The FLOTAC dual technique was performed the next morning before new samples arrived. We used flotation solution 2 (FS2; selleck kinase inhibitor saturated sodium chloride [NaCl] solution; specific gravity [s.g.] = 1.20) and FS7 (zinc sulfate [ZnSO4?7H2O] solution; s.g. = 1.35). For the DNA isolation, we followed the procedure described by Verweij and others.39 All DNA samples were stored at ?20��C and transferred on ice to the NIMR-MMRC, where PCR amplification and detection were conducted in June and November of 2012. A multiplex real-time PCR was used for the simultaneous detection of A. lumbricoides, N. americanus, S. mansoni, and S. stercoralis DNA in fecal samples.31,32,40,41 For DNA amplification, 5 ��L DNA extracted from 0.1 g stool specimens were used as a template in a final volume of 25 ��L with PCR buffer (HotstarTaq Master Mix [5 mM MgCl2 and 2.

5 ��g bovine serum albumin]; Roche Diagnostics, Almere, The Netherlands), 2 pmol each A. lumbricoides-specific primer (Thermo Fisher, Ulm, Germany), 5 pmol each N. americanus-specific primer (Thermo Fisher), 5 pmol each Schistosoma-specific primer (Thermo Fisher), 2.5 pmol each S. stercoralis-specific primer (Thermo Fisher), 1.25 pmol each N. americanus-specific double-labeled probe (Biolegio, Nijmegen, The Netherlands), A. lumbricoides-specific double-labeled probe (Thermo Fisher), S. stercoralis-specific double-labeled probe (Biolegio), and Schistosoma-specific double-labeled probe (Thermo Fisher). Amplification consisted of 15 minutes at 95��C followed by 50 cycles of 15 seconds at 95��C, 30 seconds at 60��C, and 30 seconds at 72��C.

Amplification, detection and data analysis were performed with the Corbett Rotor-Gene 6000 Real-Time PCR System (Corbett Research, Mortlake, New South Wales, Australia) and Corbett Rotor-Gene 6000 Application Software, version 1.7.87 (Corbett Life Science, Cambridge, UK). Negative and positive external control samples were included in each amplification run. The details of all primers and detection probes used in our study are described elsewhere.31,32,40,41 Data management and statistical analysis. The helminth species-specific results derived by each method were entered manually in the participant’s CRF and subsequently transferred into a Microsoft Access 2010 electronic database (Microsoft Corporation 2010, Redmond, WA). Data were analyzed using STATA, version 12 (StataCorp., College Station, TX) and R, version 2.15.2 (R Foundation for Statistical Computing, Vienna, Austria).42 For the comparison of diagnostic methods, the diagnostic results of the first stool sample collected and examined from Batimastat each participant were included in the analysis.

Supporting Information Checklist S1 CONSORT Checklist. (DOC) Click here for additional data file.(222K, doc) Axitinib melanoma Protocol S1 Trial Protocol. (DOC) Click here for additional data file.(147K, doc) Footnotes The authors have declared that no competing interests exist. The authors are grateful to the Velux Foundation and the Swiss National Science Foundation (project no. PPOOA-114941) for financial support of this study. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Increasing evidence suggests that, similar to normal tissues, cancers might also be hierarchically organised. Only a minority of tumour cells, endowed with stem cell-like features, and thus termed cancer stem cells (CSCs), might be responsible for tumour initiation and maintenance (Reya et al, 2001; Pardal et al, 2003; Dalerba et al, 2007a; Visvader and Lindeman, 2008).

Notably, owing to their high expression of DNA repair mechanisms, detoxifying enzymes, such as aldehyde dehydrogenase-1 (ALDH1), and molecular pumps, CSCs might survive radiochemotherapies; thus, possibly causing local recurrences and metastasis formation despite treatment (Dean et al, 2005; Dalerba et al, 2007a; Zhou et al, 2009). Putative CSC populations have been identified in several types of solid tumours, on the basis of the expression of specific markers and on functional stem cell-like properties, including high clonogenicity, differentiation capacity, spheroid formation, and, critically, the ability to reproduce the original tumour on transplantation in immunodeficient mice (Dalerba et al, 2007a; Visvader and Lindeman, 2008).

Phenotypic characterisation of CSCs derived from colorectal cancers is still debated. While initial works identified CD133 molecule as a reliable CSC marker in primary human colorectal cancers (O’Brien et al, 2007; Ricci-Vitiani et al, 2007), a subsequent study has shown that in both mouse and human colorectal cancers, CD133 expression is not restricted to rare cell subsets, but it is detectable in a large majority of tumour cells, irrespective of their tumourigenicity (Shmelkov et al, 2008). Alternatively, the co-expression on tumour cells of CD44, CD166, and EpCAM molecules, has been reported to identify the CSC pool more precisely than CD133 expression alone (Dalerba et al, 2007b). Despite the potentially high clinical relevance of CSCs, little is known about the prognostic value of the expression of putative CSC markers GSK-3 in colorectal cancers. Contradictory findings have been reported about the association between the expression of CD44, in particular of its v6 splicing variant, and tumour progression (Mulder et al, 1994; Herrlich et al, 1995; Weg-Remers et al, 1998).

Similarly, co-twin’s neuroticism was largely not associated with severity of withdrawal-induced depressive or anxious symptoms nor was there a significant interaction especially between this term and zygosity. Critical to our analyses, co-twin’s FTND score consistently predicted both outcomes; furthermore, when the interaction between co-twin’s FTND and zygosity was included as a predictor, this term and the main effect of co-twin’s FTND significantly predicted outcome, with monozygotic co-twin’s FTND score more strongly predicting outcome than dizygotic co-twin’s FTND score. The results of these analyses strongly suggest that the severity of nicotine withdrawal-induced depressive and anxious symptoms is a pharmacological component of the nicotine withdrawal syndrome, which is indexed by ND.

Furthermore, this association appears to be driven primarily by genetic influences on liability to ND and withdrawal, with environmental factors playing a less pronounced role: the main effect of co-twin’s FTND score, which is strongly predictive of outcome in nearly every case, is sharply reduced when the co-twin’s FTND �� zygosity interaction term is included in the model (Equations 2, 4, 7, and 8). Parameter estimates from these regressions further suggest that ND is slightly more predictive of withdrawal-induced anxious symptoms than depressive symptoms. The main effects of co-twin’s MD and GAD are not significantly associated with outcome in any regression. Co-twin’s mean neuroticism score, which is positively and significantly correlated with both GAD and MD, is associated with withdrawal-induced depressive symptoms, but not with anxious symptoms.

Nor was the interaction between zygosity and MD, GAD, or mean N associated with either outcome (Equations 1, 3, 5, and 7, respectively). However, results from the within-individual regressions differ slightly in that a history of GAD was associated with withdrawal-induced anxious symptoms, and mean N was associated with both outcomes. The results of within-individual regressions are largely consistent with previous studies that have reported that an individual’s history of anxiety or depression is predictive of their withdrawal-induced anxious or depressive symptoms (Burgess et al., 2002; Covey et al., 1990; Pomerleau et al., 2000).

However, not all studies include ND as a covariate, which could be problematic: Dacomitinib if ND is positively correlated with psychopathology (as it is in the current study), predictive value could be erroneously attributed to psychopathology. The use of co-twins�� phenotypes as instrumental variables in the current analyses enables us to distinguish between our two hypotheses regarding outcome. The intraindividual analyses indicate that people with a lifetime history of MD or GAD have significantly higher levels of ND than do individuals without such a history.

S. Environmental Protection Agency [U.S. EPA], 1992; Wigle, Collishaw, Kirkbride, & Mao, 1987; Woodward, Hill, & Blakey, 2004). TSP has been www.selleckchem.com/products/Cisplatin.html found to be a cause of lung cancer (National Research Council, 1986; U.S. Department of Health and Human Services [USDHHS], 1986) and heart disease (Glantz & Parmley, 1995; Law, Morris, & Wald, 1997; Taylor, Johnson, & Kazemi, 1992). Recently, a review of the epidemiological evidence concluded that TSP was associated with a significant increase in breast cancer among premenopausal women (California Environmental Protection Agency, Air Resources Board, 2005). Of great concern is the health hazard that TSP exposure poses to children who are still developing physically and biologically.

Compared with adults, children breathe more rapidly, absorb more pollutants because of their small size, have less developed immune systems, and are more vulnerable to cellular mutations (Bearer, 2005), making them more susceptible to the effects of TSP exposure. TSP is associated with a greater likelihood of asthma, triggering an asthma attack, and chronic lung diseases (USDHHS, 1986), and it has been recognized as a cause of sudden infant death syndrome (Anderson, & Cook, 1997; USDHHS, 1986). It has been estimated that more than 20 million children in the United States will be exposed to TSP on a daily basis, with exposure often occurring in the home or the family vehicle (Klerman, 2004). Given the restricted area within which the smoke is circulated, the levels of TSP in cars would seem to pose a significant risk to children.

In a longitudinal study, Sly, Deverell, Kusel, and Holt (2007) found that by age 14, children exposed to TSP in cars were more likely to have a current wheeze, a persistent wheeze, and decreased lung function, relative to children who were not exposed. These risks for children exposed in cars were greater than that of children exposed in the home. Furthermore, results from an observational study in New Zealand (Martin Brefeldin_A et al., 2006) suggest that children of lower socioeconomic status may receive more frequent exposure to TSP in cars, thus furthering health inequalities already being experienced by some of the most vulnerable members of society. Despite the significant health threat that TSP poses and the health benefits that reduced exposure offers, few studies have attempted to measure the levels of TSP in cars, and the methods by which research has been conducted vary in terms of quality and in their findings. Findings from tobacco industry�Caffiliated studies have reported that nicotine and particulate levels in cars vary substantially (range=0.4 ��g/m3 [negligible air quality value] to 1,010 ��g/m3 [extremely poor air quality value]; Guerin, Jenkins, & Tomkins, 2002).

5H). These results demonstrate that amino selleck kinase inhibitor acids 237L, 239S, and 245L play critical roles in the regulation of STAT3, ERK, and JNK, while amino acids 228D, 241T, and 250H are not involved in this regulation. DISCUSSION HCV persistent infection is associated with a variety of human liver diseases, including HCC. However, the mechanisms by which HCV infection causes chronic liver diseases remain unclear. Here, we demonstrated that STAT3, MMP-2, and Bcl-2 are significantly stimulated in the PBMCs of patients with HCV infection and in cell cultures infected with HCV. These results are consistent with those of previous studies showing that MMP-2 and Bcl-2 levels are elevated in the serum of patients with HCC, cirrhosis, and chronic hepatitis (15, 23, 28, 63).

It has been reported that HCV is able to infect not only hepatocytes but also PBMCs (37, 54). Immediately after discovery of the virus in 1989, different group
Intraductal papillary neoplasms of the bile duct (IPNBs) are uncommon neoplasms arising in the biliary tree, which were first described more than a century ago.1 In 1959, Caroli provided a rigorous anatomical description of this entity.2 These neoplasms can arise as either solitary or multifocal lesions within the biliary tree; in the latter scenario they are termed in classical parlance as ��biliary papillomatosis��.3�C6 Intraductal papillary neoplasms of the bile duct show considerable morphologic similarities to intraductal papillary mucinous neoplasms (IPMNs) of the pancreas.

Both entities present with a prominent intraductal papillary component within larger-diameter ducts, both are mucin-producing, both harbour varying grades of dysplasia in the lining epithelium, and both exhibit distinct patterns of differentiation, such as intestinal and pancreatobiliary manifestations.3 Finally, both IPMNs and IPNBs are bona fide precursor lesions of invasive adenocarcinomas at their respective anatomic sites. In view of these similarities, it has been proposed that IPNBs may be the biliary counterpart of pancreatic IPMNs,7 although there are also significant differences between IPMNs and IPNBs in incidence of mucin hypersecretion, relative prevalence of different papilla phenotypes and the frequency of high-grade dysplasia. Reports of IPNBs in the Asian literature also cite aetiologic associations (lithiasis, flukes) that differ from those of IPMNs.

Intraductal papillary neoplasms of the bile duct are predominantly diagnosed in the elderly population and show a slight male predominance. Characteristically, patients suffer from abdominal pain and recurrent cholangitis. The accompanying jaundice arises as a result of the physical obstruction of bile outflow by the neoplasm itself or the viscous inspissated mucin.5 There are no evident risk factors for IPNBs, although some studies have reported an association AV-951 with longstanding biliary inflammation.

No outbreaks selleck chem Brefeldin A of veno-occlusive liver disease associated with pyrrolizidine alkaloids have been reported to our knowledge in East Africa. Pyrrolizidine alkaloids are inert until dehydrogenation by cytochrome P450 3A4 (CYP3A4) in the liver [19], where reactive toxic pyrrolic and N-oxide metabolites directly damage liver sinusoidal endothelial cells and hepatocytes (zone III of the liver acinus) [20]. Pyrroles cause chromosomal damage in a dose-dependent manner, resulting in an inflammatory response that culminates in fibrin deposition [17], [20], [21]. Although plants in both the Asteraceae and Fabaceae families ingested by study participants may contain pyrrolizidine alkaloids, our data shows a strong association between significant liver fibrosis and use of herbs in the Asteraceae family but not the Fabaceae family.

The literature about African traditional herbal medicines is limited and does not explain why this difference might exist. Traditional herbal medicine remedies used in Rakai and throughout Uganda are often mixtures containing multiple herbs [8], [22]. It is possible that herbs in the Asteraceae family are taken at high doses, or potentiate the toxicity of other herbs or hepatotoxins. Two participants with fibrosis reported use of Vernonia amygdalina in the Asteraceae family. This particular herb is commonly used in Africa is thought to have hepatoprotective properties [23]. However, animal studies show that at higher doses, this member of the Asteracaae family may be hepatotoxic.

In an in-vivo rat CCl4 liver injury model, low doses (250�C500 mg/kg) of Vernonia amygdalina were hepatoprotective, but a high dose (750 mg/kg) caused increased hepatoxicity [24]. Herbs from the Lamiaceae family were associated with a 3.4 fold increase in significant liver fibrosis among all participants in our study Dacomitinib (p=0.017). Herbs in the Lamiaceae family have been associated with hepatoxicity in an in-vivo rabbit model [25]. In addition, Aloe, taken by two participants in our study, has been linked in case reports to severe hepatitis [26]. However, data about the potential hepatotoxicity of many herbs used by participants in this study do not exist, or come from animal model studies only that should be interpreted cautiously. The risk of significant fibrosis associated with herb use was similar in the overall and HIV- infected study populations. Data on herb use was limited in the HIV-infected population, and plant family specific analysis was only possible for the Asteraceae family. Only two HIV- infected participants reported using herbs in the Asteraceae family. Despite the small number of HIV-infected participants in this study who reported herb use, it is important to note that ART may alter the toxicity profile of co-administered herbs.

In this way, measurements of the Cr/C and D/V diameters were made. The transducer was then rotated 90 degrees and the procedure repeated. The second set of measurements included the L/R and a repeat of the D/V diameters. For calculating the mean tumor diameter, the PD173955? two separate measurements of the D/V diameter were averaged, followed by averaging the mean D/V diameter and the measurements of the Cr/C and L/R diameters. Tumor size also was determined by direct caliper measurements during laparotomy at week 0 and during necropsy at the end of the study. From woodchucks that were euthanized because of seizures, tumor measurements were also available during subsequent necropsy. The Cr/C, D/V, and L/R diameters were measured and averaged to calculate the mean tumor diameter.

Tumor size also was confirmed by CT prior to the start of the study, whenever possible between 6 and 10 weeks posttreatment, and at the end of the study at weeks 23 to 24. The tumor volume was calculated based on the diameters obtained by US using the formula V = ��abc/6, where a is the mean D/V diameter, b is the Cr/C diameter, and c is the L/R diameter. This formula was chosen because most large liver tumors (i.e., ��1.5 cm in diameter) in woodchucks are irregular and closely approximate an ellipsoid form. Analysis of WHV serum markers. Blood samples were obtained for WHV DNA analysis and serological testing prior to the start of the study; at week 0 prior to the administration of SFV-enhIL-12 or placebo; and then at 2, 4, 6, 10, 14, 18, and 23 to 24 weeks posttreatment.

WHV DNA in serum was determined quantitatively by dot blot hybridization (assay sensitivity, ��1.0 �� 107 WHV genome equivalents per ml (28). WHsAg in serum was determined qualitatively by enzyme-linked immunosorbent assay using dilutions of serum. (12) The cutoff value of this assay was defined as ��0.05 optical density unit. Analysis of T-cell responses. For determining T-cell responses against WHV, in vitro stimulators were used at concentrations previously determined as optimal for cultures of woodchuck peripheral blood mononuclear cells (PBMCs) (31, 32). Viral antigen stimulators consisted of native 22-nm WHsAg (2 ��g/ml) and synthetic peptides of WHsAg and WHcAg. WHs peptide S18 (10 ��g/ml; Sigma-Genosys, The Woodlands, TX) corresponded to amino acids 341 to 360 of WHsAg starting at the N terminus of the large surface protein (32).

WHc peptide C100-119 (10 ��g/ml; Sigma-Genosys) corresponded to amino acids 100 to 119 of WHcAg starting at the N terminus of WHcAg (31). Culture medium and a peptide unrelated to WHV (10 ��g/ml; Sigma-Genosys) were used as negative controls. Recombinant proteins that present tumor antigens in woodchuck liver were not available for Entinostat determining antitumoral T-cell responses.

, 2002; Forrest et al., 2004; Hale et al., 2004b), phosphothioates (Foss et al., 2005), 4(5)-phenylimidazole-containing analogs (Clemens Lenalidomide solubility et al., 2005), and conformationally constrained analogs (Hanessian et al., 2007; Zhu et al., 2007), primarily for the purposes of characterizing them in terms of S1P receptor affinity and the ability to induce lymphopenia. Additional analogs have been employed to evaluate the proapoptotic effects of sphingosine and FTY720 (Don et al., 2007) or as possible antiangiogenic agents (Nakayama et al., 2008). However, this report is the first to use this valuable pharmacological approach to explore the potential of FTY720-related compounds to regulate pulmonary vascular permeability. Our data illustrate the usefulness of this approach as the FTY720 analogs described here exhibit dramatically differential effects on lung EC barrier function.

The FTY720 phosphonate (1R and 1S) and enephosphonate (2R and 2S) compounds display in vitro barrier enhancing properties comparable or superior to S1P and FTY720, whereas the FTY720 regioisomers (3R and 3S) are barrier-disruptive despite being structurally very similar to the parent FTY720 compound (Figs. 2B and and3).3). These results suggest that three of the barrier-enhancing analogs (1R, 1S, and 2R) may be more appealing as potential clinical agents than S1P or FTY720 for blocking ALI-associated pulmonary edema because they exhibit a broader therapeutic index with increased potency in vitro (Fig. 2, B and C).

Our preliminary mechanistic studies indicate that Gi-coupled receptor signaling, tyrosine kinases, and lipid raft domains are involved in mediating the enhanced EC barrier function induced by these analogs, as they are in the S1P response (Table 1). Ongoing studies are seeking to determine the signaling events that account for the differential effects of these compounds on EC barrier function. One intriguing possibility is that FTY720 phosphonate and enephosphonate compounds may not be hydrolyzed by lipid phosphatases because a similar mechanism was noted to result in differential intracellular signaling for a S1P phosphonate analog (Zhao et al., 2007). Our data further demonstrate that orientation changes present in the regioisomers compared to FTY720 are sufficient to produce opposite effects on EC permeability. Understanding how these effects are mediated may provide important additional insights into EC barrier regulation.

The mechanism through which 3R and 3S disrupt the EC barrier does not appear to involve MLC phosphorylation, actin stress fiber formation, or actomyosin contraction (Fig. 4) as observed after thrombin (Dudek and Garcia, 2001). Our data also highlight the importance of stereoisomeric structure in determining the bioactivity Brefeldin_A of these compounds in barrier regulation.

The sections then were sequentially incubated with primary mouse antibodies against E-cadherin (Santa Cruz Biotechnology, Santa Cruz, CA, USA; dilution, 1:150; monoclonal antibody) overnight at 4��C, a biotinylated goat anti-mouse secondary antibody (Santa Cruz Biotechnology) for 30 min, and peroxidase-conjugated streptavidin for 10 min. Samples kinase inhibitor Baricitinib were colored with diaminobenzidine (Boster, Wuhan, China) and counterstained with hematoxylin. Images were captured under an Olympus-BX50F4 microscope (Olympus Corporation, Tokyo, Japan) and quantitatively analyzed using the Image-Pro Plus 6.0 software (Media Cybernetics, Bethesda, USA). Sections of normal colorectal tissue were used as positive controls for E-cadherin staining. Negative controls were prepared by replacing the primary antibody with nonimmune IgG.

Statistical analysis The distributions of the clinical characteristics of the CRC patients and normal controls were analyzed by an unpaired two-tailed t-test. This test was also used to compare E-cadherin expression between the G-allele and GA-allele. The ��2-test was used to test the differences in the allele frequencies between normal controls and CRC patients. The genotype data were further stratified by age, sex, smoking, alcohol intake, tumor location, pathologic grouping and clinical stage of CRC. The odds ratio (OR) and 95% confidence interval (CI) were calculated using an unconditional logistic regression model to evaluate the risk of the CDH1 -347G��GA polymorphism for CRC. We performed all analyses with the SPSS 15.0 software package (SPSS Inc, Chicago, USA).

Values of P < 0.05 were considered statistically significant. RESULTS All the recruited subjects were successfully genotyped. The study included 290 CRC patients and 335 normal controls with available data. The clinical characteristics of the study subjects are summarized in Table Table1.1. No significant differences were noted in the distribution frequencies for sex, smoking and drinking. As shown in Table Table2,2, the G-allele and GA-allele frequencies were 52.8% and 47.2%, respectively, in the CRC patients, and 57.9% and 42.1%, respectively, in the controls. There was no significant difference between the patients and the controls (��2 = 1.671, P = 0.198). A logistic regression analysis revealed that the GA-allele did not increase the risk of CRC (OR = 1.232, 95% CI = 0.898-1.691). However, when the CRC patients were stratified by age, sex, smoking, drinking, tumor location, pathologic grouping and clinical stage, the GA-allele frequency was higher in poorly differentiated CRC patients than in normal controls (��2-test, P = 0.002). In addition, the GA-allele frequency was higher in proximal CRC patients than in normal Carfilzomib controls (��2-test, P = 0.019).

Firstly, serum insulin level was not detected and insulin resistance was previously shown to impair response to peginterferon plus ribavirin in CHC patients [25]. Secondly, we did not analyze HBV genotypes, where it is plausible that some genotypes exhibit ��steatoviruses�� characteristics, as shown in HCV genotype 3 [26]. Thirdly, we didn’t use liver biopsy in determining hepatic steatosis, as Nintedanib purchase this test is invasive and may cause both minor and major complications [27]. In this study, as elevated HBV-DNA and ALT levels were found in all CHB patients, there might be of less value to preclude other causes of liver damage by biopsy. In contrast, the non-invasive hepatic ultrasound showed a sensitivity over 80% and specificity over 90% for steaosis [28].

Therefore, hepatic ultrasound was used to detect steatosis in daily clinical practice for the strength of the least expensive and most convenient modality [29], as was done in our study. Fourthly, we only analyzed Chinese patients and our results need verification in other ethnics. Finally, we have not observed any resistance to Entecavir until the end of this study, which may be due to the relatively short observation period and low amount of subjects. Nevertheless, those weaknesses could not overwhelm the original findings of this prospective unmatched nested case control study, which may change the standard therapy of CHB patients with NAFLD. In summary, this study demonstrated, for the first time, that hepatic steatosis is significantly associated with Entecavir treatment failure in CHB patients.

Current study also confirmed the association of metabolic factors with hepatic steatosis. These novel results raised the issue on developing specific treatment strategy in CHB patients with NAFLD, which needs investigation in the future. Further studies should also focus on the molecular mechanism of steatosis on nonresponse to Entecavir and other antiviral drugs. Supporting Information Table S1 Univariate analysis of factors associated with nonresponse to Entecavir at 24 week. (DOC) Click here for additional data file.(28K, doc) Table S2 Univariate analysis of factors associated with nonresponse to Entecavir at 48 week. (DOC) Click here for additional data file.(29K, doc) Table S3 Univariate analysis of factors associated with nonresponse to Entecavir at 96 week. (DOC) Click here for additional data file.

(28K, doc) Footnotes Competing Interests: The authors have declared that no competing interests exist. Funding: This study is sponsored by the 2010 National Natural Science Foundation of China (Project 81000169), 2010 Excellent Young Investigator Foundation of Health AV-951 Bureau of Zhejiang Province (Project 2010QNA011) and 2011 Excellent Young Investigator Natural Science Foundation of Zhejiang province (Project R2110159). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.