simple centrifugation device (OncoQuick), and 4 melanocyte target RNAs (TYR, MLANA, MITF, and MIF) were quantified using QPCR. Approximately one-third of melanoma patients had elevated MIF and MLANA transcripts (p<0.0001 and p<0.001, respectively) compared with healthy controls. In contrast, healthy controls had uniformly higher levels of TYR and MITF than melanoma patients (p<0.0001). There was a marked shift of leukocytes into the CTC-enriched fractions (a 430% increase in RNA recovery, p<0.001), and no relationship between CTC levels and stage of disease was found. CTCs were captured on microfabricated filters and cultured. Captured melanoma CTCs were large cells, and consisted of 2 subpopulations, based on immunoreactivity. One subpopulation (∼50%) stained for both pan-cytokeratin (KRT) markers and the common leukocyte marker CD-45, whereas the second subpopulation stained for only KRT. Since similar cells are described in many cancers, we also examined blood from colorectal and pancreatic cancer patients. We observed analogous results, with most captured CTCs staining for both CD-45/KRT markers (and for the monocyte differentiation marker CD-14). Our results suggest that immature melanocyte-related cells (expressing TYR and MITF RNA) may circulate in healthy controls, although they are not readily detectable without considerable enrichment. Further, as early-stage melanomas develop, immature melanocyte migration into the blood is somehow curtailed, whereas a significant proportion of patients develop elevated CTC levels (based on MIF and MLANA RNAs). The nature of the captured CTCs is consistent with literature describing leukocyte/macrophage-tumor cell fusion hybrids, and their role in metastatic progression.

Plasma netrin-1 is a diagnostic biomarker of human cancers

Context: The axon guidance cues netrin-1 is a secreted protein overexpressed in many different cancer tissues. Objectives: To determine whether plasma netrin-1 can be used as a diagnostic biomarker of human cancer. Materials and Methods: A total of 300 cancer plasma samples from breast, renal, prostate, liver, meningioma, pituitary adenoma, glioblastoma, lung, pancreatic and colon cancer patients were compared against 138 control plasma samples. Netrin-1 levels were quantified by ELISA and immunohistochemistry. Results: Plasma netrin-1 levels were significantly increased in breast, renal, prostate, liver, meningioma, pituitary adenoma, and glioblastoma cancers as compared to control samples. Discussion and Conclusion: Our results suggest that plasma netrin-1 can be used as a diagnostic biomarker for many human cancers.

Mapping genes for plant structure, development and evolution: functional mapping meets ontology

One of the fundamental tasks in biology is the identification of genes that control the structure and developmental pattern of complex traits and their responses to the environment during trait development. Functional mapping provides a statistical means for detecting quantitative trait loci (QTLs) that underlie developmental traits, such as growth trajectories, and for testing the interplay between gene action and development. Here we describe how functional mapping and studies of plant ontology can be integrated so as to elucidate the expression mechanisms of QTLs that control plant growth, morphology, development, and adaptation to changing environments. This approach can also be used to construct an evo-devo framework for inferring the evolution of developmental traits.