Powerful rare variant association testing in a copula-based joint analysis of multiple phenotypes.

In genetic association studies of rare variants, the low power of association tests is one of the main challenges. In this study, we propose a new single-marker association test called C-JAMP (Copula-based Joint Analysis of Multiple Phenotypes), which is based on a joint model of multiple phenotypes given genetic markers and other covariates. We evaluated its performance and compared its empirical type I error and power with existing univariate and multivariate single-marker and multi-marker rare-variant tests in extensive simulation studies. C-JAMP yielded unbiased genetic effect estimates and valid type I errors with an adjusted test statistic. When strongly dependent traits were jointly analyzed, C-JAMP had the highest power in all scenarios except when a high percentage of variants were causal with moderate/small effect sizes. When traits with weak or moderate dependence were analyzed, whether C-JAMP or competing approaches had higher power depended on the effect size. When C-JAMP was applied with a misspecified copula function, it still achieved high power in some of the scenarios considered. In a real-data application, we analyzed sequencing data using C-JAMP and performed the first genome-wide association studies of high-molecular-weight and medium-molecular-weight adiponectin plasma concentrations. C-JAMP identified 20 rare variants with p-values smaller than 10-5 , while all other tests resulted in the identification of fewer variants with higher p-values. In summary, the results indicate that C-JAMP is a powerful, flexible, and robust method for association studies, and we identified novel candidate markers for adiponectin. C-JAMP is implemented as an R package and freely available from https://cran.r-project.org/package=CJAMP.

Estimating and testing direct genetic effects in directed acyclic graphs using estimating equations.

In genetic association studies, it is important to distinguish direct and indirect genetic effects in order to build truly functional models. For this purpose, we consider a directed acyclic graph setting with genetic variants, primary and intermediate phenotypes, and confounding factors. In order to make valid statistical inference on direct genetic effects on the primary phenotype, it is necessary to consider all potential effects in the graph, and we propose to use the estimating equations method with robust Huber-White sandwich standard errors. We evaluate the proposed causal inference based on estimating equations (CIEE) method and compare it with traditional multiple regression methods, the structural equation modeling method, and sequential G-estimation methods through a simulation study for the analysis of (completely observed) quantitative traits and time-to-event traits subject to censoring as primary phenotypes. The results show that CIEE provides valid estimators and inference by successfully removing the effect of intermediate phenotypes from the primary phenotype and is robust against measured and unmeasured confounding of the indirect effect through observed factors. All other methods except the sequential G-estimation method for quantitative traits fail in some scenarios where their test statistics yield inflated type I errors. In the analysis of the Genetic Analysis Workshop 19 dataset, we estimate and test genetic effects on blood pressure accounting for intermediate gene expression phenotypes. The results show that CIEE can identify genetic variants that would be missed by traditional regression analyses. CIEE is computationally fast, widely applicable to different fields, and available as an R package.

The long-term survival characteristics of a cohort of colorectal cancer patients and baseline variables associated with survival outcomes with or without time-varying effects.

BACKGROUND: Colorectal cancer is the third most common cancer in the world. In this study, we assessed the long-term survival characteristics and prognostic associations and potential time-varying effects of clinico-demographic variables and two molecular markers (microsatellite instability (MSI) and BRAF Val600Glu mutation) in a population-based patient cohort followed up to ~ 19 years. METHODS: The patient cohort included 738 incident cases diagnosed between 1999 and 2003. Cox models were used to analyze the association between the variables and a set of survival outcome measures (overall survival (OS), disease-specific survival (DSS), recurrence-free survival (RFS), metastasis-free survival (MFS), recurrence/metastasis-free survival (RMFS), and event-free survival (EFS)). Cox proportional hazard (PH) assumption was tested for all variables, and Cox models with time-varying effects were used if any departure from the PH assumption was detected. RESULTS: During the follow-up, ~ 61% patients died from any cause, ~ 26% died from colorectal cancer, and ~ 10% and ~ 20% experienced recurrences and distant metastases, respectively. Stage IV disease and post-diagnostic recurrence or metastasis were strongly linked to risk of death from colorectal cancer. If a patient had survived the first 6 years without any disease-related event (i.e., recurrence, metastasis, or death from colorectal cancer), their risks became very minimal after this time period. Distinct sets of markers were associated with different outcome measures. In some cases, the effects by variables were constant throughout the follow-up. For example, MSI-high tumor phenotype and older age at diagnosis predicted longer MFS times consistently over the follow-up. However, in some other cases, the effects of the variables varied with time. For example, adjuvant radiotherapy treatment was associated with increased risk of metastasis in patients who received this treatment after 5.5 years post-diagnosis, but not before that. CONCLUSIONS: This study describes the long-term survival characteristics of a prospective cohort of colorectal cancer patients, relationships between baseline variables and a detailed set of patient outcomes over a long time, and time-varying effects of a group of variables. The results presented advance our understanding of the long-term prognostic characteristics in colorectal cancer and are expected to inspire future studies and clinical care strategies.

A genome-wide association study identifies single nucleotide polymorphisms associated with time-to-metastasis in colorectal cancer.

BACKGROUND: Differentiating between cancer patients who will experience metastasis within a short time and who will be long-term survivors without metastasis is a critical aim in healthcare. The microsatellite instability (MSI)-high tumor phenotype is such a differentiator in colorectal cancer, as patients with these tumors are unlikely to experience metastasis. Our aim in this study was to determine if germline genetic variations could further differentiate colorectal cancer patients based on the long-term risk and timing of metastasis. METHODS: The patient cohort consisted of 379 stage I-III Caucasian colorectal cancer patients with microsatellite stable or MSI-low tumors. We performed univariable analysis on 810,622 common single nucleotide polymorphisms (SNPs) under different genetic models. Depending on the long-term metastasis-free survival probability estimates, we applied a mixture cure model, Cox proportional hazards regression model, or log-rank test. For SNPs reaching Bonferroni-corrected significance (p < 6.2 × 10- 8) having valid genetic models, multivariable analysis adjusting for significant baseline characteristics was conducted. RESULTS: After adjusting for significant baseline characteristics, specific genotypes of ten polymorphisms were significantly associated with time-to-metastasis. These polymorphisms are three intergenic SNPs, rs5749032 (p = 1.28 × 10- 10), rs2327990 (p = 9.59 × 10- 10), rs1145724 (p = 3 × 10- 8), and seven SNPs within the non-coding sequences of three genes: FHIT (p = 2.59 × 10- 9), EPHB1 (p = 8.23 × 10- 9), and MIR7515 (p = 4.87 × 10- 8). CONCLUSIONS: Our results suggest novel associations of specific genotypes of SNPs with early metastasis in Caucasian colorectal cancer patients. These associations, once replicated in other patient cohorts, could assist in the development of personalized treatment strategies for colorectal cancer patients.

Correction to: A genome-wide association study identifies single nucleotide polymorphisms associated with time-to-metastasis in colorectal cancer.

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The effect of blue thermal treatment on endodontic instruments and apical debris extrusion during retreatment procedures.

AIM: To determine the effect of blue thermal treatment on Reciproc (VDW, Munich, Germany) endodontic instruments and the amount of apically extruded debris during retreatment procedures. METHODOLOGY: Thirty extracted mandibular molar teeth having mesial roots with a degree of curvature less than 20° and having an initial apical size equivalent to a size 10 K-file were selected. The mesial roots of the teeth were removed from the cementoenamel junction to obtain a 15-mm root length. The mesiobuccal canals were prepared to size F2 using the ProTaper Universal system (Dentsply Sirona, Ballaigues, Switzerland), filled with gutta-percha and 2Seal (VDW) using the lateral compaction technique and then randomly divided into two groups (n = 15). The root fillings were removed with one of the following instruments using a crown-down preparation technique: M-Wire Reciproc or Reciproc Blue (both VDW). Apically extruded debris was collected in pre-weighed Eppendorf tubes. The Eppendorf tubes were then stored in an incubator at 70 °C for five days to evaporate the distilled water. The data were analysed using the Mann-Whitney U test (P = 0.05). RESULTS: Reciproc Blue (1.42 ± 0.4491 mg) extruded significantly less debris apically than M-Wire Reciproc (2.56 ± 1.0232 mg) (P < 0.05). CONCLUSIONS: Blue thermal treatment of Reciproc instruments was associated with less debris extrusion during retreatment procedures.

Child neglect and abuse: a global glimpse within the framework of evidence perspective.

AIM: This systematic review was conducted in order to integrate evidence-based knowledge and experience related to child neglect and abuse into the nursing literature. BACKGROUND: The negative and intense effects of neglect and abuse on an individual can last into adulthood. Nurses who are in close contact with such cases have an important role to play in detecting child neglect and abuse and supporting the families involved. When nurses fulfil this role, it is important that evidence-based information and interventions are known to ensure that the process is a healthy one. DATA SOURCES: Medline/Pubmed and Cochrane Library databases, from 2012 to 2016. REVIEW METHODS: The PRISMA guide, a basic search algorithm, was used as a basis for the review. RESULTS: This systematic research involved 32 articles that met the criteria. When the characteristics of the studies were examined, it was found that one study dealt with physical abuse, seven studies dealt with sexual abuse, 21 studies with neglect and abuse and three studies with all abuse types. It was also found that 16% addressed intervention, 22% addressed the relationship between abuse and other factors, 31% addressed prevention and 31% addressed the defining dimension. CONCLUSIONS: It has been found that, in general, all types of negligence and abuse are studied together and that nurses lack the knowledge and skills needed to assess childhood neglect and abuse. IMPLICATIONS FOR NURSING AND HEALTH POLICIES: Nurses have a critical role to play in identifying the dark spots and associated factors in the story of individuals because they are health professionals who are in close contact with patients. It is recommended that guidelines be developed and used in the diagnosis and treatment of abuse and neglect. Thus, in these cases, the standardization of care will be achieved.

The oral adverse effects of isotretinoin treatment in acne vulgaris patients: A prospective, case-control study.

BACKGROUND: Isotretinoin is the most effective therapy to treat severe acne vulgaris and its systemic adverse effects have been well documented, but little is known on dental side effects over the course of treatment. OBJECTIVES: This prospective case-control study aimed to evaluate the oral adverse effects of isotretinoin in Turkish patients with acne vulgaris; compare oral conditions between patients and normal controls; and investigate the association between salivary parameters and International Caries Detection and Assessment System (ICDAS) scores. MATERIALS AND METHODS: For 6 months, the patients (n = 45) received isotretinoin daily (0.5 mg/kg). The age-matched untreated controls (n = 45) were patients without acne. Both groups were examined before the study and at 6 months for salivary flow, buffer capacity, microbiologic tests, and caries status (based on the ICDAS). Salivary parameters and ICDAS scores were analyzed by Spearman's rank correlations. Data were statistically analyzed by the Mann-Whitney U test, Wilcoxon signed rank tests, and McNemar's Chi-square tests (P < 0.05). RESULTS: Twenty-two isotretinoin-treated patients and 18 controls completed the study. At baseline, the groups were not significantly different in the evaluated parameters (P > 0.05). At 6 months in the isotretinoin-treated group, salivary flow and buffer capacity significantly decreased, and the ICDAS scores significantly increased (P < 0.05). The changes in these criteria from baseline were insignificant in the controls (P > 0.05). Intraoral pathogen counts were not significantly different between the groups, compared to baseline (P > 0.05). Stimulated salivary parameters in both groups were not correlated significantly with the ICDAS scores. CONCLUSIONS: Isotretinoin significantly affected salivary flow, buffer capacity, caries lesion activity scores for 6 months. However, salivary parameters and caries lesion activity scores had no significant correlations.

Enhancement of two photon absorption properties and intersystem crossing by charge transfer in pentaaryl boron-dipyrromethene (BODIPY) derivatives.

Novel BODIPY derivatives containing N,N-diphenylamine, 4-methoxyphenyl, 2,4-dimethoxyphenyl, triphenylamine, and 1-pyrene moieties were designed and synthesized for the first time by employing the palladium-catalyzed Suzuki-Miyaura coupling on pentaaryl boron dipyrromethene compounds. The effect of various moieties and charge transfer on linear and nonlinear optical absorption was investigated. It was found that moieties with strong electron donor properties and long conjugation lengths increase charge transfer and enhance intersystem crossing in the investigated compounds. Besides, the investigated compounds showed strong two photon absorption properties at near infrared wavelengths (800 nm and 900 nm), which is required for two photon photodynamic therapy. Two photon absorption cross section values were found to be 83, 454, 331, 472 and 413 GM for , , , and compounds at 800 nm wavelength, respectively. The highest two-photon absorption cross-section value was obtained for the compound containing a triphenylamine moiety due to its more efficient charge transfer characteristics. Strong two-photon absorption properties in the near infrared region, efficient intersystem crossing and heavy atom free nature of the investigated compounds make them good candidates for two photon photodynamic therapy applications. We believe that this work will be one of the leading studies for two-photon photodynamic therapy applications of pentaaryl BODIPY derivatives.

Nutritional status and eating habits of the institutionalised elderly in Turkey: a follow-up study.

BACKGROUND: As the elderly population increases in Turkey, so do the associated health and nutritional problems. The main purpose of the present study was to determine the nutritional status of elderly individuals who live in institutions. METHODS: A total of 102 elderly volunteers was recruited from seven residential homes of the Ministry of Family and Social Policies in Ankara. In the consecutive years of 2007, 2008 and 2009, dietary intake was assessed using a 24-h food recall. Nutritional status was screened using a questionnaire from the Mini-Nutritional Assessment, basic characteristics were determined and anthropometric measurements were assessed. RESULTS: The percentage of elderly participants who were malnourished or at risk for malnutrition increased by the completion of the follow-up (P < 0.05). It was found that energy, total protein, animal proteins, carbohydrates, niacin, vitamin C, vitamin E and zinc intake of men decreased significantly over the years studied (P < 0.05). A significant decrease occurred among women in animal protein, vitamin B1 , niacin and the percentage of energy from proteins (P < 0.05); however, an increase in energy from fat (P < 0.05) was determined. Within the years studied, the percentage of nutrients meeting the Turkish recommended daily allowances decreased from 2007 to 2009 both in men and women. During the years 2007 to 2009, the percentage of waist circumferences >102 cm for men was 46.4%, 45.6% and 48.1%, respectively, and the percentage of waist circumferences for women >88 cm was 75.6%, 75.6% and 81.8%, respectively. CONCLUSIONS: During the follow-up, significant nutritional changes were determined. To prevent malnutrition, periodical screening of nutritional status should be a priority and a standard policy for elderly people, especially for those institutionalised.

Novel visual system homeobox 1 gene mutations in Turkish patients with keratoconus.

The aim of this study was to screen the visual system homeobox 1 (VSX1) gene in Turkish patients with keratoconus (KC). The patient group consisted of 44 patients who had undergone corneal transplant surgery before the age of 30, for advanced and rapidly progressive KC. The control group comprised 250 healthy individuals. We detected two missense mutations, D144N and D295Y, in exon 2 and exon 5 of the VSX1 gene, respectively, using next-generation sequencing analysis. The pathologic effects of the D144N and D295Y missense mutations on protein function were determined with bioinformatic analysis tools, SIFT, PolyPhen, and MutationTaster. Aspartic acid at the 144th position was more preserved among species than aspartic acid at the 295th position of the VSX1 protein. In the control group, five different genetic variations were detected, two of which (rs8123716 and rs12480307) were synonymous with variations in the patient group. Our results suggested that the D144N and D295Y mutations might have a role in the pathogenesis of KC disease.

Effect of a re-wetting agent on bond strength of an adhesive to primary and permanent teeth dentin after different etching techniques.

OBJECTIVE: This study investigated the effect of a re-wetting agent on the microtensile bond strengths (mTBS) of primary and permanent dentin after acid or laser etching. MATERIALS AND METHODS: Twelve permanent and 12 primary molar teeth were ground to expose an occlusal dentin surface. Each group teeth were randomly divided into groups; I-II: 37% phosphoric acid etching with/without re-wetting agent, III-IV: Erbium: Yttrium aluminium garnet laser etching with/without re-wetting agent. An etch-and-rinse adhesive was used, and vertical sticks were obtained for the microtensile test. RESULTS: mTBS of permanent teeth was higher than that of primary teeth (P < 0.05). Re-wetting agent groups were similar with control groups in both etching groups in the permanent teeth (P > 0.05). Re-wetting agent group was similar with the control group in acid etch group (P > 0.05) and lower than the control group in laser etch group in primary teeth (P < 0.05). CONCLUSION: Acid etching in permanent teeth; laser etching in primary teeth was found more successful. The use of re-wetting agent did not provide an advance on bond strength of the adhesive in both primary and permanent teeth after acid-etch or laser-etch.

Elevated expression of podocalyxin is associated with lymphatic invasion, basal-like phenotype, and clinical outcome in axillary lymph node-negative breast cancer.

Lymphatic invasion (LVI) is associated with disease recurrence in axillary node-negative (ANN) breast cancer. Using gene expression profiling of 105 ANN tumors, we found that podocalyxin (PODXL) was more highly expressed in tumors with LVI (LVI+) than in those without LVI (LVI-). Differences in PODXL expression were validated using real-time polymerase chain reaction as well as by immunohistochemistry in an independent set of 652 tumors on tissue microarrays. Disease-free survival (DFS) analyses were conducted for association of high PODXL protein expression with risk of distant recurrence overall and within breast cancer subtypes using both Cox and cure-rate models. High PODXL expression was associated with poor prognosis features including large tumor size, high histological grade, estrogen and progesterone receptor negativity, and with clinical alterations characteristic of the basal-like breast cancer phenotype. Surprisingly, despite having other poor prognosis characteristics, women with high PODXL expressing tumors had better long-term DFS in multivariate analysis with traditional clinicopathologic factors including LVI and HER2 status (P = 0.001). PODXL has the potential to be a useful biomarker for identifying good prognosis patients in characteristically poor prognosis breast cancer groups and may impact treatment of women with this disease.

Effect of caries removal techniques on the bond strength of adhesives to caries-affected primary dentin in vitro.

AIM: The aim of this in vitro study is to evaluate the effects of three different caries removal techniques on the microtensile bond strength of adhesive materials to caries-affected dentin. MATERIALS AND METHODS: Thirty primary molar teeth were used. The teeth were randomly divided into three groups according to the caries removal technique employed: conventional steel bur (group 1); Er:YAG laser (group 2); chemomechanical method (group 3). Each group was divided into two subgroups according to bonding agents: one-step self-etch adhesive and etch-and-rinse adhesive. The teeth were restored with composite resin. Vertical sticks were obtained and subjected to tensile stress. Data were analyzed by two-way analysis of variance (ANOVA), Tukey's test and an independent samples t-test. RESULTS: The values for the laser groups were significantly lower than those of the bur groups for both bonding agents (p<0.05). There were no significant differences between the bur and chemomechanical groups (p > 0.05). CONCLUSION: Bur and chemomechanical techniques in primary teeth were found more successful. Similar results were found according to the adhesives used for each caries removal techniques.

Multifactor dimensionality reduction method identifies novel SNP interactions in the WNT protein interaction networks that are associated with recurrence risk in colorectal cancer.

Background: Interactions among genetic variants are rarely studied but may explain a part of the variability in patient outcomes. Objectives: In this study, we aimed to identify 1 to 3 way interactions among SNPs from five Wnt protein interaction networks that predict the 5-year recurrence risk in a cohort of stage I-III colorectal cancer patients. Methods: 423 patients recruited to the Newfoundland Familial Colorectal Cancer Registry were included. Five Wnt family member proteins (Wnt1, Wnt2, Wnt5a, Wnt5b, and Wnt11) were selected. The BioGRID database was used to identify the proteins interacting with each of these proteins. Genotypes of the SNPs located in the interaction network genes were retrieved from a genome-wide SNP genotype data previously obtained in the patient cohort. The GMDR 0.9 program was utilized to examine 1-, 2-, and 3-SNP interactions using a 5-fold cross validation step. Top GMDR 0.9 models were assessed by permutation testing and, if significant, prognostic associations were verified by multivariable logistic regression models. Results: GMDR 0.9 has identified novel 1, 2, and 3-way SNP interactions associated with 5-year recurrence risk in colorectal cancer. Nine of these interactions were multi loci interactions (2-way or 3-way). Identified interaction models were able to distinguish patients based on their 5-year recurrence-free status in multivariable regression models. The significance of interactions was the highest in the 3-SNP models. Several of the identified SNPs were eQTLs, indicating potential biological roles of the genes they were associated with in colorectal cancer recurrence. Conclusions: We identified novel interacting genetic variants that associate with 5-year recurrence risk in colorectal cancer. A significant portion of the genes identified were previously linked to colorectal cancer pathogenesis or progression. These variants and genes are of interest for future functional and prognostic studies. Our results provide further evidence for the utility of GMDR models in identifying novel prognostic biomarkers and the biological importance of the Wnt pathways in colorectal cancer.

Regression and data mining methods for analyses of multiple rare variants in the Genetic Analysis Workshop 17 mini-exome data.

Group 14 of Genetic Analysis Workshop 17 examined several issues related to analysis of complex traits using DNA sequence data. These issues included novel methods for analyzing rare genetic variants in an aggregated manner (often termed collapsing rare variants), evaluation of various study designs to increase power to detect effects of rare variants, and the use of machine learning approaches to model highly complex heterogeneous traits. Various published and novel methods for analyzing traits with extreme locus and allelic heterogeneity were applied to the simulated quantitative and disease phenotypes. Overall, we conclude that power is (as expected) dependent on locus-specific heritability or contribution to disease risk, large samples will be required to detect rare causal variants with small effect sizes, extreme phenotype sampling designs may increase power for smaller laboratory costs, methods that allow joint analysis of multiple variants per gene or pathway are more powerful in general than analyses of individual rare variants, population-specific analyses can be optimal when different subpopulations harbor private causal mutations, and machine learning methods may be useful for selecting subsets of predictors for follow-up in the presence of extreme locus heterogeneity and large numbers of potential predictors.

Examining SNP-SNP interactions and risk of clinical outcomes in colorectal cancer using multifactor dimensionality reduction based methods.

Background: SNP interactions may explain the variable outcome risk among colorectal cancer patients. Examining SNP interactions is challenging, especially with large datasets. Multifactor Dimensionality Reduction (MDR)-based programs may address this problem. Objectives: 1) To compare two MDR-based programs for their utility; and 2) to apply these programs to sets of MMP and VEGF-family gene SNPs in order to examine their interactions in relation to colorectal cancer survival outcomes. Methods: This study applied two data reduction methods, Cox-MDR and GMDR 0.9, to study one to three way SNP interactions. Both programs were run using a 5-fold cross validation step and the top models were verified by permutation testing. Prognostic associations of the SNP interactions were verified using multivariable regression methods. Eight datasets, including SNPs from MMP family genes (n = 201) and seven sets of VEGF-family interaction networks (n = 1,517 SNPs) were examined. Results: ∼90 million potential interactions were examined. Analyses in the MMP and VEGF gene family datasets found several novel 1- to 3-way SNP interactions. These interactions were able to distinguish between the patients with different outcome risks (regression p-values 0.03-2.2E-09). The strongest association was detected for a 3-way interaction including CHRM3.rs665159_EPN1.rs6509955_PTGER3.rs1327460 variants. Conclusion: Our work demonstrates the utility of data reduction methods while identifying potential prognostic markers in colorectal cancer.

Semiparametric estimation in copula models for bivariate sequential survival times.

Sequentially observed survival times are of interest in many studies but there are difficulties in analyzing such data using nonparametric or semiparametric methods. First, when the duration of followup is limited and the times for a given individual are not independent, induced dependent censoring arises for the second and subsequent survival times. Non-identifiability of the marginal survival distributions for second and later times is another issue, since they are observable only if preceding survival times for an individual are uncensored. In addition, in some studies a significant proportion of individuals may never have the first event. Fully parametric models can deal with these features, but robustness is a concern. We introduce a new approach to address these issues. We model the joint distribution of the successive survival times by using copula functions, and provide semiparametric estimation procedures in which copula parameters are estimated without parametric assumptions on the marginal distributions. This provides more robust estimates and checks on the fit of parametric models. The methodology is applied to a motivating example involving relapse and survival following colon cancer treatment.

Likelihood ratio procedures and tests of fit in parametric and semiparametric copula models with censored data.

Copula models for multivariate lifetimes have become widely used in areas such as biomedicine, finance and insurance. This paper fills some gaps in existing methodology for copula parameters and model assessment. We consider procedures based on likelihood and pseudolikelihood ratio statistics and introduce semiparametric maximum likelihood estimation leading to semiparametric versions. For cases where standard asymptotic approximations do not hold, we propose an efficient simulation technique for obtaining p-values. We apply these methods to tests for a copula model, based on embedding it in a larger copula family. It is shown that the likelihood and pseudolikelihood ratio tests are consistent even when the expanded copula model is misspecified. Power comparisons with two other tests of fit indicate that model expansion provides a convenient, powerful and robust approach. The methods are illustrated on an application concerning the time to loss of vision in the two eyes of an individual.

The opioid peptide biphalin modulates human corneal epithelial wound healing in vitro.

PURPOSE: Analgesic drugs, including nonselective opioids and non-steroidal anti-inflammatory drugs, should be used with great precautions to relieve pain after trauma to the corneal epithelium because of their unfavorable effects on wound healing. Biphalin is a synthetic opioid peptide that has been demonstrated to possess a strong analgesic effect on rodents. The purpose of this study is to investigate the effects of biphalin on human corneal epithelial wound healing. METHODS: An immortalized human corneal epithelial cell (HCEC) culture was used to analyze the effects of biphalin on wound healing. The toxicity of biphalin at various concentrations was measured by the MTT assay. The effects of 1µM and 10µM biphalin on wound closure, cell migration and proliferation were tested in an in vitro scratch assay of HCECs. Naloxone, a non-selective competitive opioid receptor antagonist, was also used to inhibit the effects of biphalin in all experiments. RESULTS: Biphalin did not cause any toxic effect on HCECs at concentrations lower than 100µM at various incubation time points. Biphalin significantly increased wound healing at 1µM concentration in an in vitro scratch assay of HCECs (P<0.05). It also significantly increased migration of HCECs (P<0.01). There was no significant difference between the biphalin and control groups of HCECs in the Ki67 proliferation assay. CONCLUSION: Biphalin, which is a synthetic opioid peptide, promotes corneal epithelial wound healing by increasing cell migration. This role should be evaluated in further in vivo and clinical studies.