Characterization of a multiple epigenetic marker panel for lung cancer detection and risk assessment in plasma.
Methylation patterns may be useful biomarkers of cancer detection and risk assessment. The methylation status of 6 genes, including a candidate tumor suppressor gene (BLU), the cadherin 13 gene (CDH13), the fragile histidine triad gene (FHIT), the cell cycle control gene p16, the retinoic acid receptor beta gene (RARbeta), and the Ras association domain family 1 gene (RASSF1A), was examined in plasma samples, corresponding tumor tissues, and normal lung tissues from a group of 63 patients with lung cancer and in plasma samples from 36 cancer-free individuals. The detection rate of the p16 gene was validated in a test group of 20 patients with lung cancer. The concordance of methylation in tumor tissues and plasma samples was 86%, 87%, 80%, 75%, 76%, and 84% for the BLU, CDH13, FHIT, p16, RARbeta, and RASSF1A genes, respectively. The test group showed a similar concordance for p16 methylation detection. Multiple logistic regression analysis showed that the odds ratio for having lung cancer was 10.204 for individuals with p16 methylation (P = .013) and 9.952 for individuals with RASSFIA methylation (P = .019). After several trial tests, the authors established that methylation for >/=2 of the 6 markers met the criterion for an elevated risk of cancer. Comparisons yielded a sensitivity of 73%, a specificity of 82%, and a concordance of 75% between the methylation patterns in tumor tissues and in corresponding plasma samples. The detection rate was relatively high in cigarette smokers with advanced squamous cell lung cancer. The current results indicated that multiple epigenetic markers in the plasma, especially the p16 and RASSF1A genes, can be used for lung cancer detection. This methylation marker panel should improve the detection of cancer or the risk assessment for lung cancer in combination with conventional diagnostic tools.


Dihydralazine or ketanserin for severe hypertension in pregnancy? Preliminary results.
To compare the efficacy and safety of intravenous dihydralazine with ketanserin in the management of severe hypertension in the third trimester. A double blind randomised controlled trial, comparing 5 mg dihydralazine with 10 mg ketanserin after an intravenous infusion of 500 ml of a crystalloid solution. Medication was repeated every 20 min till the therapeutic goal of 90 mm Hg was reached, to a maximum of 4 dosages. Main outcome measures were treatment failures and emergency deliveries for fetal distress. The therapeutic goal was met more often in patients receiving dihydralazine (36/38 compared to 27/42; P < 0.01). The need for delivery for fetal distress did not differ (3 after dihydralazine, 1 after ketanserin, P = 0.29) No therapy related perinatal loss occurred, but one mother with an undiagnosed phaechromocytoma died 24 h after receiving dihydralazine. Ketanserin in this dosage is less effective to lower diastolic blood pressure. The place of a fluid load prior to dihydralazine needs to be further investigated, as fetal heart rate decelerations were less common than previously reported.


Do mammographic technologists affect radiologists' diagnostic mammography interpretative performance?
The purpose of this study was to determine whether the technologist has an effect on the radiologists' interpretative performance of diagnostic mammography. Using data from a community-based mammography registry from 1994 to 2009, we identified 162,755 diagnostic mammograms interpreted by 286 radiologists and performed by 303 mammographic technologists. We calculated sensitivity, false-positive rate, and positive predictive value (PPV) of the recommendation for biopsy from mammography for examinations performed (i.e., images acquired) by each mammographic technologist, separately for conventional (film-screen) and digital modalities. We assessed the variability of these performance measures among mammographic technologists, using mixed effects logistic regression and taking into account the clustering of examinations within women, radiologists, and radiology practices. Among the 291 technologists performing conventional examinations, mean sensitivity of the examinations performed was 83.0% (95% CI, 80.8-85.2%), mean false-positive rate was 8.5% (95% CI, 8.0-9.0%), and mean PPV of the recommendation for biopsy from mammography was 27.1% (95% CI, 24.8-29.4%). For the 45 technologists performing digital examinations, mean sensitivity of the examinations they performed was 79.6% (95% CI, 73.1-86.2%), mean false-positive rate was 8.8% (95% CI, 7.5-10.0%), and mean PPV of the recommendation for biopsy from mammography was 23.6% (95% CI, 18.8-28.4%). We found significant variation by technologist in the sensitivity, false-positive rate, and PPV of the recommendation for biopsy from mammography for conventional but not digital mammography (p < 0.0001 for all three interpretive performance measures). Our results suggest that the technologist has an influence on radiologists' interpretive performance for diagnostic conventional but not digital mammography. Future studies should examine why this difference between modalities exists and determine if similar patterns are observed for screening mammography.


Production and scale-up of a monoclonal antibody against 17-hydroxyprogesterone.
The hybridoma 192 was used to produce a monoclonal antibody (MAb) against 17-hydroxyprogesterone (17-OHP), for possible use in screening for congenital adrenal hyperplasia (CAH). The factors influencing the MAb production were screened and optimized in a 2 L stirred bioreactor. The production was then scaled up to a 20 L bioreactor. All of the screened factors (aeration rate, stirring speed, dissolved oxygen concentration, pH, and temperature) were found to significantly affect production. Optimization using the response surface methodology identified the following optimal production conditions: 36.8°C, pH 7.4, stirring speed of 100 rpm, 30% dissolved oxygen concentration, and an aeration rate of 0.09 vvm. Under these conditions, the maximum viable cell density achieved was 1.34 ± 0.21 × 10(6) cells mL(-1) and the specific growth rate was 0.036 ± 0.004 h(-1) . The maximum MAb titer was 11.94 ± 4.81 μg mL(-1) with an average specific MAb production rate of 0.273 ± 0.135 pg cell(-1) h(-1) . A constant impeller tip speed criterion was used for the scale-up. The specific growth rate (0.040 h(-1) ) and the maximum viable cell density (1.89 × 10(6) cells mL(-1) ) at the larger scale were better than the values achieved at the small scale, but the MAb titer in the 20 L bioreactor was 18% lower than in the smaller bioreactor. A change in the culture environment from the static conditions of a T-flask to the stirred bioreactor culture did not affect the specificity of the MAb toward its antigen (17-OHP) and did not compromise the structural integrity of the MAb.


Comparison of everolimus-eluting and sirolimus-eluting coronary stents: 1-year outcomes from the Randomized Evaluation of Sirolimus-eluting Versus Everolimus-eluting stent Trial (RESET).
Several recent randomized trials comparing everolimus-eluting stent (EES) and sirolimus-eluting stent (SES) reported similar outcomes. However, only 1 trial was powered for a clinical end point, and no trial was powered for evaluating target-lesion revascularization. Randomized Evaluation of Sirolimus-eluting versus Everolimus-eluting stent Trial is a prospective multicenter randomized open-label trial comparing EES with SES in Japan. The trial was powered for evaluating noninferiority of EES relative to SES in terms of target-lesion revascularization. From February and July 2010, 3197 patients were randomly assigned to receive either EES (1597 patients) or SES (1600 patients). At 1 year, the primary efficacy end point of target-lesion revascularization occurred in 65 patients (4.3%) in the EES group and in 76 patients (5.0%) in the SES group, demonstrating noninferiority of EES to SES (P(noninferiority)<0.0001, and P(superiority)=0.34). Cumulative incidence of definite stent thrombosis was low and similar between the 2 groups (0.32% versus 0.38%, P=0.77). An angiographic substudy enrolling 571 patients (EES, 285 patients and SES, 286 patients) demonstrated noninferiority of EES relative to SES regarding the primary angiographic end point of in-segment late loss (0.06±0.37 mm versus 0.02±0.46 mm, P(noninferiority)<0.0001, and P(superiority)=0.24) at 278±63 days after index stent implantation. One-year clinical and angiographic outcome after EES implantation was noninferior to and not different from that after SES implantation in a stable coronary artery disease population with relatively less complex coronary anatomy. One-year clinical outcome after both EES and SES use was excellent with a low rate of target-lesion revascularization and a very low rate of stent thrombosis. URL: http://www.clinicaltrials.gov. Unique identifier: NCT01035450.


Association of miRNA-145 with the occurrence and prognosis of hydrosalpinx-induced defective endometrial receptivity.
MiR-145 is reported to facilitate inflammation and is also associated with unsuccessful embryonic implantation. Whether miR-145 mediates inflammatory response underlying hydrosalpinx-induced defective endometrial receptivity (ER) remains unclear, and this study attempted to clarify this point. Endometrium samples were collected from hydrosalpinx patients (case, n = 243) and patients with tubal patency/obstruction (control, n = 187). The peripheral blood samples of cases and controls were collected to determine the genotypes of miR-145 SNPs. The value of miR-145 expression in the diagnosis and prognostic estimation of hydrosalpinx was assessed using ROC curve and regression analysis, respectively. Lipopolysaccharide (LPS) cell model was established with endometrial cells, and cells were transfected with miR-145 mimic, inhibitor, or negative control. MiR-145 and cytokine levels were quantified by quantitative reverse transcription PCR or western blot. MiR-145 expression was significantly higher in hydrosalpinx compared to control group, and high miR-145 expression was significantly associated with moderate/severe tube lesion, high pulsatility index (>1.06), and high resistance index (>0.61) in hydrosalpinx patients. ROC curve analysis indicated that monitoring miR-145 expression may be useful for the diagnosis of hydrosalpinx (AUC = 0.704). A alleles of rs41291957 (G>A) and rs353292 (G>A) were significantly associated with an increased risk of hydrosalpinx compared to G allele (p < 0.05), yet the mutant allele of rs353291 (A>G) and rs4705343 (T>C) significantly reduced susceptibility to hydrosalpinx compared to the wild type allele. Treatments with miR-145 mimic and LPS in endometrial cells significantly increased the levels of transforming growth factor-β1, tumor necrosis factor -α, interleukin (IL)-6, and IL-8 compared to negative control, while treatment with miR-145 inhibitor decreased the cytokine levels. In conclusion, abnormally expressed miR-145 may be involved in hydrosalpinx-induced ER defects by regulating the inflammatory response.


The orexigenic effect of peripheral ghrelin differs between rats of different age and with different baseline food intake, and it may in part be mediated by the area postrema.
Ghrelin is mainly secreted during fasting. While an orexigenic effect of peripherally injected ghrelin has been reported, reproducing this effect has often proven difficult. Here, we hypothesized that ghrelin's effect to increase food intake may depend on the experimental conditions (e.g., age of animals). We therefore investigated the effect of an IP ghrelin injection (100 microg/kg) on food intake in rats of different age and at different times during the light-dark cycle, i.e. with different levels of baseline food intake. Ghrelin injected at dark onset in ad libitum fed young rats (body weight [BW] 92 g) slightly increased feeding while no such effect was observed in 12 h food deprived rats (BW 150 g). In the middle of the light phase, ghrelin significantly increased feeding up to 2 h after injection in ad libitum fed rats (BW 130 g; food intake 1 h after injection: NaCl 0.4 +/- 0.2 g versus ghrelin 1.2 +/- 0.3 g [p < 0.05]). In various subsequent experiments, older rats (BW 300-490 g) tested under the same conditions did not respond to a single ghrelin injection. However repeated ghrelin injection (15 microg/kg/day once daily at light onset) over 10 days significantly increased food intake in rats (BW 400-460 g) starting from day 4 of the experiment (24 h food intake: NaCl approx. 19.5 g, ghrelin 22.5 g). Interestingly, the latter effect was completely abolished in rats lesioned in the area postrema (AP). Cumulative food intake was also increased in SHAM but not in AP-X animals (e.g., after 7 days: SHAM/NaCl 135.1 +/- 5.3 g versus SHAM/ghrelin 149.7 +/- 3.5 g [p < 0.05], AP-X/NaCl 127.2 +/- 16.4 versus AP-X/ghrelin 127.9 +/- 5.3). We conclude that ghrelin's effect to increase food intake can best be demonstrated when basal food intake is low. Ghrelin increases feeding mainly in young, fast growing animals. Ghrelin may therefore link the high energy needs to body growth in young individuals. In older animals, peripheral ghrelin increased feeding when injected repeatedly over several days. At least under these conditions, ghrelin's effect was mediated by the AP/NTS region. Using repeated administration, ghrelin might be an interesting tool to increase feeding in patients suffering from wasting diseases such as cancer anorexia.


NTP Toxicology and Carcinogenesis Studies of 2-Mercaptobenzothiazole (CAS No. 149-30-4) in F344/N Rats and B6C3F1 Mice (Gavage Studies).
Toxicology and carcinogenesis studies of technical-grade 2-mercaptobenzothiazole (96%-97% pure), a rubber accelerant and preservative, were conducted by administering the chemical by gavage in a corn oil vehicle to groups of F344/N rats and B6C3F1 mice of each sex for 16 days, 13 weeks, or 2 years. 2-Mercaptobenzothiazole was nominated for study by the National Institute of Environmental Health Sciences and the National Institute for Occupational Safety and Health. Sixteen-Day and Thirteen-Week Studies: In 16-day studies, mean body weight gains of rats receiving 2,500 mg/kg were 6-7 g lower than those of vehicle controls; 4/5 male and 5/5 female mice dosed with 3,000 mg/kg and 4/5 female mice dosed with 1,500 mg/kg died; lethargy and prostration occurred in most of these animals after gavage. Based on these results, doses were selected for both species in the 13-week studies were 0, 94 (mice only), 188, 375, 750, and 1,500 mg/kg. In the 13-week studies, no chemical-related deaths occurred in rats, but body weight gains in males dosed with 1,500 mg/kg and in females dosed with 750 or 1,500 mg/kg were lower than those in the vehicle control groups. Hepatomegaly occurred at the two highest doses in males and at all doses in females; however, no microscopic pathologic changes were noted in any tissue. More than half the mice dosed with 1,500 mg/kg died, but no compound-related body weight changes occurred. Clinical signs in mice were dose related and included lethargy in animals dosed with 375 mg/kg and lacrimation, salivation, and clonic seizure in some dosed with 750 or 1,500 mg/kg. No association between these clinical signs of toxicity and gross or microscopic pathologic effects were observed. Doses selected for the 2-year studies were 0, 375, and 750 mg/kg for male rats and for mice of each sex and 0, 188, or 375 mg/kg for female rats. Body weight and Survival in the Two-Year Studies: Fifty animals of each species and sex were administered 2-mercaptobenzothiazole in corn oil by gavage 5 days per week for 103 weeks. Administration of 2-mercaptobenzothiazole resulted in decreased survival in dosed male rats (vehicle control, 42/50; low dose, 22/50; high dose, 20/50) and in the high dose group of female mice (37/50; 39/50; 22/50) but not in female rats (28/50; 31/50; 25/50) or in male mice (38/50; 33/50; 30/50). No effect on body weight gain in dosed rats was observed; in dosed mice, minor reductions occurred between weeks 3 and 64, withrecovery thereafter. Postgavage lethargy and prostration occurred frequently in dosed rats and mice. Nonneoplastic and Neoplastic Effects in the Two-Year Studies: The severity of nephropathy was increased in dosed male rats. Ulcers and inflammation of the forestomach were prevalent in dosed rats, as were increased incidences of epithelial hyperplasia and hyperkeratosis in male rats, but no neoplasms of the forestomach were observed. There were no increases of nonneoplastic lesions in mice which were considered to be compound related. The incidences of a variety of tumors were increased in rats dosed with 2-mercaptobenzothiazole; some of the increased incidences were not dose related. In low dose male rats, increased incidences (P<0.01) were observed for mononuclear cell leukemia (7/50; 16/50; 3/50) and pancreatic acinar cell adenomas (2/50; 13/50; 6/49). Increased tumor incidences with dose-related trends (P<0.05) included pituitary gland adenomas in females (15/49; 24/50; 25/50), preputial gland adenomas or carcinomas (combined) in males (1/50; 6/50; 5/50), adrenal gland pheochromocytomas or malignant pheochromocytomas (combined) in males (18/50; 27/50; 24/49), and pheochromocytomas in females (1/50; 5/50; 6/50). These tumors were observed at significantly greater incidences (P</=0.05) in the high dose groups than in the vehicle controls. An increased incidence (P=0.028) of hepatocellular adenomas or carcinomas (combined) was observed only in low dose female mice (4/50; 12/49; 4/50). No significant increases in tumor incidences were seen in male mice. Genetic Toxicology: 2-Mercaptobenzothgy: 2-Mercaptobenzothiazole was not mutagenic in Salmonella typhimurium strains TA98, TA100, TA1535, or TA1537 with or without metabolic activation. In the presence of rat liver S9, 2-mercaptobenzothiazole increased the frequency of chromosomal aberrations and sister chromatid exchanges (SCEs) in Chinese hamster ovary (CHO) cells, as well as mutations at the TK locus of mouse L5178Y lymphoma cells. Audit: The data, documents, and pathology materials from the 2-year studies of 2-mercaptobenzothiazole were audited at the NTP Archives. The audit findings show that the conduct of the studies is documented adequately and support the data and results given in this Technical Report. Conclusions: Under the conditions of these 2-year gavage studies, there was some evidence of carcinogenic activity of 2-mercaptobenzothiazole for male F344/N rats, indicated by increased incidences of mononuclear cell leukemia, pancreatic acinar cell adenomas, adrenal gland pheochromocytomas, and preputial gland adenomas or carcinomas (combined). There was some evidence of carcinogenic activity for female F344/N rats, indicated by increased incidences of adrenal gland pheochromocytomas and pituitary gland adenomas. There was no evidence of carcinogenic activity of 2-mercaptobenzothiazole for maleB6C3F1 mice dosed with 375 or 750 mg/kg. There was equivocal evidence of carcinogenic activity for female B6C3F1 mice, indicated by increased incidences of hepatocellular adenomas or carcinomas (combined). Synonyms and Trade Names: Captax; Dermacid; Mertax; Thiotax; 2(3H)-benzothiazolethione; 2-benzothiazolyl mercaptan


Limited drug solubility can be decisive even for freely soluble drugs in highly swollen matrix tablets.
The aim of this study was to elucidate the importance of potential limited solubility effects for the control of drug release from hydrophilic matrix tablets loaded with a freely water-soluble drug. It is often assumed that the considerable amounts of water penetrating into this type of advanced drug delivery systems are sufficient to rapidly dissolve the entire drug loading, and that limited drug solubility is not playing a role for the control of drug release. Here, we show that this assumption can be erroneous. HPMC/lactose matrix tablets were loaded with 5 to 60% diprophylline (e.g. solubility in 0.1M HCl at 37°C: 235mg/mL), and drug release was measured at low and neutral pH, respectively. A mechanistically realistic mathematical theory was applied, considering drug diffusion in axial and radial direction in the cylindrical matrices and the potential co-existence of dissolved and non-dissolved drug. Importantly, only dissolved drug is available for diffusion. It is demonstrated that during major parts of the release periods, non-dissolved drug excess exists within tablets containing 30% or more diprophylline, despite of the substantial water contents of the systems. This leads to partially almost linear drug concentration distance profiles within the tablets, and reveals a major contribution of limited drug solubility effects to the control of drug release, even in the case of freely water-soluble diprophylline. It can be expected that also in other types of drug delivery systems, e.g. microparticles and implants (containing much less water), limited drug solubility effects play a much more important role than currently recognized.


Positioning As-needed Budesonide-Formoterol for Mild Asthma: Effect of Prestudy Treatment in Pooled Analysis of SYGMA 1 and 2.
Rationale: The SYGMA (Symbicort Given as Needed in Mild Asthma) studies evaluated the efficacy and safety of as-needed budesonide (BUD)-formoterol (FORM) in patients whose asthma was uncontrolled on as-needed inhaled short-acting bronchodilators (subgroup 1) or controlled on inhaled corticosteroids (ICS) or leukotriene receptor antagonists (subgroup 2). Objectives: To assess the influence of prestudy treatment in a post hoc analysis of the SYGMA studies. Methods: In the SYGMA 1 (NCT022149199) and SYGMA 2 (NCT02224157) 52-week, double-blind, randomized, parallel-group studies, 6,735 patients with mild asthma were randomized to as-needed BUD-FORM, low-dose BUD + as-needed terbutaline (BUD maintenance), or as-needed terbutaline (SYGMA 1 only). Exacerbation rates and changes in symptom control and lung function were compared among treatments for both subgroups. Results: In a pooled analysis of SYGMA 1 and 2, the annual severe exacerbation rate in subgroup 1 was significantly lower with as-needed BUD-FORM (0.08 [95% confidence interval (CI), 0.06-0.10]) than with BUD maintenance (0.10 [95% CI, 0.09-0.13]) (rate ratio [RR], 0.74 [95% CI, 0.56-0.98]; P = 0.03), and similar results were shown in subgroup 2 with BUD-FORM (0.12 [95% CI, 0.10-0.14]) and BUD maintenance (0.10 [95% CI, 0.09-0.13]) (RR, 1.10 [95% CI, 0.86-1.41]; P = 0.44). In SYGMA 1, the annual severe exacerbation rate in both subgroups was significantly lower with as-needed BUD-FORM than with as-needed terbutaline (subgroup 1: RR, 0.34 [95% CI, 0.20-0.58]; P < 0.001; subgroup 2: RR, 0.37 [95% CI, 0.25-0.54]; P < 0.001). The number needed to treat to prevent one severe exacerbation with as-needed BUD-FORM and BUD maintenance versus as-needed terbutaline were 20 and 34 in subgroup 1 and 13 and 12 in subgroup 2, respectively. Conclusions: These findings suggest that, for patients with mild asthma currently receiving short-acting β2-agonists alone, as-needed low-dose ICS-FORM should be preferred over maintenance ICS as initial controller treatment. For patients whose asthma is controlled on maintenance low-dose ICS, as-needed BUD-FORM is an alternative to maintenance ICS without the need for daily treatment, and both of these options are safer than switching to short-acting β2-agonist-only treatment.


Modeling Parenting Programs as an Interim Service for Families Waiting for Children's Mental Health Treatment.
Using a discrete choice conjoint experiment, we explored the design of parenting programs as an interim strategy for families waiting for children's mental health treatment. Latent class analysis yielded 4 segments with different design preferences. Simulations predicted the Fast-Paced Personal Contact segment, 22.1% of the sample, would prefer weekly therapist-led parenting groups. The Moderate-Paced Personal Contact segment (24.7%) preferred twice-monthly therapist-led parenting groups with twice-monthly lessons. The Moderate-Paced E-Contact segment (36.3%), preferred weekly to twice-monthly contacts, e-mail networking, and a program combining therapist-led sessions with the support of a computerized telephone e-coach. The Slow-Paced E-Contact segment (16.9%) preferred an approach combining monthly therapist-led sessions, e-coaching, and e-mail networking with other parents. Simulations predicted 45.3% of parents would utilize an option combining 5 therapist coaching calls with 5 e-coaching calls, a model that could reduce costs and extend the availability of interim services. Although 41.0% preferred weekly pacing, 58% were predicted to choose an interim parenting service conducted at a twice-monthly to monthly pace. The results of this study suggest that developing interim services reflecting parental preferences requires a choice of formats that includes parenting groups, telephone-coached distance programs, and e-coaching options conducted at a flexible pace.


Structural basis of the fanconi anemia-associated mutations within the FANCA and FANCG complex.
Monoubiquitination of the Fanconi anemia complementation group D2 (FANCD2) protein by the FA core ubiquitin ligase complex is the central event in the FA pathway. FANCA and FANCG play major roles in the nuclear localization of the FA core complex. Mutations of these two genes are the most frequently observed genetic alterations in FA patients, and most point mutations in FANCA are clustered in the C-terminal domain (CTD). To understand the basis of the FA-associated FANCA mutations, we determined the cryo-electron microscopy (EM) structures of Xenopus laevis FANCA alone at 3.35 Å and 3.46 Å resolution and two distinct FANCA-FANCG complexes at 4.59 and 4.84 Å resolution, respectively. The FANCA CTD adopts an arc-shaped solenoid structure that forms a pseudo-symmetric dimer through its outer surface. FA- and cancer-associated point mutations are widely distributed over the CTD. The two different complex structures capture independent interactions of FANCG with either FANCA C-terminal HEAT repeats, or the N-terminal region. We show that mutations that disturb either of these two interactions prevent the nuclear localization of FANCA, thereby leading to an FA pathway defect. The structure provides insights into the function of FANCA CTD, and provides a framework for understanding FA- and cancer-associated mutations.


Long-range enhancer activity determines Myc sensitivity to Notch inhibitors in T cell leukemia.
Notch is needed for T-cell development and is a common oncogenic driver in T-cell acute lymphoblastic leukemia. The protooncogene c-Myc (Myc) is a critical target of Notch in normal and malignant pre-T cells, but how Notch regulates Myc is unknown. Here, we identify a distal enhancer located >1 Mb 3' of human and murine Myc that binds Notch transcription complexes and physically interacts with the Myc proximal promoter. The Notch1 binding element in this region activates reporter genes in a Notch-dependent, cell-context-specific fashion that requires a conserved Notch complex binding site. Acute changes in Notch activation produce rapid changes in H3K27 acetylation across the entire enhancer (a region spanning >600 kb) that correlate with Myc expression. This broad Notch-influenced region comprises an enhancer region containing multiple domains, recognizable as discrete H3K27 acetylation peaks. Leukemia cells selected for resistance to Notch inhibitors express Myc despite epigenetic silencing of enhancer domains near the Notch transcription complex binding sites. Notch-independent expression of Myc in resistant cells is highly sensitive to inhibitors of bromodomain containing 4 (Brd4), a change in drug sensitivity that is accompanied by preferential association of the Myc promoter with more 3' enhancer domains that are strongly dependent on Brd4 for function. These findings indicate that altered long-range enhancer activity can mediate resistance to targeted therapies and provide a mechanistic rationale for combined targeting of Notch and Brd4 in leukemia.


FR-Match: robust matching of cell type clusters from single cell RNA sequencing data using the Friedman-Rafsky non-parametric test.
Single cell/nucleus RNA sequencing (scRNAseq) is emerging as an essential tool to unravel the phenotypic heterogeneity of cells in complex biological systems. While computational methods for scRNAseq cell type clustering have advanced, the ability to integrate datasets to identify common and novel cell types across experiments remains a challenge. Here, we introduce a cluster-to-cluster cell type matching method-FR-Match-that utilizes supervised feature selection for dimensionality reduction and incorporates shared information among cells to determine whether two cell type clusters share the same underlying multivariate gene expression distribution. FR-Match is benchmarked with existing cell-to-cell and cell-to-cluster cell type matching methods using both simulated and real scRNAseq data. FR-Match proved to be a stringent method that produced fewer erroneous matches of distinct cell subtypes and had the unique ability to identify novel cell phenotypes in new datasets. In silico validation demonstrated that the proposed workflow is the only self-contained algorithm that was robust to increasing numbers of true negatives (i.e. non-represented cell types). FR-Match was applied to two human brain scRNAseq datasets sampled from cortical layer 1 and full thickness middle temporal gyrus. When mapping cell types identified in specimens isolated from these overlapping human brain regions, FR-Match precisely recapitulated the laminar characteristics of matched cell type clusters, reflecting their distinct neuroanatomical distributions. An R package and Shiny application are provided at https://github.com/JCVenterInstitute/FRmatch for users to interactively explore and match scRNAseq cell type clusters with complementary visualization tools.


Mechanisms that drive bone pain across the lifespan.
Disorders of the skeleton are frequently accompanied by bone pain and a decline in the functional status of the patient. Bone pain occurs following a variety of injuries and diseases including bone fracture, osteoarthritis, low back pain, orthopedic surgery, fibrous dysplasia, rare bone diseases, sickle cell disease and bone cancer. In the past 2 decades, significant progress has been made in understanding the unique population of sensory and sympathetic nerves that innervate bone and the mechanisms that drive bone pain. Following physical injury of bone, mechanotranducers expressed by sensory nerve fibres that innervate bone are activated and sensitized so that even normally non-noxious loading or movement of bone is now being perceived as noxious. Injury of the bone also causes release of factors that; directly excite and sensitize sensory nerve fibres, upregulate proalgesic neurotransmitters, receptors and ion channels expressed by sensory neurons, induce ectopic sprouting of sensory and sympathetic nerve fibres resulting in a hyper-innervation of bone, and central sensitization in the brain that amplifies pain. Many of these mechanisms appear to be involved in driving both nonmalignant and malignant bone pain. Results from human clinical trials suggest that mechanism-based therapies that attenuate one type of bone pain are often effective in attenuating pain in other seemingly unrelated bone diseases. Understanding the specific mechanisms that drive bone pain in different diseases and developing mechanism-based therapies to control this pain has the potential to fundamentally change the quality of life and functional status of patients suffering from bone pain.


Abnormalities by pulmonary regions studied with computer tomography and clinical correlation following local-regional radiotherapy for breast cancer.
Adjuvant local-regional radiotherapy (RT) is commonly recommended for breast cancer patients. Postoperative adjuvant RT for breast cancer is associated with pulmonary side effects. This study was undertaken to measure the RT-induced pulmonary radiological changes with computer tomography (CT) scan using a CT-adapted modification of the Arriagada classification system, and to correlate these changes to RT techniques, pulmonary complications, and pulmonary function. The aim of the study is to study pulmonary radiological abnormalities with CT following different RT techniques for breast cancer, and their correlation to pulmonary complications and reduction in forced vital capacity (FVC). CT scans of the lungs were performed prior to and 4 months following RT in 53 breast cancer patients treated with local-regional RT. The radiological abnormalities were analyzed with a CT-adapted modification of a classification system originally proposed by Arriagada. The patients were monitored for RT-induced pulmonary complications. FVC was measured prior to and 6 months following RT. Statistical analysis used were that increasing CT scores were correlated with pulmonary complications (P < 0.001). The correlation between density grade (0-3, 4-9) and pulmonary complications (no complication vs slight/severe) was tested using Chi-square exact test for trend (2-sided). In addition, correlation between CT scores and FVC was done. Increasing CT scores were correlated with pulmonary complications (P < 0.001). The mean reduction of FVC for patients scoring 4-9 (-220 ml) was larger than for patients scoring 0-3 (-15 ml) (Spearson correlation coefficient significant at 0.01 level 2 tailed). But the mean reduction of FVC with greater volume of lung irradiated was not statistically different than lesser volume of lung irradiated (P > 0.05). Radiological abnormalities detected on CT images and scored with a modification of Arriagada's classification system can be used as an objective endpoint for pulmonary side effects in postmastectomy RT.


Reflections on a personalized cognitive rehabilitation intervention: Experiences of people living with dementia and their carers participating in the GREAT trial.
Cognitive rehabilitation for people living with early-stage dementia improves functional ability in areas targeted in the therapy, but little is known about how participants experience this intervention. This qualitative paper investigates participants' views about a cognitive rehabilitation intervention in a randomized controlled trial (the GREAT trial) and aims to help explain and interpret the findings and to inform further intervention development. Using in-depth thematic analysis, 43 semi-structured interviews (35 individual and 8 dyadic) were conducted with 25 people living with dementia and 26 family carers from three sites. The person-centred, individualized approach was valued. Some participants' views about dementia were questioned as a consequence of taking part in the therapy; they considered the effectiveness of the intervention in the context of the progressive nature of the condition. Certain participants continued to be doubtful, focussing on the inevitability of decline, rather than the possibility of reablement. Such views may have influenced engagement. The therapeutic relationship played a vital role as it was how personalized care was provided and participants' views had changed positively. Therapists engendered greater confidence and reduced anxiety and social isolation. Positive responses support personalized rehabilitative care to address the specific needs of people living with dementia.


Congenital myasthenic syndromes in childhood: diagnostic and management challenges.
The Congenital Myasthenic Syndromes (CMS), a group of heterogeneous genetic disorders of neuromuscular transmission, are often misdiagnosed as congenital muscular dystrophy (CMD) or myopathies and present particular management problems. We present our experience of 46 children with CMS, referred to us between 1992-2007 with provisional diagnoses of congenital myopathy (22/46), CMS or limb-girdle myasthenia (9/46), central hypotonia or neurometabolic disease (5/46), myasthenia gravis (4/46), limb-girdle or congenital muscular dystrophy (4/46) and SMA (2/46). Diagnosis was often considerably delayed (up to 18y4 m), despite the early symptoms in most cases. Diagnostic clues in the neonates were feeding difficulties (29/46), hypotonia with or without limb weakness (21/46), ptosis (19/46), respiratory insufficiency (12/46), contractures (4/46) and stridor (6/46). Twenty-five children had delayed motor milestones. Fatigability developed in 43 and a variable degree of ptosis was eventually present in 40. Over the period of the study, the mainstay of EMG diagnosis evolved from repetitive nerve stimulation to stimulation single fibre EMG. The patients were studied by several different operators. 66 EMGs were performed in 40 children, 29 showed a neuromuscular junction abnormality, 7 were myopathic, 2 had possible neurogenic changes and 28 were normal or inconclusive. A repetitive CMAP was detected in only one of seven children with a COLQ mutation and neither of the two children with Slow Channel Syndrome mutations. Mutations have been identified so far in 32/46 children: 10 RAPSN, 7 COLQ, 6 CHRNE, 7 DOK7, 1 CHRNA1 and 1 CHAT. 24 of 25 muscle biopsies showed myopathic changes with fibre size variation; 14 had type-1 fibre predominance. Three cases showed small type-1 fibres resembling fibre type disproportion, and four showed core-like lesions. No specific myopathic features were associated with any of the genes. Twenty children responded to Pyridostigmine treatment alone, 11 to Pyridostigmine with either 3, 4 DAP or Ephedrine and five to Ephedrine alone. Twenty one children required acute or chronic respiratory support, with tracheostomy in 4 and nocturnal or emergency non-invasive ventilation in 9. Eight children had gastrostomy. Another 11 were underweight for height indicative of failure to thrive and required dietetic input. A high index of clinical suspicion, repeat EMG by an experienced electromyographer and, if necessary, a therapeutic trial of Pyridostigmine facilitates the diagnosis of CMS with subsequent molecular genetic confirmation. This guides rational therapy and multidisciplinary management, which may be crucial for survival, particularly in pedigrees where previous deaths have occurred in infancy.


Direct and long-range action of a wingless morphogen gradient.
Wingless (Wg), a founding member of the Wingless/Int-1 (Wnt) family of secreted proteins, acts as a short-range inducer and as a long-range organizer during Drosophila development. Here, we determine the consequences of ectopically expressing (i) a wild-type form of Wg, (ii) a membrane-tethered form of Wg, and (iii) a constitutively active form of the cytosolic protein Armadillo (Arm), which normally acts to transduce Wg, and we compare them with the effects of removing endogenous Wg or Arm activity. Our results indicate that wild-type Wg acts at long range, up-regulating the transcription of particular target genes as a function of concentration and distance from secreting cells. In contrast, tethered Wg and Arm have only short-range or autonomous effects, respectively, on the transcription of these genes. We interpret these findings as evidence that Wg can act directly and at long range as a gradient morphogen during normal development.


Human ATP-binding cassette transporter ABCC10: expression profile and p53-dependent upregulation.
Human ATP-binding cassette (ABC) transporter genes are classified into seven sub-families, where "C" subfamily comprises a total of 13 gene members. The ABCC10 cDNA was cloned in the human full-length cDNA project at the Kazusa DNA Research Institute. However, current information is limited regarding its physiological function and gene expression. In the present study, we have investigated the expression of the ABCC10 gene to gain insight into its biological nature. By quantitative PCR, ABCC10 gene expression is demonstrated to be highest in pancreas among the adult and fetal tissues and tumors presently tested. Decreased expression was observed when resting T- and B-cells were activated. Furthermore, when we examined its expression under apoptotic conditions, we found that ABCC10 mRNA levels remarkably increased in doxorubicin-treated MCF7 cells, whereas its up-regulation was suppressed in p53-dominant-negative MCF7 cells. These results suggest that expression of the ABCC10 gene is regulated in a p53-dependent manner during DNA-damage-related apoptosis.


Effects of hispolon on glioblastoma cell growth.
Hispolon is a polyphenolic compound isolated from Phellinus linteus which exhibits antitumor activity. Here, we explored the effects of hispolon on human glioblastoma cells U87MG. Cell viability was examined by MTT assay. Growth was investigated by incubating cells with various concentrations of hispolon (25 and 50 µM) for 24, 48 or 72 h and daily cell count. Cell cycle and apoptosis assay were assessed by flow cytometry. Hispolon decreased cell viability in a dose- and time-dependent manner. The cell cycle distribution showed that hispolon enhanced the accumulation of the cells in G2/M phase. Hispolon decreased the expression of G1-S transition-related protein cyclin D4 but increased the expression of CDK inhibitor p21. Additionally, hispolon enhanced the expression of p53. Moreover, hispolon treatment was effective on U87MG cells in inhibiting cell viability and inducing cell apoptosis. Our results indicate that hispolon inhibits the cell viability, induces G2/M cell cycle arrest and apoptosis in glioblastoma U87MG cells, and p53 should play a role in hispolon-mediated antitumor activity.


Systematic review of youth mental health service integration research.
Quality mental health care is based on the integration of care across organisations and disciplines. The aims of this study were, first, to assess the extent, characteristics and reported outcomes of publications concerned with youth mental health service integration in Australia and internationally; and second, to investigate the study design quality of evaluative interventions and determine whether the studies report on the cost-effectiveness of the integration in order to inform the reform of youth mental health services by Queensland Health. A systematic search of the peer-reviewed literature and a narrative synthesis were undertaken of English language publications from 21 electronic databases. Inclusion criteria were: published 1998-2014 (inclusive); peer-reviewed research; focused on mental health services integration; reported data for youth aged 12-25 years. The methodological quality of evaluative interventions was assessed using the Quality Assessment Tool for Quantitative Studies developed by the Effective Public Health Practice Project (EPHPP). Twenty-five studies met the inclusion criteria: one (4%) was classified as a measurement research, 13 (52%) as descriptive, and 11 (44%) as interventions including five (45%) evaluative interventions. Four out of the five evaluative interventions reported positive effects of youth mental health service integration. Particular problems included ambiguity of definitions, absence of economic or cost analyses and insufficient consumer involvement. The methodological quality of the interventions was variable with, on average, a moderate level of selection bias and study design. Despite a slight increase in the number of studies in the last couple of years, there are important gaps in the evidence base for youth mental health service integration processes. The relatively small number of evaluative studies and lack of economic evaluations point to the need for additional research in this important area.


Dexmedetomidine Alleviates Neurogenesis Damage Following Neonatal Midazolam Exposure in Rats through JNK and P38 MAPK Pathways.
Midazolam, a widely used anesthetic, inhibits proliferation of neural stem cells (NSCs) and induces neuroapoptosis in neonates. Dexmedetomidine, an effective auxiliary medicine in clinical anesthesia, protects the developing brain against volatile anesthetic-induced neuroapoptosis. Whether dexmedetomidine protects against neurogenesis damage induced by midazolam remains unknown. This study aims to clarify the protective effect of dexmedetomidine on midazolam-induced neurogenesis damage and explore its potential mechanism. Postnatal 7-day-old Sprague-Dawley (SD) rats and cultured NSCs were treated with either normal saline, midazolam, or dexmedetomidine combined with midazolam. The rats were sacrificed at 1, 3, and 7 days after treatment. Cell proliferation was assessed by 5-bromodeoxyurdine (BrdU) incorporation. Cell viability was determined using MTT assay. Cell differentiation and apoptosis were detected by immunofluorescent staining and terminal dUTP nick-end labeling (TUNEL), respectively. The protein levels of p-JNK, p-P38, and cleaved caspase-3 were quantified using Western blotting. Midazolam decreased cell proliferation and increased cell apoptosis in the subventricular zone (SVZ), the subgranular zone (SGZ) of the hippocampus, and cultured NSCs. Moreover, midazolam decreased cell viability and increased the expression of p-JNK and p-P38 in cultured NSCs. Co-treatment with dexmedetomidine attenuated midazolam-induced changes in cell proliferation, viability, apoptosis, and protein expression of p-JNK and p-P38 in cultured NSCs. Midazolam and dexmedetomidine did not affect the differentiation of the cultured NSCs. These results indicate that dexmedetomidine alleviated midazolam-induced neurogenesis damage via JNK and P38 MAPK pathways.


Lmx1b is required for maintenance of central serotonergic neurons and mice lacking central serotonergic system exhibit normal locomotor activity.
Central serotonergic neurons have been implicated in numerous animal behaviors and psychiatric disorders, but the molecular mechanisms underlying their development are not well understood. Here we generated Lmx1b (LIM homeobox transcription factor 1 beta) conditional knock-out mice (Lmx1b(f/f/p)) in which Lmx1b was only deleted in Pet1 (pheochromocytoma 12 ETS factor-1)-expressing 5-HT neurons. In Lmx1b(f/f/p) mice, the initial generation of central 5-HT neurons appeared normal. However, the expression of both 5-HT-specific and non-5-HT-specific markers was lost in these neurons at later stages of development. The loss of gene expression is concomitant with downregulation of Lmx1b expression, with the exception of serotonin transporter Sert and tryptophan hydroxylase TPH2, whose expression appears to be most sensitive to Lmx1b. Interestingly, the expression of Pet1 is tightly coupled with expression of Lmx1b during later stages of embryonic development, indicating that Lmx1b maintains Pet1 expression. In Lmx1b(f/f/p) mice, almost all central 5-HT neurons failed to survive. Surprisingly, Lmx1b(f/f/p) mice survived to adulthood and exhibited normal locomotor activity. These data reveal a critical role of Lmx1b in maintaining the differentiated status of 5-HT neurons. Lmx1b(f/f/p) mice with normal locomotor function should provide a unique animal model for examining the roles of central 5-HT in a variety of animal behaviors.


Emergence of Coordinated Activity in the Developing Entorhinal-Hippocampal Network.
Correlated activity in the entorhinal-hippocampal neuronal networks, supported by oscillatory and intermittent population activity patterns is critical for learning and memory. However, when and how correlated activity emerges in these networks during development remains largely unknown. Here, we found that during the first postnatal week in non-anaesthetized head-restrained rats, activity in the superficial layers of the medial entorhinal cortex (MEC) and hippocampus was highly correlated, with intermittent population bursts in the MEC followed by early sharp waves (eSPWs) in the hippocampus. Neurons in the superficial MEC layers fired before neurons in the dentate gyrus, CA3 and CA1. eSPW current-source density profiles indicated that perforant/temporoammonic entorhinal inputs and intrinsic hippocampal connections are co-activated during entorhinal-hippocampal activity bursts. Finally, a majority of the entorhinal-hippocampal bursts were triggered by spontaneous myoclonic body movements, characteristic of the neonatal period. Thus, during the neonatal period, activity in the entorhinal cortex (EC) and hippocampus is highly synchronous, with the EC leading hippocampal activation. We propose that such correlated activity is embedded into a large-scale bottom-up circuit that processes somatosensory feedback resulting from neonatal movements, and that it is likely to instruct the development of connections between neocortex and hippocampus.


Synthesis of 2-phenylnaphthalenes from styryl-2-methoxybenzenes.
A new simple and efficient method for the synthesis of 2-phenylnaphthalenes from electron-rich 1-styryl-2-methoxybenzenes has been described. The reaction proceeds via TFA catalyzed C-C bond cleavage followed by intermolecular [4+2]-Diels-Alder cycloaddition of an in situ formed styrenyl trifluoroacetate intermediate. The quantum chemical calculations identified the transition state for the cycloaddition reaction and helped in tracing the reaction mechanism. The method has been efficiently utilized for synthesis of the phenanthrene skeleton and a naphthalene-based potent and selective ER-β agonist.


Prevalence and predictors of verbal aggression in a secure mental health service: Use of the HCR-20.
Despite evidence about the negative effects of verbal aggression in mental health wards there is little research about its prevalence or about the factors that predict the behaviour among inpatients. This study aimed to determine the prevalence of verbal aggression in a secure mental health service, and to examine the relationship of verbal aggression with risk factors for aggression in the risk assessment tool HCR-20 in order to establish whether, and with which factors, the behaviour can be predicted. Verbal aggression was measured using the Overt Aggression Scale (OAS) over a 3-month period across a heterogeneous patient group (n = 613). Over half the patients (n = 341, 56%) engaged in 1594 incidents of verbal aggression. The HCR-20 total, clinical, and risk management subscale scores predicted verbal aggression, though effect sizes were not large. Item-outcome analysis revealed that impulsivity, negative attitudes, and non-compliance with medication were the best predictors of verbal aggression and, therefore, should be targeted for intervention. There are key synergies between factors predicting verbal aggression and the core mental health nursing role. Nurses, therefore, are in a prime position to develop and implement interventions that may reduce verbal aggression in mental health inpatients.


Role of Ingested Amino Acids and Protein in the Promotion of Resistance Exercise-Induced Muscle Protein Anabolism.
The goal of this critical review is to comprehensively assess the evidence for the molecular, physiologic, and phenotypic skeletal muscle responses to resistance exercise (RE) combined with the nutritional intervention of protein and/or amino acid (AA) ingestion in young adults. We gathered the literature regarding the translational response in human skeletal muscle to acute exposure to RE and protein/AA supplements and the literature describing the phenotypic skeletal muscle adaptation to RE and nutritional interventions. Supplementation of protein/AAs with RE exhibited clear protein dose-dependent effects on translational regulation (protein synthesis) through mammalian target of rapamycin complex 1 (mTORC1) signaling, which was most apparent through increases in p70 ribosomal protein S6 kinase 1 (S6K1) phosphorylation, compared with postexercise recovery in the fasted or carbohydrate-fed state. These acute findings were critically tested via long-term exposure to RE training (RET) and protein/AA supplementation, and it was determined that a diminishing protein/AA supplement effect occurs over a prolonged exposure stimulus after exercise training. Furthermore, we found that protein/AA supplements, combined with RET, produced a positive, albeit minor, effect on the promotion of lean mass growth (when assessed in >20 participants/treatment); a negligible effect on muscle mass; and a negligible to no additional effect on strength. A potential concern we discovered was that the majority of the exercise training studies were underpowered in their ability to discern effects of protein/AA supplementation. Regardless, even when using optimal methodology and large sample sizes, it is clear that the effect size for protein/AA supplementation is low and likely limited to a subset of individuals because the individual variability is high. With regard to nutritional intakes, total protein intake per day, rather than protein timing or quality, appears to be more of a factor on this effect during long-term exercise interventions. There were no differences in strength or mass/muscle mass on RET outcomes between protein types when a leucine threshold (>2 g/dose) was reached. Future research with larger sample sizes and more homogeneity in design is necessary to understand the underlying adaptations and to better evaluate the individual variability in the muscle-adaptive response to protein/AA supplementation during RET.


Integrin α6β4 identifies an adult distal lung epithelial population with regenerative potential in mice.
Laminins and their integrin receptors are implicated in epithelial cell differentiation and progenitor cell maintenance. We report here that a previously unrecognized subpopulation of mouse alveolar epithelial cells (AECs) expressing the laminin receptor α6β4, but little or no pro-surfactant C (pro-SPC), is endowed with regenerative potential. Ex vivo, this subpopulation expanded clonally as progenitors but also differentiated toward mature cell types. Integrin β4 itself was not required for AEC proliferation or differentiation. An in vivo embryonic lung organoid assay, which we believe to be novel, was used to show that purified β4+ adult AECs admixed with E14.5 lung single-cell suspensions and implanted under kidney capsules self-organized into distinct Clara cell 10-kDa secretory protein (CC10+) airway-like and SPC+ saccular structures within 6 days. Using a bleomycin model of lung injury and an SPC-driven inducible cre to fate-map AECs, we found the majority of type II AECs in fibrotic areas were not derived from preexisting type II AECs, demonstrating that SPC- progenitor cells replenished type II AECs during repair. Our findings support the idea that there is a stable AEC progenitor population in the adult lung, provide in vivo evidence of AEC progenitor cell differentiation after parenchymal injury, and identify a strong candidate progenitor cell for maintenance of type II AECs during lung repair.


Asbestos induces reduction of tumor immunity.
Asbestos-related cancers such as malignant mesothelioma and lung cancer are an important issue in the world. There are many conflicts concerning economical considerations and medical evidence for these cancers and much confusion regarding details of the pathological mechanisms of asbestos-induced cancers. For example, there is uncertainty concerning the degree of danger of the iron-absent chrysotile compared with iron-containing crocidolite and amosite. However, regarding bad prognosis of mesothelioma, medical approaches to ensure the recognition of the biological effects of asbestos and the pathological mechanisms of asbestos-induced carcinogenesis, as well as clinical trials to detect the early stage of mesothelioma, should result in better preventions and the cure of these malignancies. We have been investigating the immunological effects of asbestos in relation to the reduction of tumor immunity. In this paper, cellular and molecular approaches to clarify the immunological effects of asbestos are described, and all the findings indicate that the reduction of tumor immunity is caused by asbestos exposure and involvement in asbestos-induced cancers. These investigations may not only allow the clear recognition of the biological effects of asbestos, but also present a novel procedure for early detection of previous asbestos exposure and the presence of mesothelioma as well as the chemoprevention of asbestos-related cancers.


Modification of epithelial tight junction integrity to enhance transmucosal absorption.
Tight junctions (TJs) located between adjacent epithelial cells restrict the movement of solutes, ions, and even water. Restriction of macromolecules by TJ structures impedes one potential route of absorption for biopharmaceutical drugs, a route known as the paracellular pathway. It is anticipated that an improved understanding of TJ structure and cellular mechanisms that regulate its function will lead to successful strategies for improved transmucosal drug absorption through the paracellular pathway. Based upon clinical correlations between TJ dysfunction and several disease states, any approach to modify TJ integrity to enhance drug absorption in this way must be tempered by an appreciation of potential safety concerns that might develop through sustained or repeated TJ alterations. This review describes a current understanding of epithelial TJ organization and examines potential methods and implications for regulated modification of TJ function for the enhanced uptake of drugs without detrimental clinical outcomes.


MiTF links Erk1/2 kinase and p21 CIP1/WAF1 activation after UVC radiation in normal human melanocytes and melanoma cells.
As a survival factor for melanocytes lineage cells, MiTF plays multiple roles in development and melanomagenesis. What role MiTF plays in the DNA damage response is currently unknown. In this report we observed that MiTF was phosphorylated at serine 73 after UVC radiation, which was followed by proteasome-mediated degradation. Unlike after c-Kit stimulation, inhibiting p90RSK-1 did not abolish the band shift of MiTF protein, nor did it abolish the UVC-mediated MiTF degradation, suggesting that phosphorylation on serine 73 by Erk1/2 is a key event after UVC. Furthermore, the MiTF-S73A mutant (Serine 73 changed to Alanine via site-directed mutagenesis) was unable to degrade and was continuously expressed after UVC exposure. Compared to A375 melanoma cells expressing wild-type MiTF (MiTF-WT), cells expressing MiTF-S73A mutant showed less p21 WAF1/CIP1 accumulation and a delayed p21 WAF1/CIP1 recovery after UVC. Consequently, cells expressing MiTF-WT showed a temporary G1 arrest after UVC, but cells expressing MiTF-S73A mutant or lack of MiTF expression did not. Finally, cell lines with high levels of MiTF expression showed higher resistance to UVC-induced cell death than those with low-level MiTF. These data suggest that MiTF mediates a survival signal linking Erk1/2 activation and p21 WAF1/CIP1 regulation via phosphorylation on serine 73, which facilitates cell cycle arrest. In addition, our data also showed that exposure to different wavelengths of UV light elicited different signal pathways involving MiTF.


[Performance of screening tests in the preventive services for infants].
Early detection is one of the aims of the surveillance by the preventive services in infancy. This article presents the performance of screening for anemia in infancy, compliance with iron supplementation and treatment, screening for hearing, and abnormalities of the hip joint, and referral to ear, nose and throat (E.N.T.) and orthopedic specialists according to the screening results. The sample consisted of Jewish and Arab women who gave birth during March 2000. Mothers were interviewed after the infants reached the age of 15-19 months (667 Jewish and 211 Arab). All the women utilized the preventive services of the Ministry of Health, municipalities or H.M.O's. Arab women from East Jerusalem utilized the services of non-governmental organizations (NGO's). A total of 61% of the Jewish and 86% of the Arab infants had a hemoglobin examination. Eighty-five percent of the Jewish and 75% of the Arab infants received iron as a supplement or treatment for anemia. The performance of hearing tests was 88% and 77%, respectively. Among the infants referred to E.N.T. no hearing loss was detected. Fourteen percent were not screened nor referred by the family physician/pediatrician. Among the self referrals, one child was detected with a hearing loss. Percent referrals to the orthopedic surgeon was higher among Jewish than Arab infants. Among those not examined through the preventive services, the rate of pathology detected by the orthopedic surgeon was higher than those referred. Findings highlight the need to improve the quality of screening and increase the percent of infants screened in the Arab and Jewish population, particularly those of the N.G.O.'s, with emphasis on high risk groups. Furthermore, efforts should be directed to define and recommend routines for detection of abnormalities of the hip joint, and integrate the procedures within the health services.


Dietary fat intake and risk of epithelial ovarian cancer in the European Prospective Investigation into Cancer and Nutrition.
There are inconsistent and limited data available to assess the relationship between fat intake and risk of epithelial ovarian cancer (EOC). We examined the consumption of total fat, fat sources and fat subtypes in relation to risk of EOC and its major histologic subtypes in the European Prospective Investigation into Cancer and Nutrition which includes incident invasive (n=1095) and borderline (n=96) EOC. Cox proportional hazards regression was used to estimate hazard ratios (HRs) and 95% confidence intervals (CIs). In multivariate models, we observed no association with consumption of total fat, animal or plant fat, saturated fat, cholesterol, monounsaturated fat, or fatty fish and risk of invasive EOC. There was, however, an increased risk of invasive EOC in the highest category of intake (Quartile 4 vs. Quartile 1) of polyunsaturated fat (HR=1.22, 95% CI=1.02-1.48, P(trend)=0.02). We did not observe heterogeneity in the risk associations in comparisons of serous and endometrioid histologic subtypes. This study does not support an etiological role for total fat intake in relation to EOC risk; however, based on observations of a positive association between intake of polyunsaturated fat and invasive EOC risk in the current and previous studies, this fat subtype warrants further investigation to determine its potential role in EOC development.


Comparison of Standard and Enhanced Pulse Oximeter Auditory Displays of Oxygen Saturation: A Laboratory Study With Clinician and Nonclinician Participants.
When engaged in visually demanding tasks, anesthesiologists depend on the auditory display of the pulse oximeter (PO) to provide information about patients' oxygen saturation (SpO2). Current auditory displays are not always effective at providing SpO2 information. In this laboratory study, clinician and nonclinician participants identified SpO2 parameters using either a standard auditory display or an auditory display enhanced with additional acoustic properties while performing distractor tasks and in the presence of background noise. In a counterbalanced crossover design, specialist or trainee anesthesiologists (n = 25) and nonclinician participants (n = 28) identified SpO2 parameters using standard and enhanced PO auditory displays. Participants performed 2 distractor tasks: (1) arithmetic verification and (2) keyword detection. Simulated background operating room noise played throughout the experiment. Primary outcomes were accuracies to (1) detect transitions to and from an SpO2 target range and (2) identify SpO2 range (target, low, or critical). Secondary outcomes included participants' latency to detect target transitions, accuracy to identify absolute SpO2 values, accuracy and latency of distractor tasks, and subjective judgments about tasks. Participants were more accurate at detecting target transitions using the enhanced display (87%) than the standard display (57%; odds ratio, 7.3 [95% confidence interval {CI}, 4.4-12.3]; P < .001). Participants were also more accurate at identifying SpO2 range using the enhanced display (86%) than the standard display (76%; odds ratio, 2.7 [95% CI, 1.6-4.6]; P < .001). Secondary outcome analyses indicated that there were no differences in performance between clinicians and nonclinicians for target transition detection accuracy and latency, SpO2 range identification accuracy, or absolute SpO2 value identification. The enhanced auditory display supports more accurate detection of target transitions and identification of SpO2 range for both clinicians and nonclinicians. Despite their previous experience using PO auditory displays, clinicians in this laboratory study were no more accurate in any SpO2 outcomes than nonclinician participants.


A network of investigator networks in human genome epidemiology.
The task of identifying genetic determinants for complex, multigenetic diseases is hampered by small studies, publication and reporting biases, and lack of common standards worldwide. The authors propose the creation of a network of networks that include groups of investigators collecting data for human genome epidemiology research. Twenty-three networks of investigators addressing specific diseases or research topics and representing several hundreds of teams have already joined this initiative. For each field, the authors are currently creating a core registry of teams already participating in the respective network. A wider international registry will include all other teams also working in the same field. Independent investigators are invited to join the registries and existing networks and to join forces in creating additional ones as needed. The network of networks aims to register these networks, teams, and investigators; be a resource for information about or connections to the many networks; offer methodological support; promote sound design and standardization of analytical practices; generate inclusive overviews of fields at large; facilitate rapid confirmation of findings; and avoid duplication of effort.


DNA-PKcs phosphorylates hnRNP-A1 to facilitate the RPA-to-POT1 switch and telomere capping after replication.
The heterogeneous nuclear ribonucleoprotein A1 (hnRNP-A1) has been implicated in telomere protection and telomerase activation. Recent evidence has further demonstrated that hnRNP-A1 plays a crucial role in maintaining newly replicated telomeric 3' overhangs and facilitating the switch from replication protein A (RPA) to protection of telomeres 1 (POT1). The role of hnRNP-A1 in telomere protection also involves DNA-dependent protein kinase catalytic subunit (DNA-PKcs), although the detailed regulation mechanism has not been clear. Here we report that hnRNP-A1 is phosphorylated by DNA-PKcs during the G2 and M phases and that DNA-PK-dependent hnRNP-A1 phosphorylation promotes the RPA-to-POT1 switch on telomeric single-stranded 3' overhangs. Consequently, in cells lacking hnRNP-A1 or DNA-PKcs-dependent hnRNP-A1 phosphorylation, impairment of the RPA-to-POT1 switch results in DNA damage response at telomeres during mitosis as well as induction of fragile telomeres. Taken together, our results indicate that DNA-PKcs-dependent hnRNP-A1 phosphorylation is critical for capping of the newly replicated telomeres and prevention of telomeric aberrations.


Effects of bicarbonate on growth of Neisseria gonorrhoeae: replacement of gaseous CO2 atmosphere.
The effect of NaHCO2 on the growth of Neisseria gonorrhoeae cultures was studied in a liquid and a semisolid growth medium. With a broth culture, NaHCO3 (0.009 M) greatly reduced the lag phase and also increased the total growth. The same concentration of bicarbonate supported rapid growth when added to the semisolid medium if the plates were individually incubated in sealed plastic bags. In a container with a large air space, a higher concentration of NaHCO3 was necessary to support growth. The assimilation of 14C-labeled NaHO3 by growing cultures was also investigated.


Metformin sensitizes EGFR-TKI-resistant human lung cancer cells in vitro and in vivo through inhibition of IL-6 signaling and EMT reversal.
The EGF receptor tyrosine kinase inhibitors (EGFR-TKI) have become a standard therapy in patients with EGFR-activating mutations. Unfortunately, acquired resistance eventually limits the clinical effects and application of EGFR-TKIs. Studies have shown that suppression of epithelial-mesenchymal transition (EMT) and the interleukin (IL)-6/STAT3 pathway may abrogate this acquired mechanism of drug resistance of TKIs. This study aims to investigate the effect of metformin on sensitizing EGFR-TKI-resistant human lung cancer cells in vitro and in vivo through inhibition of IL-6 signaling and EMT reversal. The effect of metformin on reversing TKI resistance was examined in vitro and in vivo using MTT, BrdUrd incorporation assay, invasion assay, flow cytometry analysis, immunostaining, Western blot analysis, and xenograft implantation. In this study, metformin, a widely used antidiabetic agent, effectively increased the sensitivity of TKI-resistant lung cancer cells to erlotinib or gefitinib. Metformin reversed EMT and decreased IL-6 signaling activation in TKI-resistant cells, while adding IL-6 to those cells bypassed the anti-TKI-resistance effect of metformin. Furthermore, overexpression or addition of IL-6 to TKI-sensitive cells induced TKI resistance, which could be overcome by metformin. Finally, metformin-based combinatorial therapy effectively blocked tumor growth in xenografts with TKI-resistant cancer cells, which was associated with decreased IL-6 secretion and expression, EMT reversal, and decreased IL-6-signaling activation in vivo. Metformin, generally considered nontoxic and remarkably inexpensive, might be used in combination with TKIs in patients with non-small cell lung cancer, harboring EGFR mutations to overcome TKI resistance and prolong survival.


A novel Myosin essential light chain mutation causes hypertrophic cardiomyopathy with late onset and low expressivity.
Hypertrophic cardiomyopathy (HCM) is caused by mutations in genes encoding sarcomere proteins. Mutations in MYL3, encoding the essential light chain of myosin, are rare and have been associated with sudden death. Both recessive and dominant patterns of inheritance have been suggested. We studied a large family with a 38-year-old asymptomatic HCM-affected male referred because of a murmur. The patient had HCM with left ventricular hypertrophy (max WT 21 mm), a resting left ventricular outflow gradient of 36 mm Hg, and left atrial dilation (54 mm). Genotyping revealed heterozygosity for a novel missense mutation, p.V79I, in MYL3. The mutation was not found in 300 controls, and the patient had no mutations in 10 sarcomere genes. Cascade screening revealed a further nine heterozygote mutation carriers, three of whom had ECG and/or echocardiographic abnormalities but did not fulfil diagnostic criteria for HCM. The penetrance, if we consider this borderline HCM the phenotype of the p.V79I mutation, was 40%, but the mean age of the nonpenetrant mutation carriers is 15, while the mean age of the penetrant mutation carriers is 47. The mutation affects a conserved valine replacing it with a larger isoleucine residue in the region of contact between the light chain and the myosin lever arm. In conclusion, MYL3 mutations can present with low expressivity and late onset.


Ordering of ceramide formation, caspase activation, and mitochondrial changes during CD95- and DNA damage-induced apoptosis.
To evaluate the role of ceramide (Cer) in apoptosis signaling, we examined Cer formation induced by CD95, etoposide, or gamma-radiation (IR) in relation to caspase activation and mitochondrial changes in Jurkat T cells. The Cer response to all three stimuli was mapped in between caspases sensitive to benzoyloxycarbonyl-VAD-fluoromethylketone (zVAD-fmk) and acetyl-DEVD-aldehyde (DEVD-CHO). Cer production was independent of nuclear fragmentation but associated with the occurrence of other aspects of the apoptotic morphology. Caspase-8 inhibition abrogated Cer formation and apoptosis induced by CD95 but did not affect the response to etoposide or IR, placing CD95-induced Cer formation downstream from caspase-8 and excluding a role for caspase-8 in the DNA damage pathways. CD95 signaling to the mitochondria required caspase-8, whereas cytochrome c release in response to DNA damage was caspase-independent. These results indicate that the caspases required for the Cer response to etoposide and IR reside at or downstream from the mitochondria. Bcl-2 overexpression abrogated the Cer response to etoposide and IR and reduced CD95-induced Cer accumulation. We conclude that the Cer response to DNA damage fully depends on mitochondrion-dependent caspases, whereas the response to CD95 partially relies on these caspases. Our data imply that Cer is not instrumental in the activation of inducer caspases or signaling to the mitochondria. Rather, Cer formation is associated with the execution phase of apoptosis.


[The influence of occlusion on sporting performance].
The purpose of the present study is to investigate the relationship between dental occlusion and physical performance using the Ergo-jump platform, and to highlight connections, if any. For the purpose of this research, 30 patients suffering from cervico-facial pathology and 10 without temporomandibular joint dysfunction were selected from within the Dental Clinic of the University of Sassari. For the first group a resin plate was prepared to correct the malocclusion, while for the control group a bite was prepared that created it. Subjects carried out exercises with the Ergo-jump platform, a device that assesses physical capabilities and athletic performance. Patients were asked to do 2 exercises, a counter movement jump and a mechanical power test, with plate and without plate. The tests of the first group were as follows: flying times with the bite were 0.01 s longer; the average elevation in the counter movement jump test did not give statistically significant results; execution of the mechanical power test suggests an increase in average mechanical power with the plate. Execution of the same exercises in the other group showed greater flight times without the place; average elevation was not statistically significant; the execution of the mechanical power test showed a decrease in average mechanical power with the occlusion plate. Statistical analysis of individual results showed that patients do not always obtain a benefit during physical exercise using the bite. Research shows that the same pathology can cause different physical performance in different individuals; in fact, not all craniomandibular disturbances have repercussions on posture.


Recycling between cortisol and cortisone in human splanchnic, subcutaneous adipose, and skeletal muscle tissues in vivo.
11β-Hydroxysteroid dehydrogenase type 1 (11βHSD1) is a therapeutic target in metabolic syndrome because it catalyses reductase regeneration of cortisol from cortisone in adipose and liver. 11βHSD1 can also catalyze the reverse dehydrogenase reaction in vitro (e.g., if cofactor is limited). We used stable isotope tracers to test the hypothesis that both 11βHSD1-reductase and -dehydrogenase activities occur in human metabolic tissues in vivo. 1,2-[(2)H](2)-Cortisone (d2-cortisone) was validated as a tracer for 11β-dehydrogenase activity and its inhibition by licorice. d2-Cortisone and 9,11,12,12-[(2)H](4)-cortisol (d4-cortisol) (to measure 11β-reductase activity) were coinfused and venous samples obtained from skeletal muscle, subcutaneous adipose (n = 6), and liver (n = 4). Steroids were measured by liquid chromatography-tandem mass spectrometry and arteriovenous differences adjusted for blood flow. Data are means ± SEM. 11β-Reductase and -dehydrogenase activities were detected in muscle (cortisol release 19.7 ± 4.1 pmol/100 mL/min, d3-cortisol 5.9 ± 1.8 pmol/100 mL/min, and cortisone 15.2 ± 5.8 pmol/100 mL/min) and splanchnic (cortisol 64.0 ± 11.4 nmol/min, d3-cortisol 12.9 ± 2.1 nmol/min, and cortisone 19.5 ± 2.8 nmol/min) circulations. In adipose, dehydrogenase was more readily detected than reductase (cortisone release 38.7 ± 5.8 pmol/100 g/min). Active recycling between cortisol and cortisone in metabolic tissues in vivo may facilitate dynamic control of intracellular cortisol but makes consequences of dysregulation of 11βHSD1 transcription in obesity and diabetes unpredictable. Disappointing efficacy of 11βHSD1 inhibitors in phase II studies could be explained by lack of selectivity for 11β-reductase.


Bioactivity of analogs of the N-terminal region of gastrin-17.
Gastrin-17-Gly (G17-Gly) has been shown to bind to non-CCK nanomolar and micromolar affinity sites on DLD-1 and HT-29 human colonic carcinoma cells and to stimulate cellular proliferation. However, in previous studies, we showed that C-terminal truncation of the gastrin-17 (G17) to the G17 analog G17(1-12) and then to G17(1-6)-NH(2) did not remove the ability to bind to DLD-1 cells or to activate proliferation. This implies that residues and/or structural motifs required for bioactivity at these receptors rest in the N-terminal region of G17. In this work, radioligand binding studies conducted with further C-terminally truncated analogs revealed that sequences as short as G17(1-4) still bind to a single receptor with micromolar affinity. Additionally, cell proliferation assays showed that G17(1-12) stimulates proliferation of DLD-1 cells, as of HT-29 cells, but the sequences shorter than G17(1-6)-NH(2), including non-amidated G17(1-6), were incapable of stimulating proliferation. These observations indicate that the tetrapeptide pGlu-Gly-Pro-Trp is the minimum N-terminal sequence for binding to the probable growth-promoting site on DLD-1 cells. Since analogs shorter than G17(1-6) are able to bind the receptor, these peptides may be of use for developing selective antagonists.


Long-term functional outcome of repair of large and massive chronic tears of the rotator cuff.
There have been conflicting reports regarding the effect of the size of a tear of the rotator cuff on the ultimate functional outcome after repair of the rotator cuff. While some authors have reported that the size of the tear does not adversely affect the overall result of repair, others have reported that the outcome is less predictable after repair of a large tear than after repair of a small tear. The purpose of the present study was to examine the long-term functional outcome and the recovery of strength in thirty consecutive patients who had had repair of a large or massive tear of the rotator cuff. Thirty consecutive patients who had operative repair of a large or massive chronic tear of the rotator cuff had a comprehensive isokinetic assessment of the strength of the shoulder preoperatively, twelve months postoperatively, and a mean of sixty-five months (range, forty-six to ninety-three months) postoperatively. The functional outcome was assessed with the University of California at Los Angeles shoulder score. All patients reported that they were satisfied with the result and had increased strength compared with preoperatively. There was a significant decrease in pain (p < 0.01) and significant improvements in function (p < 0.01) and the range of motion (p < 0.01). The mean University of California at Los Angeles shoulder score increased significantly from 12.3 points preoperatively to 31.0 points at the most recent follow-up examination (p < 0.01). The mean peak torque in flexion, abduction, and external rotation increased significantly to 80 percent (p < 0.01), 73 percent (p < 0.01), and 91 percent (p < 0.01), respectively, of that of the uninvolved shoulder by the time of the most recent follow-up examination. Repair of a large or massive tear of the rotator cuff can have a satisfactory long-term outcome. The results of the present study suggest that more than one year is needed for complete restoration of strength. The strength of the affected shoulders still did not equal that of the unaffected, contralateral shoulders by the time of the long-term follow-up.


Toward a trophic theory of species diversity.
Efforts to understand the ecological regulation of species diversity via bottom-up approaches have failed to yield a consensus theory. Theories based on the alternative of top-down regulation have fared better. Paine's discovery of keystone predation demonstrated that the regulation of diversity via top-down forcing could be simple, strong, and direct, yet ecologists have persistently failed to perceive generality in Paine's result. Removing top predators destabilizes many systems and drives transitions to radically distinct alternative states. These transitions typically involve community reorganization and loss of diversity, implying that top-down forcing is crucial to diversity maintenance. Contrary to the expectations of bottom-up theories, many terrestrial herbivores and mesopredators are capable of sustained order-of-magnitude population increases following release from predation, negating the assumption that populations of primary consumers are resource limited and at or near carrying capacity. Predation sensu lato (to include Janzen-Connell mortality agents) has been shown to promote diversity in a wide range of ecosystems, including rocky intertidal shelves, coral reefs, the nearshore ocean, streams, lakes, temperate and tropical forests, and arctic tundra. The compelling variety of these ecosystems suggests that top-down forcing plays a universal role in regulating diversity. This conclusion is further supported by studies showing that the reduction or absence of predation leads to diversity loss and, in the more dramatic cases, to catastrophic regime change. Here, I expand on the thesis that diversity is maintained by the interaction between predation and competition, such that strong top-down forcing reduces competition, allowing coexistence.


Predictors of increase in physical activity during a 6-month follow-up period among overweight and physically inactive healthy young adults.
The beneficial effects of physical activity (PA) are well known, but it remains challenging to increase PA among physically inactive and overweight young individuals. The present study aimed to examine how selected psychological and physical characteristics assessed at baseline predict the increase in total PA over a 6-month follow-up among 51 physically inactive and overweight adults (20 women, 31 men; age 26-40 years) who participated in a lifestyle counselling study without supervised PA sessions. Baseline measurements included a questionnaire assessment of sense of coherence and psychological flexibility, heart rate monitoring-based stress/recovery from stress (stress%/recovery% during 24 hours), and body composition. PA volume was elicited through interview. Participants who increased their PA by ≥ 500 metabolic equivalent of task-minutes/week during the follow-up compared with their prebaseline PA level were regarded as able to increase PA. Logistic regression was used to analyze associations of baseline characteristics with PA increase. During the 6-month follow-up, 41% of the participants increased their total PA by ≥ 500 metabolic equivalent of task-minutes/week. The best predictors of the increase in PA were high meaningfulness subscores of the sense of coherence questionnaire (multivariate adjusted odds ratio 1.57, 95% confidence interval 1.04-2.35) and high recovery% during a day off (odds ratio 1.15, 95% confidence interval 1.02-1.30). A strong sense of meaningfulness and better recovery from stress predict an increase in PA among physically inactive and overweight young adults. Therefore, participants with a low sense of meaningfulness and low recovery from stress may require support from other interventions to be able to increase their PA.


Bioclimatic regions influence genetic structure of four Jordanian Stipa species.
Strong environmental gradients can affect the genetic structure of plant populations, but little is known as to whether closely related species respond similarly or idiosyncratically to ecogeographic variation. We analysed the extent to which gradients in temperature and rainfall shape the genetic structure of four Stipa species in four bioclimatic regions in Jordan. Genetic diversity, differentiation and structure of Stipa species were investigated using amplified fragment length polymorphism (AFLP) molecular markers. For each of the four study species, we sampled 120 individuals from ten populations situated in distinct bioclimatic regions and assessed the degree of genetic diversity and genetic differentiation within and among populations. The widespread ruderals Stipa capensis and S. parviflora had higher genetic diversity than the geographically restricted semi-desert species S. arabica and S. lagascae. In three of the four species, genetic diversity strongly decreased with precipitation, while genetic diversity increased with temperature in S. capensis. Most genetic diversity resided among populations in the semi-desert species (Φ(ST) = 0.572/0.595 in S. arabica/lagascae) but within populations in the ruderal species (Φ(ST) = 0.355/0.387 S. capensis/parviflora). Principal coordinate analysis (PCoA) and STRUCTURE analysis showed that Stipa populations of all species clustered ecogeographically. A genome scan revealed that divergent selection at particular AFLP loci contributed to genetic differentiation. Irrespective of their different life histories, Stipa species responded similarly to the bioclimatic gradient in Jordan. We conclude that, in addition to predominant random processes, steep climatic gradients might shape the genetic structure of plant populations.


Factors Associated with Multiple Biomarkers of Systemic Inflammation.
While much is known about correlates of C-reactive protein (CRP), little is known about correlates of other inflammation biomarkers. As these measures are increasingly being used in epidemiologic studies, it is important to determine what factors affect inflammation biomarker concentrations. Using age, sex, and body mass index (BMI) adjusted linear regression, we examined 38 exposures (demographic and anthropometric measures, chronic disease history, NSAIDs, dietary factors, and supplement use) of 8 inflammation biomarkers [CRP, IL1β, IL6, IL8, TNFα, and soluble TNF receptors (sTNFR) in plasma; and prostaglandin E2 metabolite (PGE-M) in urine] in 217 adults, ages 50 to 76 years. Increasing age was associated with higher concentrations of all biomarkers except IL1β. BMI was positively associated with CRP and sTNFR I and II. Saturated fat intake was associated with increased CRP, sTNFRII, TNFα, and IL1β, whereas eicosapentaenoic acid + docosahexaenoic acid (EPA+DHA) intake (diet or total) was associated with decreased CRP, TNFα, and IL1β. Results for sex were varied: CRP and IL6 were lower among men, whereas PGE-M and sTNFRI were higher. Higher CRP was also associated with smoking, hormone replacement therapy use, and γ-tocopherol intake; lower CRP with physical activity, and intakes of dietary vitamin C and total fiber. Although the associations varied by biomarker, the factors having the greatest number of significant associations (P ≤ 0.05) with the inflammation biomarkers were age, BMI, dietary saturated fat, and EPA+DHA omega-3 fatty acids. Our results suggest that potential confounders in epidemiologic studies assessing associations with inflammation biomarkers vary across specific biomarkers.


What carcinoembryonic antigen level should trigger further investigation during colorectal cancer follow-up? A systematic review and secondary analysis of a randomised controlled trial.
Following primary surgical and adjuvant treatment for colorectal cancer, many patients are routinely followed up with blood carcinoembryonic antigen (CEA) testing. To determine how the CEA test result should be interpreted to inform the decision to undertake further investigation to detect treatable recurrences. Two studies were conducted: (1) a Cochrane review of existing studies describing the diagnostic accuracy of blood CEA testing for detecting colorectal recurrence; and (2) a secondary analysis of data from the two arms of the FACS (Follow-up After Colorectal Surgery) trial in which CEA testing was carried out. The secondary analysis was based on data from 582 patients recruited into the FACS trial between 2003 and 2009 from 39 NHS hospitals in England with access to high-volume services offering surgical treatment of metastatic recurrence and followed up for 5 years. CEA testing was undertaken in general practice. In the systematic review we identified 52 studies for meta-analysis, including in aggregate 9717 participants (median study sample size 139, interquartile range 72-247). Pooled sensitivity at the most commonly recommended threshold in national guidelines of 5 µg/l was 71% [95% confidence interval (CI) 64% to 76%] and specificity was 88% (95% CI 84% to 92%). In the secondary analysis of FACS data, the diagnostic accuracy of a single CEA test was less than was suggested by the review [area under the receiver operating characteristic curve (AUC) 0.74, 95% CI 0.68 to 0.80]. At the commonly recommended threshold of 5 µg/l, sensitivity was estimated as 50.0% (95% CI 40.1% to 59.9%) and lead time as about 3 months. About four in 10 patients without a recurrence will have at least one false alarm and six out of 10 tests will be false alarms (some patients will have multiple false alarms, particularly smokers). Making decisions to further investigate based on the trend in serial CEA measurements is better (AUC for positive trend 0.85, 95% CI 0.78 to 0.91), but to maintain approximately 70% sensitivity with 90% specificity it is necessary to increase the frequency of testing in year 1 and to apply a reducing threshold for investigation as measurements accrue. The reference standards were imperfect and the main analysis was subject to work-up bias and had limited statistical precision and no external validation. The results suggest that (1) CEA testing should not be used alone as a triage test; (2) in year 1, testing frequency should be increased (to monthly for 3 months and then every 2 months); (3) the threshold for investigating a single test result should be raised to 10 µg/l; (4) after the second CEA test, decisions to investigate further should be made on the basis of the trend in CEA levels; (5) the optimal threshold for investigating the CEA trend falls over time; and (6) continuing smokers should not be monitored with CEA testing. Further research is needed to explore the operational feasibility of monitoring the trend in CEA levels and to externally validate the proposed thresholds for further investigation. This study is registered as PROSPERO CRD42015019327 and Current Controlled Trials ISRCTN93652154. The main FACS trial and this substudy were funded by the National Institute for Health Research Health Technology Assessment programme.


Association of autistic-like and internalizing traits during childhood: a longitudinal twin study.
Children with autism spectrum disorders often experience severe anxiety and depression, yet the explanation for this association remains unclear. The authors examined the longitudinal relationship between autistic-like and internalizing traits across middle to late childhood in a population-representative twin sample. Participants were approximately 6,000 twin pairs born in England and Wales from 1994 to 1996. Parental reports of autistic-like and internalizing traits were analyzed at ages 7 and 8 (timepoint 1) and again at age 12 (timepoint 2). The direction and etiology of the associations between these traits were examined within a cross-lagged design. Findings revealed an asymmetric bidirectional association between autistic-like and internalizing traits over time. Autistic-like traits at age 7 made a modest but significant contribution to the presence of internalizing traits at age 12. Earlier internalizing traits also influenced the development of later autistic-like traits, although this association was approximately one-half the magnitude. While both traits were moderately to highly heritable, they were largely independent with regard to their genetic influences. Stronger associations were found between the modest shared environmental influences on each trait. Of note, it was autistic-like communication difficulties, rather than social deficits, that made a significant contribution to later internalizing traits. The association between autistic-like and internalizing traits was attributable to reciprocal processes occurring across childhood, suggesting that these traits may serve to exacerbate each other over time. Autistic-like communication difficulties had the most notable impact. This association must now be explored within samples of children with diagnosed autism spectrum disorders and internalizing disorders, since this may help to inform the best timing and targeting of clinical intervention.


In vitro passage alters virulence, immune activation and proteomic profiles of Burkholderia pseudomallei.
Serial passage is a problem among many bacterial species, especially those where strains have been stored (banked) for several decades. Prior to banking with an organization such as ATCC, many bacterial strains were passaged for many years, so the characteristics of each strain may be extremely different. This is in addition to any differences in the original host environment. For Burkholderia pseudomallei, the number of serial passages should be carefully defined for each experiment because it undergoes adaptation during the course of serial passages. In the present study, we found that passaged B. pseudomallei fresh clinical isolates and reference strain in Luria-Bertani broth exhibited increased plaque formation, invasion, intracellular replication, Galleria mellonella killing abilities, and cytokine production of host cells. These bacteria also modulated proteomic profiles during in vitro passage. We presume that the modulation of protein expression during in vitro passage caused changes in virulence and immunogenicity phenotypes. Therefore, we emphasize the need for caution regarding the use of data from passaged B. pseudomallei. These findings of phenotypic adaptation during in vitro serial passage can help researchers working on B. pseudomallei and on other species to better understand disparate findings among strains that have been reported for many years.


Each portion of fruit or vegetable consumed halves the risk of oral cancer.
Medline was used to find relevant papers; reference lists of retrieved publications were also searched. Studies were included if they met all of the following criteria: 1) they contained original data from case-control or cohort studies; 2) the primary outcome was clearly defined as cancers of the mouth, pharynx and hypopharynx (as defined by codes 141, 143-146,148 and 149 of the ninth revision of the International Classification of Diseases or other classifications that included these sites); 3) the exposure of interest was measured as servings (per day, week or month) of fruit, vegetables or both (other exposure measurements, such as mass per day, were excluded unless data were provided to transform information to servings per day); 4) they provided relative risk (RR) estimates and their 95% confidence intervals (CI), or sufficient data to calculate these parameters; 5) were published in the English language; and 6) were published before September 2005. If a study appeared in more than one article, data from the most recent publication were used for the statistical analysis. Studies restricted to oral cancer in subjects aged under 45 years were excluded. Study quality was assessed and data were extracted independently by two investigators, differences being resolved by discussion. All studies were stratified by fruit or vegetable intake to evaluate any dose-response relations, and some of the studies were used to perform multivariate analyses to adjust for several confounders. Separate meta-analyses were conducted for fruit and vegetable consumption. Potential sources of heterogeneity between the studies were examined using the DerSimonian and Laird method. Sensitivity analyses and a multivariate metaregression analysis were performed to examine observed heterogeneity. A total of 71 potentially relevant studies were identified: 16 were included in the meta-analysis. Seven studies had been carried out in Europe, five in the Americas and four in East Asia. Three case-control studies consisted solely of men and one study consisted solely of women. The pooled data allowed 65 802 and 57 993 subjects, respectively, to be included in analysis of fruit and vegetable consumption. The combined adjusted odds ratio (OR) estimates based on 16 studies showed that each portion of fruit consumed per day had a statistically significant effect, reducing the risk of oral cancer by 49% (OR, 0.51; 95% CI, 0.40-0.65). A similar effect was seen for vegetable consumption, with a significant reduction in the risk of oral cancer of 50% (OR, 0.50; 95% CI, 0.38-0.65). Homogeneity testing found a statistically significant heterogeneity for fruit consumption and vegetable consumption studies. This was examined using sensitivity analysis which did not substantially modify the main findings of the meta-analysis. The consumption of fruit and vegetables was found to be associated with a reduced risk of oral cancer. Prospective studies are needed to confirm this result.