Challenges in Translational Research: The Views of Addiction Scientists

Objectives

To explore scientists'' perspectives on the challenges and pressures of translating research findings into clinical practice and public health policy.

Methods

We conducted semi-structured interviews with a purposive sample of 20 leading scientists engaged in genetic research on addiction. We asked participants for their views on how their own research translates, how genetic research addresses addiction as a public health problem and how it may affect the public''s view of addiction.

Results

Most scientists described a direct translational route for their research, positing that their research will have significant societal benefits, leading to advances in treatment and novel prevention strategies. However, scientists also pointed to the inherent pressures they feel to quickly translate their research findings into actual clinical or public health use. They stressed the importance of allowing the scientific process to play out, voicing ambivalence about the recent push to speed translation.

Conclusions

High expectations have been raised that biomedical science will lead to new prevention and treatment modalities, exerting pressure on scientists. Our data suggest that scientists feel caught in the push for immediate applications. This overemphasis on rapid translation can lead to technologies and applications being rushed into use without critical evaluation of ethical, policy, and social implications, and without balancing their value compared to public health policies and interventions currently in place.     

Blood autoantibody and cytokine profiles predict response to anti-tumor necrosis factor therapy in rheumatoid arthritis

Introduction

Anti-TNF therapies have revolutionized the treatment of rheumatoid arthritis (RA), a common systemic autoimmune disease involving destruction of the synovial joints. However, in the practice of rheumatology approximately one-third of patients demonstrate no clinical improvement in response to treatment with anti-TNF therapies, while another third demonstrate a partial response, and one-third an excellent and sustained response. Since no clinical or laboratory tests are available to predict response to anti-TNF therapies, great need exists for predictive biomarkers.

Methods

Here we present a multi-step proteomics approach using arthritis antigen arrays, a multiplex cytokine assay, and conventional ELISA, with the objective to identify a biomarker signature in three ethnically diverse cohorts of RA patients treated with the anti-TNF therapy etanercept.

Results

We identified a 24-biomarker signature that enabled prediction of a positive clinical response to etanercept in all three cohorts (positive predictive values 58 to 72%; negative predictive values 63 to 78%).

Conclusions

We identified a multi-parameter protein biomarker that enables pretreatment classification and prediction of etanercept responders, and tested this biomarker using three independent cohorts of RA patients. Although further validation in prospective and larger cohorts is needed, our observations demonstrate that multiplex characterization of autoantibodies and cytokines provides clinical utility for predicting response to the anti-TNF therapy etanercept in RA patients.     

Current Practice of Public Involvement Activities in Biomedical Research and Innovation: A Systematic Qualitative Review

Background

A recent report from the British Nuffield Council on Bioethics associated ‘emerging biotechnologies’ with a threefold challenge: 1) uncertainty about outcomes, 2) diverse public views on the values and implications attached to biotechnologies and 3) the possibility of creating radical changes regarding societal relations and practices. To address these challenges, leading international institutions stress the need for public involvement activities (PIAs). The objective of this study was to assess the state of PIA reports in the field of biomedical research.

Methods

PIA reports were identified via a systematic literature search. Thematic text analysis was employed for data extraction.

Results

After filtering, 35 public consultation and 11 public participation studies were included in this review. Analysis and synthesis of all 46 PIA studies resulted in 6 distinguishable PIA objectives and 37 corresponding PIA methods. Reports of outcome translation and PIA evaluation were found in 9 and 10 studies respectively (20% and 22%). The paper presents qualitative details.

Discussion

The state of PIAs on biomedical research and innovation is characterized by a broad range of methods and awkward variation in the wording of objectives. Better comparability of PIAs might improve the translation of PIA findings into further policy development. PIA-specific reporting guidelines would help in this regard. The modest level of translation efforts is another pointer to the “deliberation to policy gap”. The results of this review could inform the design of new PIAs and future efforts to improve PIA comparability and outcome translation.     

Prognostic Value of miR-21 in Various Cancers: An Updating Meta-Analysis

Background

Recently, more and more studies investigated the value of microRNA (miRNA) as a diagnostic or prognostic biomarker in various cancers. MiR-21 was found dysregulated in almost all types of cancers. While the prognostic role of miR-21 in many cancers has been studied, the results were not consistent.

Methods

We performed a meta-analysis to investigate the correlation between miR-21 and survival of general cancers by calculating pooled hazard ratios (HR) and 95% confidence intervals (CI).

Results

The pooled results of 63 published studies showed that elevated miR-21 was a predictor for poor survival of general carcinomas, with pooled HR of 1.91 (95%CI: 1.66–2.19) for OS, 1.42 (95% CI: 1.16–1.74) for DFS and 2.2 (95% CI: 1.64–2.96) for RFS/CSS. MiR-21 was also a prognostic biomarker in the patients who received adjuvant therapy, with pooled HR of 2.4 (95%CI: 1.18–4.9) for OS.

Conclusions

Our results showed that miR-21 could act as a significant biomarker in the prognosis of various cancers. Further studies are warranted before the application of the useful biomarker in the clinical.     

Diabetes Care Provision in UK Primary Care Practices

Background

Although most people with Type 2 diabetes receive their diabetes care in primary care, only a limited amount is known about the quality of diabetes care in this setting. We investigated the provision and receipt of diabetes care delivered in UK primary care.

Methods

Postal surveys with all healthcare professionals and a random sample of 100 patients with Type 2 diabetes from 99 UK primary care practices.

Results

326/361 (90.3%) doctors, 163/186 (87.6%) nurses and 3591 patients (41.8%) returned a questionnaire. Clinicians reported giving advice about lifestyle behaviours (e.g. 88% would routinely advise about calorie restriction; 99.6% about increasing exercise) more often than patients reported having received it (43% and 42%) and correlations between clinician and patient report were low. Patients’ reported levels of confidence about managing their diabetes were moderately high; a median (range) of 21% (3% to 39%) of patients reporting being not confident about various areas of diabetes self-management.

Conclusions

Primary care practices have organisational structures in place and are, as judged by routine quality indicators, delivering high quality care. There remain evidence-practice gaps in the care provided and in the self confidence that patients have for key aspects of self management and further research is needed to address these issues. Future research should use robust designs and appropriately designed studies to investigate how best to improve this situation.     

Gender differences in plasma biomarker levels in a cohort of COPD patients: a pilot study

Rationale

Little is known about gender differences in plasma biomarker levels in patients with chronic obstructive pulmonary disease (COPD).

Hypothesis

There are differences in serum biomarker levels between women and men with COPD.

Objective

Explore gender differences in plasma biomarker levels in patients with COPD and smokers without COPD.

Methods

We measured plasma levels of IL-6, IL-8, IL-16, MCP-1, MMP-9, PARC and VEGF in 80 smokers without COPD (40 males, 40 females) and 152 stable COPD patients (76 males, 76 females) with similar airflow obstruction. We determined anthropometrics, smoking history, lung function, exercise tolerance, body composition, BODE index, co-morbidities and quality of life. We then explored associations between plasma biomarkers levels and the clinical characteristics of the patients and also with the clinical and physiological variables known to predict outcome in COPD.

Results

The plasma biomarkers level explored were similar in men and women without COPD. In contrast, in patients with COPD the median value in pg/mL of IL-6 (6.26 vs 8.0, p = 0.03), IL-16 (390 vs 321, p = 0.009) and VEGF (50 vs 87, p = 0.02) differed between women and men. Adjusted for smoking history, gender was independently associated with IL-16, PARC and VEGF levels. There were also gender differences in the associations between IL-6, IL-16 and VEGF and physiologic variables that predict outcomes.

Conclusions

In stable COPD patients with similar airflow obstruction, there are gender differences in plasma biomarker levels and in the association between biomarker levels and important clinical or physiological variables. Further studies should confirm our findings.     

An integrated quantification method to increase the precision,robustness, and resolution of protein measurement in human plasma samples

Background

Current quantification methods for mass spectrometry (MS)-based proteomics either do not provide sufficient control of variability or are difficult to implement for routine clinical testing.

Results

We present here an integrated quantification (InteQuan) method that better controls pre-analytical and analytical variability than the popular quantification method using stable isotope-labeled standard peptides (SISQuan). We quantified 16 lung cancer biomarker candidates in human plasma samples in three assessment studies, using immunoaffinity depletion coupled with multiple reaction monitoring (MRM) MS. InteQuan outperformed SISQuan in precision in all three studies and tolerated a two-fold difference in sample loading. The three studies lasted over six months and encountered major changes in experimental settings. Nevertheless, plasma proteins in low ng/ml to low μg/ml concentrations were measured with a median technical coefficient of variation (CV) of 11.9% using InteQuan. The corresponding median CV using SISQuan was 15.3% after linear fitting. Furthermore, InteQuan surpassed SISQuan in measuring biological difference among clinical samples and in distinguishing benign versus cancer plasma samples.

Conclusions

We demonstrated that InteQuan is a simple yet robust quantification method for MS-based quantitative proteomics, especially for applications in biomarker research and in routine clinical testing.

Electronic supplementary material

The online version of this article (doi:10.1186/1559-0275-12-3) contains supplementary material, which is available to authorized users.     

Methylome analysis identifies a Wilms tumor epigenetic biomarker detectable in blood

Background

Wilms tumor is the most common pediatric renal malignancy and there is a clinical need for a molecular biomarker to assess treatment response and predict relapse. The known mutated genes in this tumor type show low mutation frequencies, whereas aberrant methylation at 11p15 is by far the most common aberration. We therefore analyzed the epigenome, rather than the genome, to identify ubiquitous tumor-specific biomarkers.

Results

Methylome analysis of matched normal kidney and Wilms tumor identifies 309 preliminary methylation variable positions which we translate into three differentially methylated regions (DMRs) for use as tumor-specific biomarkers. Using two novel algorithms we show that these three DMRs are not confounded by cell type composition. We further show that these DMRs are not methylated in embryonic blastema but are intermediately methylated in Wilms tumor precursor lesions. We validate the biomarker DMRs using two independent sample sets of normal kidney and Wilms tumor and seven Wilms tumor histological subtypes, achieving 100% and 98% correct classification, respectively. As proof-of-principle for clinical utility, we successfully use biomarker DMR-2 in a pilot analysis of cell-free circulating DNA to monitor tumor response during treatment in ten patients.

Conclusions

These findings define the most common methylated regions in Wilms tumor known to date which are not associated with their embryonic origin or precursor stage. We show that this tumor-specific methylated DNA is released into the blood circulation where it can be detected non-invasively showing potential for clinical utility.

Electronic supplementary material

The online version of this article (doi:10.1186/s13059-014-0434-y) contains supplementary material, which is available to authorized users.     

Identification of a seven glycopeptide signature for malignant pleural mesothelioma in human serum by selected reaction monitoring

Background

Serum biomarkers can improve diagnosis and treatment of malignant pleural mesothelioma (MPM). However, the evaluation of potential new serum biomarker candidates is hampered by a lack of assay technologies for their clinical evaluation. Here we followed a hypothesis-driven targeted proteomics strategy for the identification and clinical evaluation of MPM candidate biomarkers in serum of patient cohorts.

Results

Based on the hypothesis that cell surface exposed glycoproteins are prone to be released from tumor-cells to the circulatory system, we screened the surfaceome of model cell lines for potential MPM candidate biomarkers. Selected Reaction Monitoring (SRM) assay technology allowed for the direct evaluation of the newly identified candidates in serum. Our evaluation of 51 candidate biomarkers in the context of a training and an independent validation set revealed a reproducible glycopeptide signature of MPM in serum which complemented the MPM biomarker mesothelin.

Conclusions

Our study shows that SRM assay technology enables the direct clinical evaluation of protein-derived candidate biomarker panels for which clinically reliable ELISA’s currently do not exist.     

Cost-Effectiveness of a Community Pharmacist-Led Sleep Apnea Screening Program – A Markov Model

Background

Despite the high prevalence and major public health ramifications, obstructive sleep apnea syndrome (OSAS) remains underdiagnosed. In many developed countries, because community pharmacists (CP) are easily accessible, they have been developing additional clinical services that integrate the services of and collaborate with other healthcare providers (general practitioners (GPs), nurses, etc.). Alternative strategies for primary care screening programs for OSAS involving the CP are discussed.

Objective

To estimate the quality of life, costs, and cost-effectiveness of three screening strategies among patients who are at risk of having moderate to severe OSAS in primary care.

Design

Markov decision model.

Data Sources

Published data.

Target Population

Hypothetical cohort of 50-year-old male patients with symptoms highly evocative of OSAS.

Time Horizon

The 5 years after initial evaluation for OSAS.

Perspective

Societal.

Interventions

Screening strategy with CP (CP-GP collaboration), screening strategy without CP (GP alone) and no screening.

Outcomes measures

Quality of life, survival and costs for each screening strategy.

Results of base-case analysis

Under almost all modeled conditions, the involvement of CPs in OSAS screening was cost effective. The maximal incremental cost for “screening strategy with CP” was about 455€ per QALY gained.

Results of sensitivity analysis

Our results were robust but primarily sensitive to the treatment costs by continuous positive airway pressure, and the costs of untreated OSAS. The probabilistic sensitivity analysis showed that the “screening strategy with CP” was dominant in 80% of cases. It was more effective and less costly in 47% of cases, and within the cost-effective range (maximum incremental cost effectiveness ratio at €6186.67/QALY) in 33% of cases.

Conclusions

CP involvement in OSAS screening is a cost-effective strategy. This proposal is consistent with the trend in Europe and the United States to extend the practices and responsibilities of the pharmacist in primary care.     

Asymmetrical dimethylarginine--more sensitive than NT-proBNP to diagnose heart failure in adults with congenital heart disease

Background

Chronic heart failure is an important cause for morbidity and mortality in adults with congenital heart disease (ACHD). While NT-proBNP is an established biomarker for heart failure of non-congenital origin, its value in ACHD has limitations. Asymmetrical dimethylarginine (ADMA) correlates with disease severity and independently predicts adverse clinical events in heart failure of non-congenital origin. Its role in ACHD has not been investigated.

Methods

In 102 patients ADMA and NT-proBNP were measured and related to NYHA class, systemic ventricular function and parameters of cardiopulmonary exercise testing.

Results

In contrast to NT-proBNP ADMA differentiated between NYHA classes I-III. Both, ADMA and NT-proBNP showed a good correlation with parameters of cardiopulmonary exercise testing with comparable receiver-operating characteristic curves for identifying patients with severely limited cardiopulmonary exercise capacity.

Conclusion

ADMA seems to be a better biomarker than NT-proBNP for the assessment of NYHA class and as a good as NT-proBNP for the estimation of maximum exercise capacity in adults with congenital heart disease. Its use in clinical routine should be evaluated.     

Reference Intervals for N-Terminal Pro-B-Type Natriuretic Peptide in Amniotic Fluid between 10 and 34 Weeks of Gestation

Background

In adult and pediatric cardiology, n-terminal pro-B-type natriuretic peptide (nt-proBNP) serves as biomarker in the diagnosis and management of cardiovascular dysfunction. Elevated levels of circulating nt-proBNP are present in fetal conditions associated with myocardial pressure or volume load. Compared to fetal blood sampling, amniocentesis is technically easier and can be performed from early pregnancy onwards. We aimed to investigate amniotic fluid (AF) nt-proBNP concentrations in normal pregnancies between 10 and 34 weeks of gestation.

Methods

Nt-proBNP and total protein (TP) was measured in AF by chemiluminescence assay (photometry, respectively). To adjust for a potential dilutional effect, the AF-nt-proBNP/AF-TP ratio was analyzed. Reference intervals were constructed by regression modeling across gestational age.

Results

132 samples were analyzed. A negative correlation between AF-nt-proBNP/AF-TP ratio and gestational age was observed. Curves for the mean and the 5% and 95% reference interval between 10 and 34 weeks of gestation were established.

Conclusion

In normal pregnancy, nt-proBNP is present in AF and decreases during gestation. Our data provide the basis for research on AF-nt-proBNP as biomarker in fetal medicine.     

Placebo Use in the United Kingdom: Results from a National Survey of Primary Care Practitioners

Objectives

Surveys in various countries suggest 17% to 80% of doctors prescribe ‘placebos’ in routine practice, but prevalence of placebo use in UK primary care is unknown.

Methods

We administered a web-based questionnaire to a representative sample of UK general practitioners. Following surveys conducted in other countries we divided placebos into ‘pure’ and ‘impure’. ‘Impure’ placebos are interventions with clear efficacy for certain conditions but are prescribed for ailments where their efficacy is unknown, such as antibiotics for suspected viral infections. ‘Pure’ placebos are interventions such as sugar pills or saline injections without direct pharmacologically active ingredients for the condition being treated. We initiated the survey in April 2012. Two reminders were sent and electronic data collection closed after 4 weeks.

Results

We surveyed 1715 general practitioners and 783 (46%) completed our questionnaire. Our respondents were similar to those of all registered UK doctors suggesting our results are generalizable. 12% (95% CI 10 to 15) of respondents used pure placebos while 97% (95% CI 96 to 98) used impure placebos at least once in their career. 1% of respondents used pure placebos, and 77% (95% CI 74 to 79) used impure placebos at least once per week. Most (66% for pure, 84% for impure) respondents stated placebos were ethical in some circumstances.

Conclusion and implications

Placebo use is common in primary care but questions remain about their benefits, harms, costs, and whether they can be delivered ethically. Further research is required to investigate ethically acceptable and cost-effective placebo interventions.     

Leukocyte Mitochondrial DNA Copy Number in Blood Is Not Associated with Major Depressive Disorder in Young Adults

Background

Major depressive disorder (MDD) is the leading cause of disability worldwide, and has significant genetic predisposition. Mitochondria may have a role in MDD and so mitochondrial DNA (mtDNA) has been suggested as a possible biomarker for this disease. We aimed to test whether the mtDNA copy number of peripheral blood leukocytes is related to MDD in young adults.

Methods

A case-control study was conducted with 210 MDD patients and 217 healthy controls (HC). The mtDNA copy number was measured by quantitative polymerase chain reaction (qPCR) method. Depression severity was assessed by the Hamilton-17 Depression Rating Scale (HDRS-17).

Results

We found no significant differences in mtDNA copy number between MDD patients and HC, though the power analysis showed that our sample size has enough power to detect the difference. There were also no significant correlations between mtDNA copy number and the clinical characteristics (such as age, age of onset, episodes, Hamilton Depression Rating Scale (HDRS) score and Global Assessment of Function Scale (GAF) score) in MDD patients.

Conclusion

Our study suggests that leukocyte mtDNA copy number is unlikely to contribute to MDD, but it doesn’t mean that we can exclude the possibility of involvement of mitochondria in the disease. Further studies are required to elucidate whether mtDNA can be a biomarker of MDD.     

Diagnostic Value of Urinary Kidney Injury Molecule 1 for Acute Kidney Injury: A Meta-Analysis

Background

Urinary Kidney Injury Molecule 1 (KIM-1) is a proximal tubular injury biomarker for early detection of acute kidney injury (AKI), with variable performance characteristics depending on clinical and population settings.

Methods

Meta-analysis was performed to assess the diagnostic value of urinary KIM-1 in AKI. Relevant studies were searched from MEDLINE, EMBASE, Pubmed, Elsevier Science Direct, Scopus, Web of Science, Google Scholar and Cochrane Library. Meta-analysis methods were used to pool sensitivity and specificity and to construct summary receiver operating characteristic (SROC) curves.

Results

A total of 2979 patients from 11 eligible studies were enrolled in the analysis. Five prospective cohorts, two cross-sectional and four case-control studies were identified for meta-analysis. The estimated sensitivity of urinary KIM-1 for the diagnosis of AKI was 74.0% (95% CI, 61.0%–84.0%), and specificity was 86.0% (95% CI, 74.0%–93.0%). The SROC analysis showed an area under the curve of 0.86(0.83–0.89). Subgroup analysis suggested that population settings and detection time were the key factors affecting the efficiency of KIM-1 for AKI diagnosis.

Limitation

Various population settings, different definition of AKI and Serum creatinine level used as the standard might have influence on AKI diagnosis. The relatively small number of studies and heterogeneity between them also affected the evaluation.

Conclusion

Urinary KIM-1 may be a promising biomarker for early detection of AKI with considerable predictive value, especially for cardiac surgery patients, and its potential value needs to be validated in large studies and across a broader scope of clinical settings.     

All Nations Depend on the Global Knowledge Pool – Analysis of Country of Origin of Studies Used for Health Technology Assessments in Germany

Background

Health Technology Assessments (HTAs) are used to inform decision-making and their usefulness depends on the quality and relevance of research and specific studies for health-policy decisions. Little is known about the country of origin of studies used for HTAs.

Objective

To investigate which countries have made the largest contributions to inform health policy decisions through studies included in HTAs in Germany.

Methods

The country of origin was extracted from all studies included in HTAs of the German Institute for Quality and Efficiency in Health Care, (IQWiG), published from 2/2006 to 9/2010. Studies were ranked according to the total number of studies per country, adjusted for population size, gross domestic product (GDP), and total health expenditure.

Results

1087 studies were included in 54 HTA reports. Studies were assigned to 45 countries. Most of the studies (27%) originated from the United States (USA), 18% were multinational, followed by 7% from the United Kingdom (UK) and 5% from Germany. Nordic countries led the ranking when adjusting for population size/million (ranks 1-3,6,9/45 countries), GDP/billion US$ (1,2,5,9,14/45), or health expenditure/billion US$ (1,3,5,12,13/45). The relative contribution of the UK was stable in the analyses when adjusted for population size (7/45), GDP (7/45), and health expenditure (9/45), whereas the USA (13, 18, and 30/45) and Germany (17, 19, and 21/45) dropped in the ranking.

Conclusions

More than half of the studies relevant for evidence-informed decision-making in Germany originated from the USA, followed by multinational research and the UK. Only 5% of the studies originated from Germany. According to our findings, there appears to be some discrepancy between the use of globally generated evidence and the contribution to the knowledge pool by individual countries.     

Access to Cancer Screening in People with Learning Disabilities in the UK: Cohort Study in the Health Improvement Network, a Primary Care Research Database

Objectives

To assess whether people with learning disability in the UK have poorer access to cancer screening.

Design

Four cohort studies comparing people with and without learning disability, within the recommended age ranges for cancer screening in the UK. We used Poisson regression to determine relative incidence rates of cancer screening.

Setting

The Health Improvement Network, a UK primary care database with over 450 General practices.

Participants

Individuals with a recorded diagnosis of learning disability including general diagnostic terms, specific syndromes, chromosomal abnormalities and autism in their General Practitioner computerised notes. For each type of cancer screening, a comparison cohort of up to six people without learning disability was selected for each person with a learning disability, using stratified sampling on age within GP practice.

Main outcome measures

Incidence rate ratios for receiving 1) a cervical smear test, 2) a mammogram, 3) a faecal occult blood test and 4) a prostate specific antigen test.

Results

Relative rates of screening for all four cancers were significantly lower for people with learning disability. The adjusted incidence rate ratios (95% confidence intervals) were Cervical smears: Number eligible with learning disability = 6,254; IRR = 0.54 (0.52–0.56). Mammograms: Number eligible with learning disability = 2,956; IRR = 0.76 (0.72–0.81); Prostate Specific Antigen: Number eligible = 3,520; IRR = 0.87 (0.80–0.96) and Faecal Occult Blood Number eligible = 6,566; 0.86 (0.78–0.94). Differences in screening rates were less pronounced in more socially deprived areas. Disparities in cervical screening rates narrowed over time, but were 45% lower in 2008/9, those for breast cancer screening appeared to widen and were 35% lower in 2009.

Conclusion

Despite recent incentives, people with learning disability in the UK are significantly less likely to receive screening tests for cancer that those without learning disability. Other methods for reducing inequalities in access to cancer screening should be considered.     

The Potential Biomarker Panels for Identification of Major Depressive Disorder (MDD) Patients with and without Early Life Stress (ELS) by Metabonomic Analysis

Objective

The lack of the disease biomarker to support objective laboratory tests still constitutes a bottleneck in the clinical diagnosis and evaluation of major depressive disorder (MDD) and its subtypes. We used metabonomic techniques to screen the diagnostic biomarker panels from the plasma of MDD patients with and without early life stress (ELS) experience.

Methods

Plasma samples were collected from 25 healthy adults and 46 patients with MDD, including 23 patients with ELS and 23 patients without ELS. Furthermore, gas chromatography/mass spectrometry (GC/MS) coupled with multivariate statistical analysis was used to identify the differences in global plasma metabolites among the 3 groups.

Results

The distinctive metabolic profiles exist either between healthy subjects and MDD patients or between the MDD patients with ELS experience (ELS/MDD patients) and the MDD patients without it (non-ELS/MDD patients), and some diagnostic panels of feature metabolites'' combination have higher predictive potential than the diagnostic panels of differential metabolites.

Conclusions

These findings in this study have high potential of being used as novel laboratory diagnostic tool for MDD patients and it with ELS or not in clinical application.