Background: Moderate-severe traumatic brain injury (TBI) is increasingly being understood as a progressive disorder, with growing evidence of reduced brain volume and white matter (WM) integrity as well as lesion expansion in the chronic phases of injury. The scale of these losses has yet to be investigated, and pattern of change across structures has received limited attention.Objectives: (1) To measure the percentage of patients in our TBI sample showing atrophy from 5 to 20 months post-injury in the whole brain and in structures with known vulnerability to acute TBI, and (2) To examine relative vulnerability and patterns of volume loss across structures.Methods: Fifty-six TBI patients  underwent MRI at, on average, 5 and 20 months post-injury; 12 healthy controls underwent MRI twice, with a mean gap between scans of 25.4 months. Mean monthly percent volume change was computed for whole brain , corpus callosum (CC), and right and left hippocampi (HPC).Results: (1) Using a threshold of 2 z-scores below controls, 96% of patients showed atrophy across time points in at least one region; 75% showed atrophy in at least 3 of the 4 regions measured. (2) There were no significant differences in the proportion of patients who showed atrophy across structures. For those showing decline in VBR, there was a significant association with both the CC and the right HPC (P < 0.05 for both comparisons). There were also significant associations between those showing decline in (i) right and left HPC (P < 0.05); (ii) all combinations of genu, body and splenium of the CC (P < 0.05), and (iii) head and tail of the right HPC .Conclusions: Atrophy in chronic TBI is robust, and the CC, right HPC and left HPC appear equally vulnerable. Significant associations between the right and left HPC, and within substructures of the CC and right HPC, raise the possibility of common mechanisms for these regions, including transneuronal degeneration. Given the 96% incidence rate of atrophy, a genetic explanation is unlikely to explain all findings. Multiple and possibly synergistic mechanisms may explain findings. Atrophy has been associated with poorer functional outcomes, but recent findings suggest there is potential to offset this. A better, understanding of the underlying mechanisms could permit targeted therapy enabling better long-term outcomes. Traumatic brain injury (TBI) is increasingly being understood as a chronic and possibly progressive disease, rather than an injury with a circumscribed period of recovery and a static course thereafter  to evaluate risk factors for degeneration; ubiquitous atrophy would suggest that TBI itself is a degenerative disorder. The central aim of this study, therefore, was to begin to shed light on this question of scale.To address the question, we undertook volumetric MRI measurements in TBI patients at approximately 5 and 20 months post-injury and compared the extent of volume loss across time for each patient to a normative control sample, also measured at two time points. We called patients showing significant volume loss “decliners.” In both samples, we examined the whole brain using ventricle-to-brain ratio (VBR), as well as the hippocampi and corpus callosum (CC) and their sub-structures, areas with demonstrated vulnerability to the acute mechanical and neurochemical effects of injury  for each structure in order to ascertain whether one structure showed a greater frequency of decliners than another structure.acute injury would show the greatest chronic atrophy. This is because of growing evidence that while “use” increases brain  volume .Patient participants were 56 males and females with clinically confirmed TBI. See Table The Toronto Rehab Traumatic Brain Injury Recovery Study), which includes cognitive, motor, and neuro-imaging assessments at multiple time points. Inclusion criteria for the larger study comprise: clinically confirmed TBI with central (as opposed to orthopedic) injuries severe enough to warrant in-patient neurorehabilitation; out of PTA by 3 months post-injury; aged between 17 and 75; able to use at least one upper extremity; and, functional command of English. An additional inclusion criterion for the present study was completion of two or more MRIs. Exclusion criteria for the larger study include: past history of TBI; history of psychotic or neurological illness; and, TBI sustained secondary to another neurological event . An additional exclusion criterion for the current study was an intervening neurological event between the first and follow-up MRI with the potential to influence the structural status of the brain .All patient participants were recruited at a large, urban rehabilitation teaching hospital in Toronto from the in-patient Acquired Brain Injury service. The patients were part of an ongoing, prospective study on recovery from TBI , and a mean education of 17.5 years .Twelve healthy adult control participants were recruited to the current study. These individuals were students and staff members at the rehabilitation hospital or friends or family members of students and staff members. Controls were excluded if they had a previous history of TBI, including concussion, or any other disease affecting the central nervous system. The control group was 50% male, with a mean age of 36.3 years . The second scan was at a mean of 20 months post-injury , with a mean gap between scans of 14.8 months . For control participants, the mean gap between scans was 25.4 months . The relatively greater gap in the controls biased hypotheses against Type I errors. The longer gap between control scans allowed for greater non-specific decline to transpire, thereby increasing the threshold for reaching decline in the patients.The study was a prospective, repeated measures design. The normative control group was used to establish the level of decline in each patient for each structure. This was computed for each subject for each structure, as described below. MRIs were administered at two time points. The first was completed for all patients at a mean of 5.2 months post-injury, (The MRI outcome measures were as follows: computed monthly percent change for ventricle to brain ratio (VBR), volumes of the left hippocampus , volumes of the right hippocampus , and CC volumes .All participants were required to pass a rigorous, clinical screening procedure prior to the first MRI assessment. All MRIs were conducted at a separate site , which is part of the same center. All equipment and acquisition parameters were identical for the initial and follow-up assessments. One of two MRI technologists performed all MRIs.TR/TE = 300/13 ms), slice thickness = 5 mm, space 2.5 mm, matrix 256 × 128 axial gradient recalled echo (GRE) TR/TE = 450/20, flip angle = 20°, slice thickness = 3 mm no gap, matrix 256 × 192 axial fluid-attenuated-inversion-recovery (FLAIR) TR/TE = 9000/45 ms, TI (inversion time) = 2200 ms, slice thickness = 5 mm no gap, matrix 256 × 192 axial fast spin echo (FSE) proton density (PD)/T2 TR/TE 5500/30,90 ms, slice thickness = 3 mm no gap, matrix 256 × 192. All above mentioned sequences were obtained with a 22 cm field of view (FOV). The high-resolution isotropic T1 weighted, three-dimensional IR prepped radio-frequency spoiled-gradient recalled-echo (3D IRSPGR) images TI/TR/TE = 12/300/5,TI, FA = 20, slice thickness = 1 mm no gap, matrix = 256 × 256 were acquired in the axial plane utilizing a 25 cm FOV. The entire scanning session lasted approximately 55 min.MRI scans were acquired on a General Electric (GE) Signa-Echospeed 1.5 Tesla HD scanner , using an eight channel head coil. Sequences included sagittal T1  file format and were subsequently converted into  file format that was created at McConnell Brain maging Centre of the Montreal Neurological Institute. Following this procedure, the files were anonymized.A number of image processing steps were performed in order to make MRI data usable for image analysis. The first step was the intensity non-uniformity correction , gray matter (GM), and WM using an automated tissue classification algorithm  by an experienced tracer (JM) from coronally orientated MR images in the anterior-posterior direction. Calculations of volumes were computed automatically by multiplying the number of voxels traced in each slice, by their depth . As described by Watson et al. , 1997, tThe CC was manually traced on the midline sagittal slice of the T1 images using anatomical landmarks in an hierarchical order on a Windows XP Professional workstation  using the Region of Interest module within analyzetm 8.1 . The landmarks based on the midline sagittal slice were, first, no WM or only minimal WM in the cortical mantle surrounding the CC, second, the interthalamic adhesion, and third, the transparent septum and the visibility of the aqueduct of Sylvius. To adjust for total brain volume, total midsagittal CC area and every subregional area in the analyses data were normalized by dividing by each individual's total intracranial volume (ICV). ICV was calculated from the total of GM, WM, and cerebrospinal fluid volumes which were estimated from processing the T1-weighted scans through FSL 4.0  We calculated a z-score for each patient participant using the following method: First, for each structure and substructure, percent change from time 1 to time 2 was calculated for patients and controls using the following formula: [t2 − t1/(t1 + t2)] * 100. Second, monthly percent change was then calculated in order to compare subjects with differing temporal gaps between scans. Third, a z-score was calculated for each patient using his/her observed score , the expected score , and the standard deviation for monthly percent change in the control group. We used a conservative z-score cut-off to classify decliners. Only if the patient was at least 2 z-scores below that of controls was he/she classified as a decliner. This enabled us to compute the percentage of decliners in our sample, in order to address the primary objective of our study.Relative vulnerability of decline of structures/sub-structures and pattern of deterioration. To examine whether structures were equally vulnerable to decline or whether the number of decliners varied across structures, we calculated 95% confidence intervals for each proportion of decliners across structures, and substructures. To examine whether decline in one structure was associated with decline in another, we calculated PHI coefficients and their 95% confidence intervals between structures and between substructures. This enabled us to measure the degree of overlap within subjects in decliner classification across the structures . The PHI coefficient is a measure of association for two binary variables, interpreted similarly to the Pearson Correlation Coefficient.Presentation of findings. Results are presented by each of the three research questions: (1) the overall percentage of people showing decline on at least one structure, and the number of structures on which subjects declined; (2) the relative vulnerability of structures , and (3) the pattern of atrophy .t = 0.897 (df = 72), p = 0.373, Cohen's d = 0.211. The controls had significantly more education than the patients, t = −4.25 (df = 72), p < 0.001, Cohen's d = −1.00.There was no significant difference between the patients and controls for age, percentage of decliners in our sample, we have included patient means with unpaired t-test results  to permit group mean comparisons]. These illustrate highly significant group differences.Table Table In Table Examining similarities and differences across structures in the number of decliners, Table Examining whether atrophy across structures and sub-structures was related, we examined the association between decliners. If a patient showed atrophy across time on one structure, were they likely to also show atrophy on another structure? Table Table The primary purpose of the current study was to gain a better understanding of the scale of atrophy in the chronic phase of TBI. Our cohort was a Canadian sample of patients with brain injuries ranging from complicated mild to severe. More than 96% of our sample showed atrophy over time in at least one region and the large majority showed atrophy in at least three of the four regions measured. Therefore, these findings indicate substantive atrophy—we employed a threshold of at least two z-scores below that of controls—across several structures. Given the relationship between sub-acute atrophy and behavioral and functional outcomes  the extent to which the number of decliners in each structure and sub-structure differed, allowing us to evaluate preliminarily the relative vulnerability of structures examined, and (ii) the extent to which decline in one structure was associated with decline in another. With regard to the first question, we found few differences in the number of decliners across structures and sub-structures, with only the genu differing significantly from the other sub-structures of the CC. Thus, contrary to our hypothesis, the findings in the small number of regions examined in our study do not suggest that one region is more vulnerable to chronic volume loss than another, with the exception of the genu. However, it is possible that limited power may have elevated Type II errors; given the rather large confidence intervals secondary to sample size, the results may under-represent differences between structures.underestimate the degree of association between the structures.Regarding the second question, we found more associations across structures than we had predicted. Subjects showing atrophy in the whole brain also showed atrophy in other structures measured  as expected, which likely does not speak to underlying mechanisms, but rather to VBR's inclusive relationship with the other structures measured. However, we also found that those who declined in one HPC were more likely to also decline in the other. Within the sub-structures, those who declined in the right HPC head were more likely to decline in the tail, and those who declined in genu of the CC were more likely to decline in the body and the splenium. It is important to note that wide confidence intervals secondary to sample size bias the findings in favor of Type II errors, and that these findings may actually acute primary and secondary mechanical, chemical, and electrophysiological changes in the brain following TBI, which in more serious injuries are ultimately associated with necrotic or apoptotic death (Povlishock and Katz, These findings offer some direction for future research into mechanisms. A great deal of research has examined the In broad strokes, atrophy in the chronic stages of injury may reflect volume loss, neuronal death or a reduction in neuronal proliferation and/or survival. Cell death may occur due to delayed apoptotic mechanisms (Colicos et al., Importantly, these candidate mechanisms of atrophy are empirically testable. Ultimately, an understanding is needed of whether a single mechanism or multiple mechanisms influence atrophy, whether different mechanisms affect different regions, and if multiple mechanisms do influence atrophy, whether they are additive or synergistic.ubiquity of atrophy across patients would not support an exclusive genetic explanation of atrophy; for example, base rates of the e-4 allele that has been implicated in the relationship between TBI and dementia are considerably lower than the 96% incidence of patients who showed volume loss in this study.The findings in the current study favor some mechanisms over others, helping at least to constrain hypotheses for future research. The With regard to neuroinflammation, Johnson et al.  showed eIn humans, the identification of behavioral factors that may exacerbate or buffer against volume loss is of high importance given the potential for clinical intervention. It has been postulated that a downward spiral of negative neuroplastic change secondary to disuse may play a role in chronic decline Evans, . Supportnegative association between hippocampi. The positive association between hippocampi suggests a common mechanism deleteriously affecting both. One such mechanism is reduction in new neuronal growth, survival and integration. As is the case for many TBI patients, many of the patients in our cohort at 5 months post-injury and later, had residual physical impairments, reduced volition, had neither returned to work or school, were less socially engaged, and had limited access to resources (Frasca et al., However, other aspects of our findings do not support the interpretations above. For example, we found that hippocampal volume loss on the right was positively associated with volume loss on the left. Experience-dependent volume increases in the hippocampus (Draganski et al., The array of possible interpretations for these findings indicates that much further research is needed to understand mechanisms of atrophy in sub-acute and chronic TBI. Such an understanding is critical for the development of treatment research to avert or abate this atrophy.There were limitations of the current study. The size of the control group was relatively small and may have compromised the reliability of our findings. As well, because the timelines of the two assessments of controls and patients differed, we calculated monthly percent change to compare patients with controls. This calculation assumes a linear month-to-month change, which is not substantiated. There were significant education, but not age, differences between the patient and control groups. However, we speculate that this difference did not contribute to our findings. In a previous study by our group Miller et al.  using ovIn the chronic stage of moderate-severe atrophy, loss of volume is substantive and ubiquitous across patients. Changes may be attributable to tissue shrinkage—the result of lost neuropil, protein and/or fluids—or to cell death, with disconnection and disuse, inflammation and delayed apoptosis contributing independently or interactively. Environmental enrichment could play a role in offsetting these changes, and in the chronic stages of injury is a “no-harm” intervention that warrants investigation. Further research is needed to identify precise mechanisms of atrophy that would help us to develop targeted clinical interventions.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Prostate cancer (PCa) is a global disease causing large numbers of deaths every year. Recent studies have indicated the RTK/ERK pathway might be a key pathway in the development of PCa. However, the exact association and evolution-based mechanism remain unclear. This study was conducted by combining genotypic and phenotypic data from the Chinese Consortium for Prostate Cancer Genetics (ChinaPCa) with related databases such as the HapMap Project and Genevar. In this analysis, expression of quantitative trait loci (eQTLs) analysis, natural selection and gene-based pathway analysis were involved. The pathway analysis confirmed the positive relationship between PCa risk and several key genes. In addition, combined with the natural selection, it seems that 4 genes  with five SNPs  especially rs17172432, might be pivotal factors in the development of PCa. The results indicate that the RTK/ERK pathway under natural selection is a key link in PCa risk. The joint effect of the genes and loci with positive selection might be one reason for the development of PCa. Dealing with all the factors simultaneously might give insight into prevention and aid in predicting the success of potential therapies for PCa. As one of the most destructive cancers worldwide Considering the complexity of the factors inducing disease, especially cancer, the interlacing signaling pathways and cell signal transmission involved in biological activities such as cell survival, development, and apoptosis PCa is one of the most common malignancies in the world. Its high rate of morbidity and mortality bring about enormous losses. So studying the pathogenesis of PCa seems extremely urgent, including determining which genetic factors might be significant in the process. Summarizing recent studies, the pathway might be a vital factor in the development and progression of PCa. In 2007, Caraglia M et al. Meanwhile, recent studies have discovered natural selection in genes and networks based on populations This study was based on the key role function of the RTK/ERK pathway, the HapMap database and our own GWA study data about PCa risk. In this analysis, we explored the pathway via several methods: (1) association analysis between gene polymorphisms in the RTK-ERK pathway and PCa; (2) gene-based pathway testing; and (3) natural positive selection and expression quantitative trait loci (eQTLs) analysis. Combining all of these effective analyses, this study might give us a more comprehensive understanding of the RTK/ERK pathway based on genes and SNP analysis, which would help us discover potential crucial loci or genes and give a wide view on the treatment, prevention, and forecasting of Pca.http://www.hapmap.org), and other databases (NCBI36). Subjects in the ChinaPCA were male Han Chinese recruited from the southeastern in China. They came from different areas, including Shanghai, Suzhou, Guangxi, Nanjing, etc. There were 1417 hospital-based cases that were confirmed to have primary prostate cancer histologically and 1008 health controls that had undergone routine physical examination in local hospitals. Their characteristics are showed in the The analyses in this study were mainly based on the Chinese Consortium for Prostate Cancer Genetics (ChinaPCa) GWAS, the HapMap Project  and JPT (Japanese from Tokyo) were pooled with 89 non-affinity Asian individuals, referred to as the ASN. In this study, the numbers of SNPs in each population were 791,208 (ASN), 849,575 (CEU), and 885,926 (YRI) As an important pathway in the development of cancer, the RTKs-ERK pathway could easily induce cancer when its proteins become mutated. Many genes are known to be involved in this pathway, with some related ones as yet undiscovered. In our study, we included almost all the genes known in the pathway for analysis, comprising 40 genes: EGF, EGFR, PDGF, VEGF, MAP2K1, CCND1, and others located from every chromosome. In order to ensure all the SNPs in the pathway were included, we amplified the length of the genes in both directions for 100 kb. Finally, nearly 50,000 SNPs were involved. With these SNPs, systematic association tests for the risk of PCa were conducted, including single-SNP association tests and a gene-based pathway association test. A logistic regression model implemented in Plink P<0.05) were collected in the gene-based ARTP analysis. In addition, 10,000 permutations were included in the analysis in order to make the statistics more convincing.In the gene-based pathway test, the adaptive rank truncated product (ARTP) method was applied. ARTP method was proposed by Yu K et al. A and iHHB with the limitation of computing with respect to an ancestral or derived core allele. In addition, considering the influence of allele frequency on the core SNP, Voight et al. improved the data analysis. And iHS score was conducted with all SNPs with minor allele frequency >5% that were treated as core SNPs. As to the results, both extremely positive and extremely negative iHS scores would be potentially interesting.Although the GWA study indicated that a number of SNPs were associated with prostate cancer, an area analysis of the haplotype could not be included, which might neglect possible related loci. However, the interactions in terms of the functional consequences and evolutionary history of these loci remain largely unknown. Recently, several studies have proposed natural positive selection based on haplotype analysis in various populations In our study, the web-based tool Haplotter http://www.sanger.ac.uk/resources/software/genevar/). The HapMap III with 726 individuals , Geneva with 75 individuals  and MuTHER healthy female twins with three tissue types  were collected in this analysis. A linear regression model was used take the distribution of normalized expression levels between genotypes into consideration. And a significance threshold of P<0.05 with 10,000-fold permutations was applied to avoid false positive associations.When it came to the expression quantitative trait loci (eQTLs) analysis, not only cis- but tran-, the left SNPs with |iHs|>2 were included. Single cis-eQTL considered all association signals from SNPs within 1 Mb up- and down-stream, and trans-eQTLs were identified as being associated with SNPs located greater than 1 Mb from the probe set. The correlation with nearby gene expression was available in the eQTL database Genevar as measured by probes  software  and Plink. All statistical tests were two sided.As a key pathway in carcinogenesis, the RTKs-ERK pathway could influence the cell, tissue, and organism directly by mutations in its proteins, which would block normal signal transmission from the extracellular milieu to the DNA. In recent years, many studies have focused on this pathway for the pathogenesis and treatment of diseases, particularly cancers. On the basis of the genes and SNPs of this pathway, the study was conducted with the positive selection loci and eQTL analysis involved.P<0.05). When it came to the genes in the RTKs-ERK pathway, three SNPs  in IGF1R presented with ideal P-values , which were in the exon in utr-variant-3-prime. In addition, ERBB2 with six loci  and CCND3 with three loci  were said to be significant in PCa risk considering their P-values.We tested the association between each SNP and PCa risk using a logistic regression model after adjusting for age . The resP<0.05) with 10,000 permutations. The gene-based P-values derived from ARTP methods were significant for genes ERBB2 (P = 0.0034), PTK2 (P = 0.0043), FLT1 (P = 0.0251), RAF1 (P = 0.0173), BIRC2 (P = 0.0089), KDR (P = 0.0477), and CCND1 (P = 0.0394). Meanwhile, the pathway-level test statistic was statistically significant with a P-value of 0.0065 . In addition, the results suggested the association with PCa risk was mainly focused on EGFR. After selecting the potential natural selection loci, we identified the genes with selection in On the basis of the web-based tool Haplotter, iHs values for all three different ethnic groups  in HapMap phase II were obtained. Considering our Asian samples, iHs values in ASN were selected according to the number of SNP in the two data sets. In order to analyze the data comprehensively, we defined the threshold as 0.05. In addition, |iHs|>1.65 was used in the next selection. Then, 26 SNPs came into view, which were mainly focused on two genes, EGFR (in the pathway) and MKRN2, located on chromosomes 7 and 16, respectively . The maxeQTL analysis was conducted with the proposed SNPs according to the Sanger method based on three different databases: HapMapIII, MuTHER healthy female twins, and Geneva GenCord individuals. The positive results are presented in PCa is a worldwide disease that causes enormous losses each year considering its high morbidity and mortality. Recently, many studies have been focused on the function of regulatory pathways in the development and progression of related diseases, especially for cancers, As one of the significant genes, EGFR was proven to be important in PCa risk. The gene located on Chr7p12 was also determined to be ERBB1, a member of the HER/ERBB/EGFR family of receptors. Recent studies had revealed its close relationship with cancers In this analysis, the four genes  we proposed were under natural selection, which supported the views we presented above. There are three different types of natural selections: positive selection, negative selection, and balancing selection. Different types of selection would guide the development or purification of the species, in which either positive or negative selection could be more important. At the level of loci and genes, we presented the conditions of evolution for the RTK/ERK pathway. According to their suggestive thresholds (|iHS|>1.65), positive loci were selected. After combining the associated analysis and the eQTL method, five SNPs  were identified as the significant loci that might play a key role in the Pca risk. Tracking recent research, these loci were said to be the possible key factors in the cancer risk. In 2009, Dong et al. With the natural selection effects, the four genes seemed to be more important in regulating the development and progression of Pca based on human population analysis using linkage disequilibrium (LD)-based methods. Explanations for the development of PCa might be associated with evolution at the gene- and locus-level. In addition, combined with the gene-based pathway analysis and molecular evolution method, we did not only explain the key role of the pathway in the development of PCa, but also gained new insight into the potential role of molecular evolution in the pathway.The presented study combining various analyses has some limitations. First, there are a lot of criteria to evaluate natural selection in a population. As just one of these measurements, iHs might be limited in the estimation. Second, the eQTL and positive selection were based on the available databases, which made it difficult to avoid deficiencies in the results. Finally, every statistical analysis should confirm the deviations. So the real association needs further research.In conclusion, prostate cancer is a worldwide disease that brings about inestimable losses for health and the economy. The RTKs-ERK pathway was said to be an important link in this cancer's development and progression. Combining natural selection, gene-based pathway analysis, and other associated analysis, we proposed that the pathway was significantly associated with PCa risk, in which the mutation of EGFR, ERBB2, PTK2, RAF1, and related SNPs in the genes might be the key link in the evolution of the cancer. However, given that the specific locus in the EGFR with positive selection was located in a non-functional intron, the real function of this intron in the evolution of cancers requires further attention.Table S1The results of association analysis for Pca risk on the SNPs-level with logistic regression model adjusting for age.(DOC)Click here for additional data file.Table S2Expression of quantitative trait loci (eQTLs) analysis with Geneva in three different databases . *the results presented in the tables were significant collecting from the total eQTLs analysis for all SNPs in (DOC)Click here for additional data file.


A cross-sectional exploratory study was conducted to describe the psychosocial health status of persons seeking health care for exposure to Libby amphibole asbestos (LAA). Health indicators including depression, stress, acceptance of illness, and satisfaction with access and financial aspects of care were obtained via electronic and paper-pencil survey. The exposure pathway and demographic data were gleaned from the health record. Of the 386 participants, more than one-third (34.5%) demonstrated significant levels of psychological distress. The oldest group of women had the lowest levels of depression and stress and the highest acceptance of illness. Gender, age, and satisfaction with financial resources were significantly related to depression, stress, and acceptance of illness. Satisfaction with access to care was significant only for stress. No differences in depression, stress, and acceptance of illness were found based on residence, exposure pathway, or insurance status. Professional response to the psychological needs of individuals exposed to acute, sudden, and catastrophic natural disasters or terrorism has been the focus of recent research  and has Libby, a small rural mountain town (population 2600), was contaminated with amphibole asbestos as a result of decades of mining tainted vermiculite ore. In 2002, with the resolve of politicians, medical providers and community members, Libby was placed on the National Priorities List as a superfund site by the United States Environmental Protection Agency (EPA). The community-wide environmental and occupational contamination left Libby with a legacy of economic and insidious health-related problems across generations and a reputation as a place with “air that kills” . In 2009Exposure to microscopic asbestos fibers is linked to several malignant and nonmalignant illnesses including lung cancer, mesothelioma, asbestosis, pulmonary fibrosis, and pulmonary effusions . PersonsIn the latest asbestosis mortality statistics, Lincoln County, where Libby is located, had the highest age-adjusted asbestosis death rate per million people in the USA for residents age 15 and older . As of MIn addition to physical and ecologic damage, technological disasters can affect the psychosocial health of individuals, families, and communities. Anxiety, chronic stress, depression, loss of control, and uncertainty among disaster victims have been documented , while cDepression results in sadness, loss of interest in a person's usual activities, feelings of worthlessness or hopelessness, disturbed sleep or appetite, low energy, and poor concentration , 22. OccIndividuals living in rural areas are particularly vulnerable to depression. According to the 1999 National Health Interview Survey, 2.6 million rural adults in the USA suffer from depression . The preStress results when external forces impact individuals and physical and behavioral responses are triggered. Any agent or stimulus that challenges adaptive capabilities can be considered a stressor , for exaRealistic acceptance of illness is proposed to have a direct influence on how well individuals manage and adapt to living with their health condition . AcceptaFor persons exposed to a deadly toxin, such as those living in areas where amphibole asbestos-contaminated vermiculite was mined and processed, the probability of chronic illness is high along with its negative impact on psychosocial health. Thus, it is important to recognize the antecedents of negative psychosocial outcomes, such as depression, stress, and poor acceptance of illness, in this population and identify these individuals so that appropriate interventions and care management can be initiated. The research reported here was conducted to explore the biopsychosocial health status and health service needs of a national cohort of persons seeking care for exposure to Libby amphibole asbestos (LAA). The purposes of this paper are to (a) describe the psychosocial health status  of the participants, and (b) explore differences in their psychosocial health status based on age, gender, residence, exposure pathway, insurance status, and access, availability, convenience, and financial aspects of health care among this sample. A descriptive cross-sectional design was used for the study. Psychosocial health status and demographic data were collected via electronic and a paper-pencil questionnaire. The exposure pathway and physical health data were gleaned from the participants' health record. Approval for the study was granted by the Institutional Review Board at Montana State University. Data were collected from February through September, 2007, two years prior to the declaration of a public health emergency in Libby, Montana. Written informed consent was obtained, and coded, deidentified data were used in the analysis. Participants were clients of a specialty care clinic in Libby, Montana which has been providing asbestos health screening, follow-up health care, and consultation for exposed individuals since its opening in 2000. At the time of this study, the clinic was caring for more than 1500 patients from 32 states in the USA. To publicize the study, a notice was placed in the clinic newsletter and descriptive posters and brochures were displayed in the clinic waiting room. As clients presented to the clinic, they were asked to participate in the study. Those who consented completed either an electronic (via computer) or paper version of the questionnaire during their clinic visit. In addition, a group of individuals who were exposed to LAA and lived elsewhere in the USA, but continued to seek their specialty asbestos care at the clinic in Libby, were invited to participate in the study via normal clinic correspondence that included a packet containing a letter describing the study, consent form, and a paper copy of the questionnaire.Data collected using the electronic methods were sent through a secure Internet connection to a protected database at the university research office. The paper/pencil data and health status information collected from the health record were electronically recorded and transmitted via the secure Internet site. Summary results from the psychosocial measures were reported back to the clinic weekly as they had a potential for impacting the plan of care. All questionnaire data collected became a part of the client's health record at the clinic.The measures used to describe the psychosocial health status included Center for Epidemiological Studies-Depression Scale (CES-D) ; PerceivThe CES-D is a 20-item self-report designed for initial screening in settings where psychological distress is not routinely assessed and is intended for use with research conducted in the general population . PotentiThe PSS consists of 14 items that measure the degree to which situations in one's life are seen as stressful  and is rThe AOIS was designed to measure persons' acceptance of chronic illness . The 14-The PSQ-III is a survey, with seven subscales, designed to evaluate health services delivery . The 12-The purposes of this study were to (a) descriptively examine depression, stress, and acceptance of illness among persons seeking care for exposure to LAA, and (b) explore differences in those variables based on age, gender, education, asbestos exposure route, residence, health insurance status, and satisfaction with access and financial aspects of healthcare. Data were analyzed using SPSS (version 16). For the first purpose, means (observed) and standard deviations were produced to allow for general comparisons with other well and ill samples described in the literature. Minimal subgroup analysis based on age, gender, and education allowed for a more complete understanding of observed values. P-values for post hoc tests were adjusted using the Bonferroni correction with the exception of the age∗gender interaction where cell frequencies dropped, for example, n = 22 for women 0–49 years, n = 76 for women 50–64 years, leading to a violation of the equality of variance assumption and the use of the Games-Howell procedure. General linear modeling (GLM) was used to evaluate the second purpose with main effects as stated and a single a priori determined interaction of age∗gender. A cohort of 386 patients of a specialty care clinic living in the Libby area, across Montana, and throughout the USA, with a history of environmental or occupational amphibole asbestos exposure, participated in the study. The majority were 50–64 years of age (46.9%); 17.9% had less than a high school education; 69.9% were married; and a slight majority were men (57.3%). Most resided in or near Libby, Montana (73.8%). The primary health insurance was from public sources such as the Centers for Medicare & Medicaid Services, Veterans Administration, and Social Security Administration (24.1%); private insurers (29%), and specialty asbestos health insurance called Health Network America (HNA)/Libby Asbestos Medical Plan (46.9%). N = 386) exposed to LAA. On The first study purpose was to describe the psychosocial health status defined by depression, stress, and acceptance of illness among patients  = 7.37, P < .05, and the age∗gender interaction F = 4.96, P < .05. There initially appeared to be an omnibus difference in depression based on primary insurance status, but in post hoc tests significant differences were not demonstrated for the three means. Multiple comparisons were performed for satisfaction with healthcare access and indicated that those scoring in the lowest quartile were significantly different than the 2nd, 3rd, and 4th quartiles on depression and that the 2nd and 3rd quartiles were different than the highest 4th quartile. Similarly, for the age∗gender interaction, group 6 (women aged 65+) was significantly different from all other groups of both women and men on depression. It is demonstrated in The intent of the second study purpose was to explore differences in depression, stress, and acceptance of illness based on age, gender, education, asbestos exposure route, residence, insurance status, and satisfaction with access and financial aspects of healthcare. For depression, using an ANOVA analysis , omnibusF = 2.97, P < .05, satisfaction with healthcare access F = 5.48, P < .05, and the age∗gender interaction F = 4.81, P < .05. Multiple comparisons were performed for satisfaction with financial resources as well as healthcare access and indicated that those scoring in the lowest quartile were significantly different from the 3rd and 4th quartiles on stress. Additionally, for satisfaction with financial resources, the lowest quartile was different from the second quartile, and for both variables the 2nd and 4th quartiles differed. Mirroring the findings from depression, the age∗gender interaction showed that women in the 65+ age category differed from all other men and women on stress. These differences can be clearly seen in An identical GLM procedure was repeated examining stress with the same grouping variables and interaction effect of age∗gender . OmnibusF = 7.93, P < .05 and the main effect of gender F = 4.20, P < .05. Multiple comparisons were performed for satisfaction with financial resources, and scores in the lowest and 2nd quartiles were significantly different from those in the 3rd and 4th quartiles on acceptance of illness. Although there was no significant age∗gender interaction, the main effect of overall differences between men and women can be seen in Figures 1, 2 and 3. At every age level, women had higher scores on acceptance of illness. Last, the GLM procedure was repeated, examining acceptance of illness with the same grouping variables and interaction effect of age∗gender . OmnibusTo our knowledge, this is the first exploration of the psychosocial health status of persons seeking treatment for exposure to LAA. For depression, stress, and acceptance of illness, no differences in means were found based on residence, exposure pathway, or insurance. Gender, age, and satisfaction with financial resources were significantly related to depression, stress, and acceptance of illness. Satisfaction with access to care was significant only for stress.n = 243; 17.4; sd = 13.5) [n = 236; 16.6; sd = 13.1) [n = 100; 18.3; sd = 13.2) [Based on the severity, length, and all-prevailing nature of the Libby environmental disaster, it could be anticipated that the overall depression level would be high. When comparing the mean depression scores of the Libby cohort to those for groups dealing with a serious chronic illness, such as HIV ( = 13.5) , rheumat = 13.2) , the Lib = 13.2)  and Mont = 13.2) . Reporten = 70; 25.1; sd = 7.6) [n = 181; 22.8; sd = 8.4) [n = 113; 18.1; sd = 6.9) [The Libby cohort mean stress score was not exceptionally high. It had a lower overall stress score when compared to individuals with Parkinson's Disease (d = 7.6)  and thosd = 8.4) , and a sd = 6.9) . Similarn = 1603; 53.6; sd = 11.9) [n = 59; 26.6; sd = 8.4) [n = 786; 34.58; sd = 9.0) [The impact of the disaster and the subsequent aftermath of resulting chronic illness and deaths in the community appear to be somewhat reflected in the scores on the AOI scale. Higher scores are indicative of a higher degree of acceptance. The Libby cohort had a lower mean acceptance score than a group of individuals with post-polio syndrome ( = 11.9) . Howeverd = 8.4)  and a grd = 9.0) , 53 and d = 9.0) . At everIn the multivariable analysis, a cluster of variables was examined to explore potential differences in psychosocial indicator scores. The analysis for depression yielded two significant findings: those with higher satisfaction with health care access were less depressed; and age and gender had an interaction effect with older women being much less depressed than older men. For stress, those with higher satisfaction with health care access and with financial resources had lower levels of stress. Again, age and gender had an interaction effect with older women being less stressed than older men. For acceptance of illness, men were significantly different than women; however, differences based on age were not identified. As with stress, those with higher satisfaction with financial resources for health care had higher degrees of acceptance of their illness.In evaluating the findings, it should be recognized that participants were recruited from a single specialty clinic and not from the Libby community at large. Recruiting from the community at large may have resulted in the inclusion of participants who were not associated with the clinic, were not being treated for LAA exposure or illness, and who may have had different health and illness experiences. Most participants were “local,” for example, recruited from Lincoln County and across Montana potentially limiting the ability to generalize the study findings to other groups of exposed persons.In a study of patients seeking care for exposure to LAA, participants demonstrated moderate levels of stress and acceptance of illness however more than one-third (34.5%) had depression scores indicating a clinically significant level of psychological distress. Gender, age, and satisfaction with financial resources were significantly related to depression, stress, and acceptance of illness. Satisfaction with access to care was significant only for stress.Depression can be reliably diagnosed and effectively treated by primary care providers . The useFurther research is needed to gain a better understanding of the impacts of a disaster and its resulting health effects on men that specifically targets their personal identity and social roles. Understanding the resilience demonstrated by the women, a quality that might account for their adaptability, could be useful to health care providers and community-based nurses working with clients who live in an area affected by an environmental disaster. Studies are also needed that explore the psychosocial health status of exposed persons not affiliated with the specialty clinic in Libby to tease out the clinic providers' influence on the psychosocial health status and access and satisfaction with care.Psychological distress identification, prevention, and intervention strategies, including self-management skills, are needed for persons exposed to environmental and workplace contamination such as the LAA disaster. With the declaration of a public health emergency in June of 2009, federal funding became available to address health issues resulting from the Libby environmental disaster; however, these resources were set up around a one-year recovery program as opposed to the long-term needs and duration of an ongoing environmental disaster. Population-based nurses and other health care providers are uniquely positioned to examine the biopsychosocial human response to an ongoing environmental disaster and intervene appropriately. They are obligated to work with the community to design and integrate best practice self-management interventions to reduce the effect of inordinate adversity from the event's lingering aftermath.


In rapidly developing Internet-user societies, eHealth literacy has become important in promoting wellness. Although previous studies have observed that poor health literacy is associated with less knowledge and screening practice of colorectal cancer (CRC), little is known about whether eHealth literacy is associated with these variables.The present study examined associations between eHealth literacy, knowledge of CRC, and CRC screening practices.Data were analyzed for 2970 Japanese adults  who responded to an Internet-based cross-sectional survey. Knowledge of the definition of CRC, its risk factors and screening practice, previous experience of CRC screening, score on the Japanese version of the eHEALS (J-eHEALS), sociodemographic attributes , and frequency of Internet usage were obtained. Sociodemographic attributes and frequency of Internet usage were used as control variables in the multiple regression and logistic regression models.β = .116, < .001), when the covariables of both eHealth literacy and CRC knowledge were used in the multiple regression model. Moreover, after controlling for sociodemographic factors, which were significantly associated with eHealth literacy and CRC screening practice, an increase of 1 point in the eHEALS score signified that participants were 1.03 times (95% CI = 1.01–1.05) more likely to undergo CRC screening.eHealth literacy was positively associated with CRC knowledge (Internet users with high eHealth literacy are more likely to have knowledge and previous screening practice related to CRC compared to those with low eHealth literacy. The Internet has become a powerful source of information for health and medicine ,2. ApproColorectal cancer (CRC) is the third most common cause of cancer death in the Japanese population, although it is largely preventable . ScreeniThree studies in the US showed that low health literacy was associated with less knowledge and screening practices of CRC -24. In aPrevious studies of eHealth literacy have focused on particular populations: older people , collegeParticipants were recruited in 2009 from registrants of a Japanese Internet research service company and asked to answer a cross-sectional Internet-based survey. The research company had approximately 1,150,000 voluntarily registered participants and obtained detailed sociodemographic data  from each participant at the time of registration. The survey requirement in the present study was to collect data from 3000 men and women aged 20–59 years.To remove selection bias caused by the proportional differences between gender or age group, the participants were classified by gender and placed into four age groups , and they were allocated equally to the eight sample groups . Potential respondents  were randomly and blindly invited from the registered samples in accordance with the set sample size and attributes, and they were invited to participate in the survey via email. The number of potential respondents in each stratified sample group was determined by dividing the quota (n = 375) by the response rate for the corresponding sociodemographic group. This rate was computed from the results of numerous previous surveys conducted by the research company . Internet-based questionnaires were placed in a protected area of a website, and the potential respondents received a specific URL in their invitation email. Potential respondents could log on to the protected area of the website using their unique log-on ID and password. After 375 participants in each group had voluntarily signed an online informed consent form, which had been approved by the institutional review board, and completed the sociodemographic data information form, acceptance of further participants was stopped in each group. The response rate of the total sample was 24.1% . In addition, to remove the influence of CRC diagnosis, 14 participants diagnosed with the condition were excluded from the analyses. The present study received prior approval from the Ethics Committee of the Faculty of Sports Sciences, Waseda University, Japan.The research company provided data on sex, age, marital status, educational attainment, and household income level. These data were collected following the participants’ registration with the research company. The participants were asked to select the category that best described their current condition for sex , age group , marital status , education level , and household income . Moreover, since few participants answered the questions on graduate school, two-year college, career college, and junior high school, the category groups for education level were divided into the following three categories: ≤ high school graduate (high school and junior high school); two-year college or career college (two-year college and career college); and ≥ college graduate (graduate school and college).Daily frequency of information searches on the Internet was assessed by the following four response categories: Every day, 4–5 times/week, 2–3 times/week, or ≤1 time/week.P < .01).The Japanese version of eHEALS (J-eHEALS) was used to assess eHealth literacy levels of participants . J-eHEALKnowledge of CRC was assessed by 20 true/false questions regarding the definition, risk factors, and screening of CRC. This self-administered test was adapted from previous studies of knowledge and attitudes of CRC ,37. The The participants were asked whether they had ever undergone CRC screening by answering “Yes” or “No”.t test was used to examine the differences in eHEALS score and CRC knowledge score between male and female and between married and unmarried individuals. In addition, the differences in eHEALS score and CRC knowledge score among three or more category groups  were examined using one-way ANOVA. Moreover, a chi-square test was employed to evaluate the proportional differences in CRC screening practice for sociodemographic variables and frequency of Internet searching. In accordance with the analytical methodology adopted in previous studies of health literacy and CRC knowledge and practice [P < .05 was considered statistically significant in all analyses. Adjusted ORs and 95% confidence intervals (CI) were calculated for each variable. PASW Statistics 18.0 was used to compute the statistics.Data were analyzed for 2970 adults (response rate: 24.1%) who provided complete information for the study variables. Patients excluded from the data analysis included 16 participants with incomplete information and 14 participants diagnosed with CRC. The practice ,24,38, tt test. Also, P = .07). Moreover, education level (P = .136) and frequency of Internet searching (P = .08) were not statistically significantly related to CRC knowledge test score. Since sex, age group, marital status, and household income were statistically associated with both eHealth literacy level and CRC knowledge level, these variables were included in the multiple regression model as controlling factors.2 = .046, P < .001). When all the controlled variables were entered into the regression model, eHealth literacy was found to be positively associated with CRC knowledge . In addition, all the controlled variables were significant contributors to the knowledge score of CRC. Moreover, age was a stronger contributor than the other controlled variables.P = .38) and frequency of Internet searching (P = .173) were not related to CRC screening practice. By contrast, participants who had undergone CRC screening were more likely to be older adults (P < .001), be married (P < .001), have a higher education level (P = .03), and have higher household income (P < .001) than reference groups.The present study is the first to examine the association between eHealth literacy and knowledge and screening practice of CRC. The present study found that high eHealth literacy was associated with high knowledge about CRC and CRC screening practice. Considering a great increase in the number of Internet users, the Internet may be an important channel for providing information about CRC screening among the general population -21. ThusThe present study found that higher eHealth literacy was associated with higher knowledge and screening practices of CRC even after controlling covariables. These findings are consistent with those observed in the previous studies with respect to associations of health literacy with knowledge and practice about CRC -24. HoweSince the present study demonstrates the positive association between eHealth literacy and the knowledge about CRC and previous CRC screening practice, both designing the CRC information websites for those with low eHealth literacy levels and developing an intervention for enhancing eHealth literacy might be required strategies in order to improve the knowledge and enhance screening practice of CRC. First, the previous studies found that CRC information websites were often too difficult for American adults with limited literacy to use and understand ,21. For Future studies should identify subcomponents of eHealth literacy such as specific skills or health literacy among at-risk subgroups, in order to design interventions that improve eHealth literacy in Japan. For example, although people in Japan with higher frequency of Internet searching have high eHealth literacy, young adults who use the Internet more than 30−60 minutes have lower eHealth literacy level than older adults . This suThe present investigation has some limitations. First, the analysis was cross-sectional, thereby making determinations of cause and effect not feasible. Second, participants were recruited from one Japanese Internet research service company, and thus the relationships assessed may be biased because of the potentially nonrepresentative nature of this sample as general Japanese Internet users . Also, bAmong Japanese adult Internet users, individuals with low eHealth literacy have less knowledge about CRC and are less likely to undergo CRC screening practice. To promote information about CRC screening on the Internet for individuals who need to undergo CRC screening, it is important to improve eHealth literacy among the appropriate populations. In addition, it is essential to design websites containing CRC information specifically for those with low eHealth literacy.


Furthermore, this method can be extended for individual estimation of FOR, TRI, and CLS in various commercially available pulmonary dosage forms.A stability-indicating reversed-phase high performance liquid chromatography (RP-HPLC) method was developed for the simultaneous determination of Formoterol fumarate (FOR), Tiotropium bromide (TRI), and Ciclesonide (CLS) in a pulmonary drug product. The desired chromatographic separation was achieved on the Zorbax SB C8, 5 μm (150 × 4.6 mm) column, using gradient elution at 230 nm detector wavelength. The optimized mobile phase consisted of a 0.2 % v/v perchloric acid as solvent-A and acetonitrile as solvent-B. The developed method separated FOR, TRI, and CLS in the presence of its five unknown degradation products within 10 minutes. The stability-indicating capability was established by forced degradation experiments and the separation of unknown degradation products. The developed RP-HPLC method was validated according to the International Conference on Harmonization (ICH) guidelines. This validated method was applied for the simultaneous estimation of FOR, TRI, and CLS in commercially available Triohale This is a significant improvement over the current therapy involving two to three inhalers which can be confusing for some patients. Although there is no cure for COPD, the use of combination inhaler drug product will reduce symptoms, increase lung function, decrease the number of times patients are hospitalized, and lead to improved quality of life [The combination inhaler drug combines three essential medicines that are recommended for use in patients with severe Chronic Obstructive Pulmonary Disease (COPD). It contains a long-acting anticholinergic (Tiotropium), a long-acting beta-2-agonist (Formoterol), and an inhalable corticosteroid (Ciclesonide). Recent research shows that COPD is a multi-component disease and the individual components of combination pulmonary drug product target different aspects of the disease. Patients suffering from COPD who were prescribed combination inhaler drug product  determination of FOR, TRI, and CLS in pulmonary drug product formulations, by HPLC and other techniques –10. A deet al. , describd method  is not sterature , limit oterature .®, Cipla Ltd.). The chemical structures, UV spectrums, and chemical names of FOR, TRI, and CLS are presented in The combination of FOR, TRI, and CLS is not official in any pharmacopoeia. So far, no RP-HPLC stability-indicating method has been reported for the rapid simultaneous determination of FOR, TRI, and CLS in pulmonary drug product. Therefore, it is necessary to develop a new rapid and stability-indicating method for simultaneous determination of three compounds  in pulmonary drug product. The proposed method is able to separate FOR, TRI, and CLS from one another and from all five unknown degradation products within 10 minutes. Thereafter, this method was validated according to the ICH guidelines  and succThe main objectives of the RP-HPLC method development for the rapid and simultaneous determination of FOR, TRI, and CLS in the pressurized metered-dose inhaler were: the method should be able to determine assay of the three compounds in a single run and should be accurate, reproducible, robust, stability-indicating, linear, free of interference from blank/placebo/degradation products, and straightforward enough for routine use in quality control laboratories.The spiked solution of FOR (0.6 μg/mL), TRI (0.9 μg/mL), and CLS (20 μg/mL) was subjected to separation by RP-HPLC. Labeled claim of compounds and its working concentration is presented in FOR, TRI, and CLS are well-resolved with each other and also well-resolved with all five unknown degradation products in the reasonable time of 10 minutes. There was not any chromatography interference due to blank (diluent) and placebo at the retention time of FOR, TRI, and CLS which is presented in After satisfactory development of the method, it was subjected to method validation as per ICH guideline .Specificity is the ability of the method to measure the analyte response in the presence of its potential impurities . Forced Degradation was observed when the drug product was subjected to acid hydrolysis , base hySystem suitability parameters were measured so as to verify the system performance. System precision was determined on six replicate injections of the standard preparation . All impThe precision of the assay method was evaluated by carrying out six independent determinations of FOR, TRI, and CLS  test samples against the qualified working standard. The method precision study shows the repeatability of the results obtained by the testing method. The % RSD (n=6) was 0.3 % for FOR, 0.5 % for TRI and 0.7 % for CLS, which are well within the acceptable limit of 2.0%. It was confirmed from results that the method is precise for the intended purpose .The purpose of this study was to demonstrate the reliability of the test results with variations. The reproducibility was checked by analyzing the samples with a different analyst, using a different chromatographic system and column, on a different day. The analysis was conducted in the same manner as the method precision and the % RSD of all six sets of sample preparations was determined . The % RThe accuracy of an analytical method is the closeness of test results obtained by that method compared with the true values. To confirm the accuracy of the proposed method, recovery experiments were carried out by the standard addition technique. The accuracy of the method was carried out by adding known amounts of each drug corresponding to three concentration levels; 50, 100, and 150% of the label claim  along wiThe linearity of an analytical method is its ability to elicit test results that are directly, or by a well-defined mathematical transformation, proportional to the concentration of analyte in a sample within a given range. The nominal concentrations of the standard and test solutions for FOR, TRI, and CLS were 0.6, 0.9 and 20 μg/mL, respectively. The response function was determined by preparing standard solutions at five different concentration levels ranging from 0.3–0.9 μg/mL for FOR, 0.45–1.35 μg/mL for TRI, and 10–30 μg/mL for CLS . The response was found to be linear from 50% to 150% of the standard concentration. The regression statistics are shown in The robustness of an analytical procedure is a measure of its capacity to remain unaffected by small, but deliberate variations in method parameters and provides an indication of its reliability during normal usage. The effect of change in flow rate (± 0.2 mL/min) and column oven temperature (± 5°C) on resolution (between FOR and TRI), theoretical plates, and tailing factor were studied. It was confirmed from results that the method is robust with respect to variability in above conditions .Drug stability in pharmaceutical formulations is a function of the storage conditions and chemical properties of the drug and its impurities. The condition used in stability experiments should reflect situations likely to be encountered during actual sample handling and analysis. Stability data is required to show that the concentration and purity of analyte in the sample at the time of analysis corresponds to the concentration and purity of analyte at the time of sampling. Stability of the sample solution was established by storage of the sample solution at ambient temperature (25°C) for 24 hours. The sample solution was re-analyzed after 12- and 24-hour time intervals, and assays were determined for the compounds  and compared against the fresh sample. The sample solution did not show any appreciable changes in assay value when stored at ambient temperature up to 24 hours, which is presented in The present method is applied to the estimation of drugs in the commercially available metered-dose inhaler .®, Milford, MA, USA).The working standards and placebo of the drug product were provided by Dr. Reddy’s laboratories Ltd., Hyderabad, India. HPLC grade acetonitrile was obtained from J. T. Baker . GR grade perchloric acid was obtained from Qualigens Ltd. 0.45 μm nylon membrane filter, and nylon syringe filters were purchased from Pall Life Science limited (India). High purity water was generated by using Milli-Q Plus water purification system . The thermal stability study was performed in a dry air oven .TM system , consisting of a binary solvent manager, sample manager, and PDA (photo diode array) detector. System control, data collection, and data processing were accomplished using Waters EmpowerTM-2 chromatography data software. The chromatographic condition was optimized using the Zorbax SB C8,  column. 0.2% v/v perchloric acid was used as solvent-A and acetonitrile was used as solvent-B. The separation of FOR, TRI, CLS, and all unknown impurities was achieved by gradient elution using solvent-A and B  kept inside the 100 mL glass beaker, had about 50 mL of diluent added to it, which insured that the discharge volume did not drop below 25mm under the surface of the solvent. The pressurized canister was shaken for 15 seconds in an inverted position. The canister was placed invertedly in the hole of the stainless steel base plate, discharged one spray, and then waited for two seconds. The container was shaken for at least five seconds. Once again, the canister was discharged. The same procedure was repeated another eight times. The pressurized container was removed and washed along with the valve and stem with five to ten mL of diluent. The washing solution was collected in the same 100 mL beaker. The entire beaker (holding solution from two to three washings) content was transferred to the 100 mL volumetric flask. This solution was then diluted up to the mark with diluent and mixed well.The placebo pressurized canister was taken and same procedure was followed as per sample preparation.The sample solution preparation was followed for the marketed sample preparation.A rapid, RP-HPLC method was successfully developed for the simultaneous estimation of FOR, TRI, and CLS in a pressurized metered-dose inhaler. The developed method is selective, precise, accurate, linear, and robust. Forced degradation data proved that the method is specific for the analytes and free from the interference of placebo and unknown degradation products. The run time (10 min) enables rapid determination of the pressurized metered-dose inhaler. Moreover, it may be applied for individual and simultaneous determination of FOR, TRI, and CLS compound in the pharmaceutical drug product (pulmonary drug formulation) and drug substance. Also, it can be utilized for determination of assay, content uniformity (μg/spray), emitted dose by TSI (Twin stage impinger), and Aerodynamic particle size distribution by ACI (Anderson cascade impactor) of pulmonary drug products.


Schizophrenia is a severe psychotic disorder characterized by significant disturbances in thinking, perception, emotions and behavior. Even if it is not a very frequent disorder, but it is the most burdensome and costly illnesses worldwide. The total population was approximate 1.3 billion and there are approximate 8 million schizophrenic patients in China. Despite the wide-ranging financial and social burdens associated with schizophrenia, but there have been few cost-of-illness studies of this illness in China.To evaluate the economic cost of schizophrenic patients in China.356 schizophrenic patients who met with DSM-IV criteria were enrolled and investigated with the Economic Burden Questionnaire(EBQ), 299 schizophrenic patients completed the study for 12 months. All the data were combined and classified by researcher. EBQ include all kinds of cost such as direct cost, indirect cost and total cost as well. It was filled in by patients and their close caregivers. Comparison of cost was made between not only out-patients and in-patients but also urban patients and rural patients. Multiple stepwise regression analysis was made to identify the main influence factors of total cost.P < 0.05). (ii) There was significant difference in per case per annum total cost, direct cost, direct medical cost, cost due to lost working-days and disability between urban and rural schizophrenic patients (P < 0.05), the former is higher than the latter. (iii) The results of multivariate stepwise regression analysis show that five variables were significantly correlated with higher cost: professional status(cadre), diagnostic subtype, urban or rural patients(urban patients), in-patients or out-patients(in-patients) and researcher centre(southern center). The standardized regression coefficient were 0.308, 0.218, 0.212, 0.156 and 0.149 respectively, the correlation of determination R square was 0.2741, F = 15.651, P < 0.0000. These characteristics explain 27.41% of the variability in the total cost.(i) The per case per annum total costs, direct costs and indirect costs of schizophrenia amounted to US$2586.21, US$862.81(33.4%) and US$1723.40(66.6%) respectively. The per case total cost, direct cost and direct medical cost of in-patients were more higher than out-patients ((i) Economic cost of schizophrenia were serious, we must pay close attention to it. (ii) The indirect cost are the majority of the total cost. The cost of urban patients are more higher than the cost of rural patients, the cost of in-patients are more higher than the cost of out-patients. Schizophrenia is a chronic, devastating mental disorder characterized by distortions of thinking and perception. About 1.1 percent of the population age 18 and older have schizophrenia. It is very costly for families and society. The expensive costs of schizophrenia imposes a disease burden on themselves, health care providers, their families and the wider society . The totThe treatment for schizophrenia is still a major clinical challenge to psychiatrists. The characterized psychotic symptoms of schizophrenia such as hallucination and delusion, inappropriate affect, loss of association induced the treatments more complex. The prognosis of schizophrenia is poor for at least one third of patients. Schizophrenia patients have a higher risk of comorbidity and social exclusion and the mortality rate is more than twice that in the overall population. As a result, schizophrenia is the most costly psychiatric illness currently . An ItalInternational cost of illness (COI) studies for schizophrenia show a wide variation of annual costs -16. MangIn Canada, Goeree used a prevalence-based cost-of-illness (COI) approach to estimate the financial burden of schizophrenia in 2004. Results showed that the direct healthcare and non-healthcare costs were estimated to be 2.02 billion Canadian dollars (CAN dollars) in 2004. There were 374 deaths attributed to schizophrenia. The cost of lost productivity due to unemployment, absence from work and an additional productivity morbidity estimate of 4.83 billion CAN dollars, for a total cost estimate of 6.85 billion CAN dollars. The largest part of the total cost estimate was for productivity losses associated with morbidity in schizophrenia . Their results also showed that total cost estimates were most sensitive to alternative assumptions regarding the additional unemployment due to schizophrenia .In Australia , Carr esSchizophrenia accounts for 2.5% of total health care expenditures in the US. The cost of all mental illness in the US has been estimated at US$103.7 billion, of which schizophrenia alone accounts for US$22.7 billion .Lee investigated the economic costs of outpatients with schizophrenia in Taiwan China, and surveyed the influence factors of costs. The average annual total cost was approximately US$16,576 per patient. The direct and indirect costs were 13% and 87% of the total costs, respectively, the indirect costs were higher than the direct costs. The productivity loss of both the patients and their caregivers was the major component of the indirect costs. The main influence factors of costs were patient’s age, global functions, severity of extrapyramidal symptoms, type of antipsychotics and patient’s illness duration [The total population was approximate 1.3 billion in China. There are approximate 8 million schizophrenic patients in China . DespiteThe present paper aims at: (i) evaluating per case per annum total economic costs, direct costs and indirect costs of schizophrenia using a bottom-up approach; (ii) comparing the differences of the cost between in-patients and out-patients; (iii) comparing the difference of the cost between urban-patients and rural-patients; (iv) empirically determining the explanatory variables of total cost variations. The final purpose being that of offering useful information to policies makers and highlighting the economic importance of schizophrenia.The present study is a prospective prevalence-based ,18 multiPatients eligible for the study had a diagnosis of schizophrenia, defined according to the Diagnostic and Statistical Manual of Mental Disorders-fourth edition (DSM-IV) criteria.Each centre required to recruit patients, meeting the inclusion and exclusion criteria.Inclusion criteria were:i. Meet with the diagnostic criteria of schizophrenia of DSM-IV;ii. Age between 16 and 65 years old;iii. There is one caregiver at least and knew well the information of the illness, treatment and costs during the period of study;iv. No plan to emigrate during the period of study;v. Age of caregiver is more than 18 years old,educational time is more than 5 years, caregiver can understand the content of the Economic Burden Questionnaire (EBQ);vi. Informed consent.Exclusion criteria were:i. With serious physical disease;ii. Information is not particular or not credible;iii. Plan to emigrate in 1 year;iv. The caregiver is suffering from mental disorder;v. Drug or alcohol abusers.Each of the centers provided the list of all patients meeting the inclusion and exclusion criteria. Researchers selected patients randomly according to random number chart and inclusion criteria. 356 schizophrenic patients were enrolled and 299 schizophrenic patients completed the study and their data was integrated. 57 case of patients drop out for different reasons, 28 revoked the informed consent, 12 had concurrent illness, 5 emigrated, 12 could not provide complete information.Specifically instruments EBQ were design to collect data and measure costs. EBQ consisted of Part A and Part B, Part A gathered data on: (1) socio-demographic data referred to gender, age and marriage as well; and (2) clinical data referred to duration of illness, diagnostic subtype, time in contact with hospital, previous hospitalizations in mental hospitals as well. Part B gathered data on: economic costs and time costs and other resources use as well.Part A was filled by psychiatrists who followed up the patients during the period of study. Part B was submitted by psychiatrist to the patients’ main caregiver  who filled Part B.The total cost components included in the present study can be classified as direct costs and indirect costs.Direct costs components concerned:i. Direct medical costs--treatment costs and other costs of medical services;ii. Direct non-medical costs--all other resource use related to a disease.iii. Indirect costs components concerned: (i) Value of damaged properties; (ii) The costs due to lost working-days and disability incurred by patients and caregivers; (iii) Mortality costs--loss of productivity to suicide and related mortality.A bottom-up approach ,18,19 waTo analyze per case per annum total costs, direct costs, indirect costs and the proportion of each part. To compare the differences of the cost not only between in-patients and out-patients but also between urban-patients and rural-patients, to determine the explanatory variables of cost variations. As variables were not normally distributed, we used the non-parameter Mann–Whitney U test.We set up a multi-variable regression model to identify the factors that could explain the variability of average total cost of a patient. As regressions of total cost we used the following socio-demographic and medical variables recorded in our questionnaire.Demographic variable ;Socio-economic variable ;Geographic variable;Clinical variable and variable related with illness .Because total cost had a slightly skewed distribution, a logarithmic transformation was used in analyzing the relationship between total cost and the other independent variables. Thus the dependent variable presented an approximately normal distribution. Polytomous variables (i.e. discrete variables which have more then two options) like occupational status, educational status and diagnostic subtype were transformed into dummy variable.All variables were entered into a stepwise backward regression model, the final model being that which offered the best explanation for total costs, using the set of available variables.Statistical analysis was performed with SPSS 12.0 for Windows Statistical Package.Sample characteristics are presented in Table The per case per annum total cost, direct cost and indirect cost are shown in Table Average annual total cost per patient was of US$ 2586.21, the per case per annum direct cost and indirect cost amounted to US$862.81 (33.4%) and US$1723.40 (66.6%) respectively. Direct medical cost was US$714.86, it was the great majority of direct cost, direct non-medical cost was only 5.8% of total cost. Cost due to lost working-days and disability was the great majority of indirect cost. Indirect cost can be split up patients’ (39.1%) and caregivers’ (25.6%) cost. We have not found suicide cases because of the researcher methods, objective reasons and the traditional idea of Chinese and culture background in China, hence mortality cost was 0. Value of damaged properties was US$49.63 (1.9%).The comparison of costs between out-patients and in-patients are given in Table P < 0.05). There was no difference in other cost between in-patients and out-patients (P > 0.05).With non-parameter Mann–Whitney U test, we found that the per case total cost, direct cost and direct-medical cost of in-patients were more higher than out-patients . There was no difference in other cost between in-patients and out-patients (P > 0.05).With non-parameter Mann–Whitney U test, we found that the per case total cost, direct cost, direct medical cost and cost due to lost working day and disability of urban-patients were more higher than rural-patients .Table The burden of schizophrenia is large and multifaceted. Economic costs are one of the most important components. This study provided an overall estimate of the costs of schizophrenia, but it can not capture all costs, as noted by other researchers, two types of the costs were always underestimated: the cost to families and the costs of publicly owned capital.In the present study, the sample were selected from two areas, one of which was from northern China and another was from southern China, the two centers were not selected randomly, hence, the results of the study reflected only the cost of schizophrenia in two areas of China. Centers taking part in were located in the north and south of China, but none in the west and east of the country. The sample seems not to be representative of the cost of schizophrenic patients in China (mainland). The sample was not quite large, only patients, not centre, were randomly selected, and only two centers were selected. We have not found suicide cases, however, the cost due to suicide are the important parts.We study the economic cost of schizophrenia with human capital approach(HCP), however, HCP has many limitations, it has many assumptions that are not likely to hold, e.g. it assumes that there is full employment. Goeree  valuatedDespite these limitation, the results from the present study show that schizophrenia cause a substantial economic burden to healthcare systems, community, other caregivers and society. The per case per annum total cost, direct cost and indirect cost amounted to US$2586.21, US$862.81 (33.4%) and US$1723.40 (66.6%) respectively. Indirect costs were the great majority of the economic cost of schizophrenia. In the indirect cost, cost due to lost working days and disability were the great majority. Schizophrenia is correlated to loss of working days, lack of well-being and poor levels of social functioning. Theses are the main results drawn from the analysis of indirect costs that appear to be exclusively borne by patients, caregivers and ultimately society as a whole. These findings are consistent with the majority of results of other cost of illness studies on schizophrenia where indirect costs vary from 48% to 86.5% of total cost . Hence wChina is a large developing country, the per case pure income of rural residents was about US$450, about 60% patients of our sample were peasants, students or unemployed, the total cost, amounted to US$2586.21, was heavy for them and their families who had been very poor because of the chronic illness.A study carried out by Andlin-Sobock  shown thThe total cost, direct cost and direct medical cost of per case of in-patients was higher than out-patients of schizophrenia. The reason, might be stated that the medical model in China was different from other countries. There are various medical model for the management of schizophrenia such as day-care center, day-hospital, patients’home, community mental health centers (CMHCs), sheltered workshops, psychiatric ambulatories and national health service as well in developed countries . HoweverThere was significant difference in per case per annum total cost, direct cost, direct medical cost, cost due to lost working-days and disability of patients between urban and rural schizophrenic patients, the former is higher than the latter. In China, expensive new antipsychotics were mainly applied to urban patients but few to rural patients, drug cost was the main part of direct medical cost to a turn, lead to the increase of direct cost and total cost. Cost due to lost working days and disability of urban patients are higher than rural patients because the economic income of urban residents was higher than rural residents in China.Multivariate regression analysis shows that cadre professional, paranoid type schizophrenia, urban patients, in-patients and southern researcher centre were correlated with higher total costs.The reason why the cost of urban patients and in-patients were higher is in line with the explanation above. Cadres (civil servants) are a special population with higher economic level in China. In general, their income was higher than peasants and people being unemployed, they would pay more money to the treatment of schizophrenia. It is differs from the finding of Percudani in Italy ,25, theySimilar with cadres (civil servants), most urban schizophrenic patients had regular income, good family economic condition, more medical knowledge and saw doctros in the early stage of the illness. They usually took new and expensive medicine. Conversely, most rural patients were peasants or people being unemployed, they had lower economic income and poor family economic condition, they could not afford to enormous economic pressure. These patients could not be hospitalized in time and they usually took cheaper medicine, so their costs were lower. Residual type schizophrenic patients had longer duration of illness and poor prognosis, with more difficulties in treatment and rehabilitation and higher probability of hospitalization, so their costs were higher.The reason why southern researcher centre correlated with higher costs was complex. The main reason was associated with economic development.Economic cost was a complex index which was affected by many factors, not just related with the medical factors. In addition to the five factors getting into the regression equation, there were a lot of other factors influencing the economic cost of the schizophrenic patients. So the five factors can only explain the dependent variable  27.41%, other factors and accidental factors accounted for 72.59%.The major strengths of this study are that of having clearly showed that (i) the economic burden of schizophrenia was serious in China, (ii) the highest proportion of the economic cost of schizophrenia was indirect cost, the cost of urban patients are more higher than the cost of rural patients, the cost of in-patients are more higher than the cost of out-patients; (iii) We must pay close attention to the economic burden of schizophrenia.The authors declare that they have no competing interests.JZ, XG and MC contributed equally to the study. JZ, MC, ZS and XG carried out the collection and analysis of data, JZ and ZS contributed in drafting the manuscript. JZ contributed to the design of the study. JZ had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. All authors read and approved the final manuscript.