Functional studies confirmed that PRAME facilitated the proliferation, invasion, migration, and epithelial–mesenchymal transition of LSCC cells, and PRAME also promoted tumor growth in vivo. HDAC5 was identified as an upstream regulator that can affect the expression of PRAME. Moreover, PRAME played the role at least partially by activating PI3K/AKT/mTOR pathways. The above findings elucidate that PRAME may be a valuable oncogene target, contributing to the diagnosis and therapy of LSCC.Laryngeal squamous cell carcinoma (LSCC) is an aggressive and lethal malignant neoplasm with extremely poor prognoses. Accumulating evidence has indicated that preferentially expressed antigen in melanoma (PRAME) is correlated with several kinds of cancers. However, there is little direct evidence to substantiate the biological function of PRAME in LSCC. The purpose of the current study is to explore the oncogenic role of PRAME in LSCC. PRAME expression was analyzed in 57 pairs of LSCC tumor tissue samples through quantitative real-time PCR, and the correlation between PRAME and clinicopathological features was analyzed. The result indicated that PRAME was overexpressed in the LSCC patients and correlated with the TNM staging and lymphatic metastasis. The biological functions and molecular mechanism of PRAME in LSCC progression were investigated through  MoreoveIn recent years, our group has been devoted to exploring the pathogenesis of LSCC from clinical study to molecular basis. Previously, we reported several potential biomarkers such as TGF-beta, HO-1, and CEACAM1 that were closely related to the head and neck squamous cell carcinoma ,7,8. DeeEpigenetic regulation, like ubiquitin, methylation, and acetylation, plays an important biological role in many tumors, including laryngeal carcinoma . SeveralPI3K/Akt pathway is one of the most important signaling pathways that is often reported to be involved in human cancers . RecentlIn vitro, we have found that PRAME overexpression has the ability to stimulate LSCC-related AMC-HN-8 and TU177 cell proliferation, migration, invasion, and epithelial–mesenchymal transition (EMT), and knock-down of PRAME can reduce the proliferative, migratory and invasive capabilities of TU177. In vitro, we have also found that HDAC5 as a potential upstream regulator might mediate PRAME expression, contributing to the progression of LSCC. In vitro and in vivo, our data further revealed that the proliferation, invasion, and migration of LSCC cells that are induced by PRAME may be at least partially dependent on the activation of PI3K/AKT/mTOR signaling pathways. The above findings elucidate the function of PRAME in LSCC development and progression and provide the new evidence for PRAME as an oncogene that probably serves as a valuable target for the diagnosis and management of LSCC.This study demonstrated that PRAME is overexpressed in LSCC tissues. High expression of PRAME is also significantly correlated with poor prognosis and survival rate in the LSCC patients. 22.1A total of 57 pairs of LSCC tumor tissues and corresponding normal tissues were provided by the Second Hospital of Hebei Medical University  from October 2017 to September 2019. In this study, none of the LSCC patients received chemotherapy, radiotherapy, or other anti-tumor treatment before surgery. All of the tissue specimens were frozen and stored at −80°C at the Institute of Otolaryngology of Hebei Medical University to perform further experiments. All clinical features were acquired from the hospital records and pathological diagnoses, as listed in Ethics approval and consent to participate: All procedures in this study were performed were in agreement with the ethical standards of the Research Committee of Hebei Medical University  as well as with the Declaration of Helsinki (2008). The approval of this study was obtained from the Ethics Committee of Hebei Medical University and the Second Hospital of Hebei Medical University. Informed consent was obtained from all patients.2.22 atmosphere at 37°C . The PI3K inhibitor (LY294002) and mTOR inhibitor (Rapamycin) were purchased from MCE . LSCC cells were incubated with LY294002 or Rapamycin to perform functional assays and Western blot assay.The human LSCC cell lines of AMC-HN-8 and TU177 were purchased from Beijing Beina Chuanglian Institute of Biotechnology . The AMC-HN-8 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM), containing 10% foetal bovine serum (FBS). The TU177 cells were cultured in RPMI-1640 medium, supplemented with 10% FBS. All of the above cell culture reagents were provided by GIBCO . Accordingly, the culture of two cells was performed in a humidified incubator with 5% CO2.3®Super Total RNA Extraction Kit  following the experimental instructions. RNA reverse transcription into cDNA was performed with Transcriptor First Strand cDNA Synthesis Kit . A GoTaq®qPCR Master Mix  was used for the quantitative real-time PCR (qRT-PCR). Relative expression was normalized using the 2−∆∆Ct method and the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was assigned into the endogenous control. Primer sequences were as follows:Total RNA was extracted from LSCC specimens and cell lines of AMC-HN-8 and TU177 with EastepPRAME, forward: 5′-CAGGACTTCTGGACTGTATGGT-3′;reverse: 5′-CTACGAGCACCTCTACTGGAA-3′.GAPDH, forward: 5′-CTCACCGGATGCACCAATGTT-3′;reverse: 5′-CGCGTTTCACAATGTTCAT-3′.All the samples were run in triplicate.2.4®2000  in accordance with the manufacturer’s instructions when the cells had 75–80% confluence. Moreover, the empty vector, as control, was transfected under the same condition. In addition, small interfering RNA (siRNA) was adopted to knock-down PRAME expression in TU177. The siRNA of PRAME (siRNA sequence: 5′-GGTCATGCTGACCGATGTA-3′) and negative control (si-NC) were obtained by RiboBio . After the transfection for 48 h, the transfection efficiency was determined through qRT-PCR, and the cells were harvested and employed in subsequent experiments. The expression of PRAME in the treated cells was examined using qRT-PCR as well as western blot assay.AMC-HN-8 and TU177 were grown in six-well plates and transfected with 3 μg overexpression of PRAME  or 3 μg the PcDNA3.1 vector  using 6 μl Lipofectamine2.53 cells/well were cultured in 96-well plates containing 100 μl of complete medium, respectively. 20 µl MTS reagent was introduced into the respective well, followed by the incubation of the plates for 2.5 h at 37°C according to the instructions of the CellTiter96®AQueousOne Solution Cell Proliferation Assay Kit . Furthermore, the optical density (OD) was examined at 490 nm using a Spark® multimode microplate reader . Proliferation rates were examined at 0, 24, 48, 72, and 96 h after the transfection. Each assay was performed in triplicate.The proliferation ability of cells was examined through 3--5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt (MTS) assay. AMC-HN-8 and TU177 transfected at 2 × 102.63 AMC-HN-8- and TU177-transfected cells were seeded into the respective well of a six-well plate and incubated at 37°C in a humidified incubator containing 5% CO2. Ten days later, the cell colonies were rinsed gently with PBS in triplicate, fixed with 4% paraformaldehyde for 20 min, and then stained with 0.5% crystal violet for 20 min. Lastly, the number of colonies formed with more than 50 cells per well was calculated using a microscope  at 200× magnification.A total of 2 × 102.75 AMC-HN-8 and TU177 transfected cells were seeded into the respective well of a six-well plate and were cultured at 37°C overnight in a humidified incubator containing 5% CO2. When the cell monolayers had 100% confluence, a straight wound line was made by a scratch using a 200 μl pipette tip. Subsequently, the images at the same position of the respective well were captured. The relative distance between cell migration and the scratched area was measured at 0, 24, and 48 h, respectively, under a fluorescence microscope . The experiment was repeated in triplicate.In total, 5 × 102.85 cells in 100 μl culture medium free of FBS were seeded into the upper chamber, followed by supplementation with 650 ul of culture medium with 10% FBS, in the bottom chamber. Following the incubation for 24 h at 37°C and the removal of the transwell chamber, the samples were rinsed with PBS in triplicate, fixed with 4% paraformaldehyde for 20 min and subsequently stained with 0.5% crystal violet for 20 min. For the invasion assay, the membrane was coated with 50 µl Matrigel to develop a matrix barrier first. Subsequently, the same protocol was employed for transwell assay. Lastly, the number of cells that experienced migration or invasion was observed and calculated in five microscopic fields selected randomly by a microscope  at a fixed 200× magnification.Cell migration and invasion abilities were examined through transwell assay . For the migration assay, a total of 1 × 102.9By lysing the cells AMC-HN-8 and TU177 transfected with the overexpression of PRAME or the PcDNA3.1 empty vector, RIPA lysis buffer  was adopted for protein extraction, supplemented with PMSF  as well as with a protease inhibitor cocktail . The above step was followed by ultrasonication  for higher acquisition efficiency. The protein concentration was examined using the BCA Protein Assay Kit . Protein lysates were then mixed with SDS-PAGE loading buffer  and boiled at 95°C for 5 min. Equal amounts of protein (20 µg) were employed for electrophoresis on 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and probed with the following primary antibodies. The signals were visualized with enhanced chemiluminescence  using ChemiDoc™ XRS + . All primary antibodies used are listed in 2.10https://www.ncbi.nlm.nih.gov/gds/?term=)  was adopted to obtain the gene matrix expression profiles of LSCC (P < 0.05 were considered DEGs. All genes in each dataset were sorted by logFC and integrated using the RobustRankAggreg R package. The GEPIA online database (http://gepia.cancer-pku.cn/) was adopted to analyze the PRAME expression in an enlarged sample size. In addition, a Kaplan–Meier plot (http://kmplot.com/) was employed to analyze the correlation of the expression of PRAME with the overall survival (OS) rate of LSCC patients. The NCBI online database (https://www.ncbi.nlm.nih.gov/) was applied to analyze the expression levels of PRAME in various normal tissues. To further explore the potential mechanism underlying the function of PRAME, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were conducted for the prediction of the downstream signaling pathway.The GEO online database  mice  were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. . The nude mice were randomly assigned into four groups, including the empty vector group; the transiently transfected PRAME group; the stably transfected PRAME group; and the LY294002 (an inhibitor of PI3K) group (n = 5 in the respective group). 1 × 107 AMC-HN-8 cells transfected with overexpressed PRAME were mixed with 100 μl culture medium free of FBS, followed by the subcutaneous injection into the right flanks of the BALB/c nude mice. The tumor volume was documented every 3 days with a caliper by the formula: (A × B2)/2, where A denotes the maximum diameter and B represents the diameter perpendicular to A. Four weeks later, all nude mice were sacrificed, and the tumor xenografts were harvested immediately and weighed. The animal study was conducted at the Institute of Otolaryngology of Hebei Medical University following the NIH guidelines. All procedures of animal experiments were approved by the Hebei Medical University and were conducted in agreement with international guidelines for the maintenance and care of laboratory animals.An animal xenograft model was employed to examine the effect of PRAME on tumor growth 2.12t-test and chi-square test. All statistical tests were two-sided, and P < 0.05 indicated a difference achieving statistical significance. GraphPad Prism7.0  was used for relevant diagrams.Statistical analysis was conducted using SPSS21.0 software . The qRT-PCR results are expressed as the mean ± standard deviation (S.D), and significance analysis between different groups was conducted using Student’s 33.1P values versus normal control, and PRAME was identified as the greatest fold change of 305.222 in upregulated DEGs. Moreover, we used the GEO database to further validate the expression of PRAME in the LSCC. The GEO online database (https://www.ncbi.nlm.nih.gov/gds/?term=) including GSE143224, GSE117005, GSE84957, GSE59652, GSE59102, and GSE51985 were applied to obtain the gene matrix expression profiles of LSCC using the RobustRankAggreg R package , as well as with lymphatic metastasis (P < 0.05)  . However < 0.05) . Further < 0.05)  and the  < 0.05)  of patie3.2in vitro. The expression levels of PRAME in LSCC were characterized in AMC-HN-8 and TU177. A higher mRNA expression levels of PRAME were observed in the cell lines of AMC-HN-8 and TU177 compared with the pool group . In addition, the number of TU177 colonies showed an apparent decrease after the knock-down of PRAME . The above findings reveal that PRAME can stimulate the proliferation of LSCC cells.After the transfection of PRAME in the AMC-HN-8 and TU177, the role of PRAME in the proliferation of LSCC was also investigated ll lines . In addill lines . Since t3.3Our clinicopathologic correlation analysis suggested that a high expression level of PRAME was correlated with the TNM staging and lymphatic metastasis. As a result, the following study was to explore the potential involvement of PRAME in the migration and invasion in LSCC cells via regulating the EMT. Transwell assays and wound healing assay were employed to determine the functions of PRAME on the migration and invasion capabilities of those cells. It can be seen that overexpression of PRAME obviously enhanced the invasive and migratory capabilities of LSCC cell lines in both of AMC-HN-8 and TU177, compared with the vector group, respectively . FurtherExisting research reveals that the migration and invasion of cells are primarily dependent on the EMT process ,35, in w3.4P = 0.00)  . To be s = 0.00) . Moreove = 0.00) . Further = 0.00) . In addi = 0.00) . In brie3.5in vitro  and KEGG pathway analyses were conducted to predict downstream signaling pathways. This study was conducted based on GO term analysis with respect to biological process (BP) and cellular component (CC), as well as MF. The functional enrichment results revealed that 12 BP terms, 6 MF terms, and 2 CC terms were correlated with the overexpression of PRAME . Interesin vitro .Furthermore, PRAME overexpression-induced mRNA or protein expression levels of EMT markers of E-cadherin, β-catenin, N-cadherin, Snail, Vimentin, and ZEB1 were also partially abrogated in this pathway . In the 3.6in vivo, the mice were subcutaneously inoculated with AMC-HN-8 cells stably transfected with overexpressed PRAME. Importantly, there was a significant increase in the relative expression levels of PRAME in AMC-HN-8 cells after the transfection with overexpressed PRAME  cell lines of Huh-BAT and HepG2 [in vivo study further strengthens the view that PI3K/AKT/mTOR signaling plays a role in LSCC in the development and progression of LSCC.Studies have found that EMT process is regulated by a wide variety of signaling pathways. The PI3K/AKT/mTOR signaling pathways have been known to be implicated in tumorigenic roles in various cancers via the EMT process . Existinnd HepG2 . TGFβ-innd HepG2 . In addind HepG2 . It is nnd HepG2 ,17,55. Tin vitro study in human acute myeloid leukemia cells, a selective HDAC inhibitor chidamide can upregulate PRAME mRNA expression [Histone acetylation is a typical type of epigenic modification, and the level and state are regulated by both HATs and HDACs ,57. HATspression . It was pression . Thus, ipression . Furtherpression  that couIn vitro studies confirmed that PRAME facilitated the proliferation, invasion, migration, and EMT of human LSCC cell lines. In vivo also substantiated that PRAME promoted tumor growth. In addition, HDAC5 was identified as an upstream regulator that can affect the expression of PRAME and contribute to LSCC progression. Moreover, both in vitro and in vivo studies further confirmed that PRAME can facilitate the proliferation, migration, invasion, and EMT of LSCC cells and promote tumor growth, at least partially by activating PI3K/AKT/mTOR pathways (In the present study, we found that PRAME was overexpressed in the LSCC patients, and which was also significantly correlated with the TNM staging and lymphatic metastasis. pathways . In brieLSCClaryngeal squamous cell carcinoma;ESCCesophageal Squamous cell carcinomaPRAMEpreferentially expressed antigen in melanomaGOGene OntologyKEGGKyoto Encyclopedia of Genes and GenomesEMTepithelial–mesenchymal transitionHATshistone acetyltransferasesHDACshistone deacetylases


Hyperspectral imaging represents a suitable, non-invasive method to evaluate tissue morphology and organ perfusion during NMP. Liver allografts were subjected to NMP prior to LT. Serial image acquisition of oxygen saturation levels (StO2), organ hemoglobin (THI), near-infrared perfusion (NIR) and tissue water indices (TWI) through hyperspectral imaging was performed during static cold storage, at 1h, 6h, 12h and at the end of NMP. The readouts were correlated with perfusate parameters at equivalent time points. Twenty-one deceased donor livers were included in the study. Seven (33.0%) were discarded due to poor organ function during NMP. StO2 (p < 0.001), THI (p < 0.001) and NIR (p = 0.002) significantly augmented, from static cold storage (pre-NMP) to NMP end, while TWI dropped (p = 0.005) during the observational period. At 12–24h, a significantly higher hemoglobin concentration (THI) in the superficial tissue layers was seen in discarded, compared to transplanted livers (p = 0.036). Lactate values at 12h NMP correlated negatively with NIR perfusion index between 12 and 24h NMP and with the delta NIR perfusion index between 1 and 24h . Furthermore, NIR and TWI correlated with lactate clearance and pH. This study provides first evidence of feasibility of hyperspectral imaging as a potentially helpful contact-free organ viability assessment tool during liver NMP.Normothermic machine perfusion (NMP) allows for   While this concept is uniquely appealing, the identification of techniques and biomarkers for a meaningful determination of the quality and function of an organ remains to be established. Essentially, NMP mimics physiologic liver perfusion. During a period of up to 24 h, the liver is accessible for inspection, biopsy, perfusate and bile sampling  continues to rise. This poses a risk of increased rates of early allograft dysfunction (EAD), primary non-function (PNF) and biliary complications –10. Compsampling . Contempsampling –17. Furtsampling , 12. Novsampling –20. Thissampling –27. We hLiver allografts accepted for transplantation were procured and subjected to NMP. The decision to apply NMP at our center was based on a previously developed concept . NMP wasThe study protocol was approved by the local institutional review board.A total of 21 donor livers were enrolled in this study between December 2020 and May 2021. The majority of these livers were ECD livers. For definition of ECD, the Eurotransplant criteria were applied . These iFor the acquisition of HSI data, a contactless and non-ionizing radiation imaging system  was used under standardized conditions and previously reported settings , 30. TheWe primarily assessed the dynamic change of perfusion and oxygenation of liver tissue during NMP. Further to this, we have investigated 1) the differences in HSI dynamics between livers discarded and transplanted as well as 2) the correlations between HSI indices and perfusion parameters.The clinical follow-up of transplanted patients included the assessment of patient survival, graft survival, Clavien Dindo post-operative complications rate, EAD, Model of Early Allograft Function (MEAF) score, Liver Graft Assessment Following Transplantation (L-Graft) score, biliary and vascular complications, 90 days readmission rate, ICU and total hospital stay.p-values < 0.05 were considered significant throughout the entire analysis. Statistical analysis was performed using SPSS Statistics Version 27.0 for Macintosh .To examine the Gaussian distribution, we used the D’Agostino-Pearson normality test. The data were analyzed as proportions and medians with interquartile ranges (IQR), because they were consistent with a skewed distribution. Chi-square and Fisher’s exact tests for categorical variables and Mann-Whitney-U tests for continuous variables were used to compare the HSI values in the transplanted and non-transplanted groups. Ordinal variables were analyzed as continuous variables. Using the t or F distributions, Mann-Whitney-U tests were approximated for ordinal variables. The Friedman test and Sing tests were applied for paired non-parametric tests. To correlate HSI parameters and laboratory values measured in the perfusate during NMP, Spearman rank correlation tests were performed. Two-tailed p = 0.031). All discarded liver allografts were from male donors, while 8 (57.1%) transplanted liver allografts were from female donors (p = 0.011).During the study period, a total of 21 deceased donor livers were preserved via NMP. Night-time procedures were avoided and NMP time did not exceed 24 h. Seven (33.0%) were discarded after NMP, due to insufficient organ quality and performance. The median donor age was 61 years (48–70 years) and the median Donor Risk Index was 2.119 (1.610–2.435). Cold ischemia time (CIT) was 6 h (5–8 h) and total NMP time was 20 h (17–27 h). Six (28.6%) grafts derived from DCD donors (Maastricht category III), the remaining grafts from DBD donors. Median recipient MELD and Balance of risk (BAR) scores were 17 (8–21) and 7 (7–10), respectively. The median recipient age was 62 years (58–65 years). The median donor age of transplanted vs. discarded livers was 66 (56–70 years) vs. 46 years (43–56 years)  and 28 days (21–46), respectively. Six patients (42.9%) developed EAD, the median MEAF and L-Graft scores were 5.67 (4.02–6.90) and −0.73 (−1.33 – (−0.07)). Clavien-Dindo grade ≥3 complications occurred in 11 (78.6%) of 14 patients. Arterial complications occurred in two (14.3%) patients . Early (≤30 days) biliary complications were detected in six (42.9%) while late biliary complications (>30 days) in three (21.4%) patients. No patients developed non-anastomotic strictures, ischemic type biliary lesions (ITBL) or primary non-function. No patients were listed for re-transplantation. Two patients died due to multi-organ failure. The median follow-up was 106 (82–163) days. Recipient and donor demographics, as well as post-operative outcome parameters are described in p < 0.001, p < 0.001 and p = 0.002 respectively), while the TWI drastically decreased (p = 0.005) during the observational period , while the TWI dropped . Contrarily, StO2 mainly remained constant. The dynamics of perfusion and oxygenation over the entire NMP period (between 1 h and 12–24 h) illustrated a significant augmentation of StO2 , while the remaining HSI parameters remained stable. A longitudinal assessment of the tissue during NMP showed a substantial increase of the relative blood oxygenation StO2 , the NIR perfusion index  and the water distribution (TWI)  during the first 6 h of NMP, while HSI values remained stable after this time  over the entire observational period, while the tissue water concentration (TWI) drastically decreased (p = 0.016). Livers undergoing NMP without subsequent transplantation also displayed a significant augmentation of the relative blood oxygenation (StO2%) (p = 0.033). However, the other HSI parameters remained mainly constant during the study period. Notably, at the end of perfusion (12–24 h), a significantly higher hemoglobin concentration (THI) in the superficial tissue layers was seen in discarded, compared to transplanted livers (p = 0.036). In contrast, StO2, THI, NIR perfusion index and TWI parameters did not differ during the early course of NMP . The perfusate pH has been introduced as a viability criterium in the context of NMP, given the association with lactic acidosis, most commonly resulting from an imbalance between oxygen delivery and oxygen demand (p = 0.016). The TWI concomitated a decrease in lactatemia and the rising pH. Liver oedema and the related parenchymal damage as detected with HSI during cold storage decreased during NMP. In accordance, the TWI between 6 and 12 h and between cold storage and 1 h NMP were negatively associated with the pH at 12 h , while a high TWI during static cold storage correlated with a high pH at 6 h , , . These fIn vivo, HSI can detect alterations at the early stages of NMP. While intra-operative haemodynamic monitoring has been limited to systemic measurements, a more organ-specific approach reflecting local oxygen delivery and microcirculatory perfusion has gained interest (This pilot study was conducted with the intent to investigate HSI in the clinical setting of liver NMP. NMP allows to push the boundaries of organ transplantation, including the use of ECD grafts and longer preservation times , 35–37. interest –40. In tinterest , 42. Indinterest . Intraopinterest . Sidestrinterest . In continterest , 23, 30.interest . Moreoveinterest –51. Histinterest , 12. It interest . For livinterest . In an aThe strengths of the HSI technology as applied during NMP are the immediate applicability and the comprehensive assessment of the perfusion state of an organ over the entire exposed surface . Integrap-values ≤ 0.05 were termed significant for descriptive reasons only.In addition to the small cohort analyzed in this study, the different perfusion times and the overall heterogeneity of the liver grafts represent apparent limitations. Further to these, HSI has a relatively low tissue penetration depth, which precludes the detection of injuries in deeper regions, or the potential transcutaneous measurement after transplantation. All in all, HSI during NMP appears promising and feasible and its apparent simplicity makes it attractive for clinical use, but validation in large clinical trials is needed before establishing routine application. All analyses are explorative and To the best of our knowledge, HSI has not yet been applied previously in the field of liver NMP. We herein proved the technical feasibility of the combination of HSI and NMP. This real-time perfusion imaging may contribute to pre-transplant viability assessment.


Spectrum-based Fault localization have proven to be useful in the process of software testing and debugging. However, how to improve the effectiveness of software fault localization has always been a research hot spot in the field of software engineering. Dynamic slicing can extract program dependencies under certain conditions. Thus, this technology is expected to benefit for locating fault.We propose an improved dynamic slicing for spectrum-based fault localization under a general framework. We first obtain the dynamic slice of program execution. Secondly, we construct a mixed slice spectrum matrix from the dynamic slice of each test case and the corresponding test results. Finally, we compute the suspiciousness value of each statement in the mixed-slice spectram matrix.To verify the performance of our method, we conduct an empirical study on 15 widely used open-source programs. Experimental results show that our approach achieves significant improvement than the compared techniques.Our approach can reduce approximately 1% to 17.79% of the average cost of code examined significantly. Where S is the node set of CFG, E is the set of edges, and edges represent the data dependency or control dependency between two nodes.System Dependence Graph: System Dependence Graph (SDG), SDG = {GPDGs, EInterpro} of multi-process program P is a directed graph. Where GPDGs is the PDG set, and each process is represented as a PDG; EInterpro is the set of inter-procedure calling edges and dependent edges. Data-dependent edges represent data flow between actual and formal parameters.The Static Slicing: Given a slicing criterion s is the interesting points in the program and V is the set of variables. A static slice of a program P is the set of all statements affecting the set of variables V in a point of interest s (a statement or block of statements).The Dynamic Slicing: Given a slicing criterion I is the input to the program, the s is the interest in the program, and V is the set of variables. Based on the input I, a dynamic slice is the set of all statements of the program P that affect the variable set V in the point of interest s (a statement or block of statements) in this execution.The concept of program slicing was first proposed by Weiser in his doctoral dissertation in 1979 . SubsequTo solve this issue, Dataflow-based program slicing algorithm. The basic idea is to first calculate the control dependencies in the program, and then according to the data flow transfer in the control flow analysis program, iteratively construct slices by calculating the statements of the direct and indirect dependencies of the variables. The first program slice proposed by Graphreach-based program slicing algorithm. The basic idea is to obtain program slices by traversing the constructed Program Dependency Graph (PDG) or System Dependency Graph (SDG), and finding other nodes that the program can reach from the slicing criterion according to the dependencies. For example, At present, the typical program slicing algorithm is based on the data flow equation and the graph reach ability algorithm, and other algorithms are extended or improved from these two types of algorithms. The following briefly introduces the program slicing algorithm based on data flow and the program slicing algorithm based on graph reach ability.By program slicing, all statements can be classified into correct set of statements and suspicious set of statements. Therefore, most of the slicing-based fault location techniques use different slices , executiSoftware fault localization has a problem that the search domain is too large and the accuracy is not high. Fault localization approaches often use dynamic slicing techniques to narrow down the scope of the fault. However, the existing forward computing dynamic slicing approaches  calculatFor the example program in arantula  approacharantula  and imprHowever, the existing forward-calculated dynamic slice side  calculatAs can be seen from columns 2 and 3 of In this section, we present an improved dynamic slicing for fault localization. Our approach collects the dynamic slice information of program execution. Next, we construct a mixed slice spectrum matrix from the dynamic slice information of each test case execution and the corresponding test results. Finally, we compute the suspiciousness ranking of each statement in the mixed slice spectrum matrix. The framework of our approach is shown in Dynamic slicing is an effective debugging technique, which has been widely used in fault localization. When program execution results in a fault output or exception, the debugger usually wants to quickly ascertain the range of possible fault statements. By calculating the backward dynamic slice of the fault output statement, the dynamic slice can narrow the range of fault localization. Compared with the backward calculation, after the occurrence of the fault, the forward approach can generate a dynamic slice, which is according to the direct dynamic dependency of bug output statement. The forward approach does not need to traverse the dynamic dependency in the back, so it can calculate the dynamic slice result more quickly when a program runs incorrectly.Dynamic slicing technology  reduces Therefore, the forward calculation approach is used to calculate the program’s dynamic slicing, and the influence set analysis of the defined variables is carried out to improve the slicing accuracy, and the obtained dynamic slicing results are used for fault localization. The forward calculation method obtains the dynamic slice of interest points in the program through the direct dynamic dependencies of the interest points in the program. This method needs to maintain the slice results of each execution instance of the program . The forDefinition1. Influence set Influence[v]. For a given statement s, the variable Influence[v]= {v′| The v′ is the variabl e actually referenced when v is defined, Def[s] is the set of variables defined in statement s, and the reference set Use[s] is the set of variables referenced in statement s.In this section, the influence set analysis is performed on the defined variables to remove irrelevant variables in order to reduce the redundancy of the slicing results.Definition2. Hybrid Slicing Spectrum Matrix (HSSM). For a program P consisting of n statements s and a given m test cases, each execution of the test case yields a dynamic slice (slice) and the corresponding execution result. The mixed slice spectrum matrix is a two-dimensional matrix composed of multiple dynamic slices and execution results.This hybrid slice spectrum matrix uses the program dynamic slice information to extend the program coverage information and combines it with the corresponding program execution results to generate the hybrid slice spectrum matrix.call), dynamic control dependency of slicePred). The final output is the statement s refers to the dynamic slice of the variable use and the statement s defines the dynamic slice of the variable def. Lines 1–3 calculate the dynamic slice[use] of the variable use in the reference set Use[s] of statement s. slice[use] of the referenced variable based on the dynamic data dependency of the current execution instance def according to definition 1: Influence[def]. Lines 11–18 compute the dynamic slice[def] of the variable def in the definition set of Def[s]. Line 11 executes the dynamic control dependency slicePred of the control dependent predicate pred. Lines 12–18 calculate the dynamic slice slice[def] of the defined variable, which is consist of four parts: the current execution statement s, the call statement call, the control dependent predicate slice slicePred, and the slice of the variables influence in the defined variable influence set Influence[def].Program slicing can extract a set of statements from a program according to a certain slicing criterion, we use the fault-related criteria of failed runs by program slicing to collect the slices. Our approach computes the slices according to the Program Dependence Graph (PDG) and the slice of statements with a forward computing algorithm. Dynamic slicing of programs can narrow the search for software fault localization by obtaining statements related to the slicing criterion. Then, we compute the suspiciousness of entities in the dynamic slice by the formulas of fault localization to generate a fault localization report. For passed tests and failed tests, more uncovered entities with passing executions and covered entities with failed executions should be assigned a higher degree of suspiciousness. Moreover, the covered entities by more passed executions and the uncovered entities by the failed executions should be assigned a lower suspiciousness. Therefore, the number of covered entities should have more weight than the number of uncovered entities in the suspiciousness calculation. Over the past few decades, dozens of doubtful formulas have been proposed and evaluated through empirical research. In our approach, we apply Tarantula , Naish1 i.e., Tarantula, Naish1, GP02, Ochiai, Jaccard, and GP13) and compare our approach and other approaches using the same evaluation formula, respectively. For example, Tarantula is applied to the original dynamic slice for locating faults, which is denoted as D-Tarantula; Tarantula is applied to the improved dynamic slice to locate faults, which is denoted as DS-Tarantula. Whereas, Tarantula is applied to ADBS to locate faults, which is denoted as ADBS-Tarantula.To verify the effectiveness of our approach, we compare our approach with both the original dynamic slice and ADBS on the fault localization. In this Section, 15 Java programs are selected as the benchmark for testing, and the detailed description is shown in Our approach applied to single-bug version programs to locate fault. We removed two versions with no test case execution failure and eight versions with multiple faults from the 41 fault versions of jtcas, and selected the remaining 31 versions for testing. We randomly selected 10 of the 23 incorrect versions of the totAs shown in fourth column of In order to evaluate the effectiveness of various fault localization approaches, we use the following four criteria to evaluate the effectiveness from different perspectives.Cumulative Number of Statements Examined: The number of cumulative examined statements for each program is the number of total statements that need to be checked when m faults are located in m fault versions. For m fault versions of any given program, M(i) and N(i) represent the number of statements that need to be checked when approach M and approach N locate a fault. If Cost Score: Cost criterion is to evaluate the effectiveness of fault localization from the perspective of relative index, which was first proposed by Because the faulty statement may share the same suspiciousness with others, the programmer might need to examine only one of these statements fortunately (best cases) or to examine all these statements to locate the real fault unfortunately (worst cases) . In the EXAM Score: To more intuitively compare the effectiveness of different approaches, we also use EXAM indicators for evaluation. The EXAM indicator is the ratio of the fault detection rate (% of faults located) to the code examined rate (% of code examined) (xamined) , as showIn the early research, Recall at Top-N: This dependent variable measures the number of faults with at least one faulty element within Top-N in the ranked list. This metric emphasizes earlier fault detection and has been widely used in fault localization work (ion work . KochharWe can observe the cumulative number of statements examined between our technique and the original dynamic slice in Furthermore, we can observe the cumulative number of statements examined between our technique and ADBS in %, 4.79%, 4.87%, 4.53%, 4.76% and 5.08%, respectively. Furthermore, the cost of DS-Tarantula is much smaller than that of D-Tarantula on schedule2, which is 6.27%, whereas the cost of DS-GP02 is much smaller than that of D-GP02 on schedule2, which is 8.52%.As shown in Than, we compare the average cost of fault localization our method and ADBS on each topic, as shown in Additionally, to more intuitively show the improvement of our approach over the compared techniques, we use the Exam metric to evaluate the effectiveness. As shown in As shown in In this section, we evaluate the time cost of the improved slice, the ADBS slice, and the original slice fault localization using the tarantula formula. As shown in In this subsection, we discuss the potential threats to the validity of our empirical study.Threats to external validity are about whether the observed experimental results can be generalized to other subjects. To guarantee the representativeness, we choose a large number of programs from the widely used Simens, Linux, and Defects4J suites. Our experiments consider both C and Java programs and include both seeded and real bugs. We realize that there is no perfect empirical study and there must be some tricky programs and bugs not considered in our experiments. We plan to enlarge our subjects in our future work.The internal validity of this work lies in the accuracy of the slicing result computed by our approach. To avoid faults in our tool implementation, we prepared our data carefully and tested our approach with simple programs.Threats to construct validity are about whether the performance metrics used in the empirical studies reflect the real-world situation. In the experiment, we used the cumulative number of statements examined, Cost, EXAM, and Top-N other indicators. We realize some indicators were not considered in our experiments. Therefore, using more widely metrics is our future work.alias analysis on sliced programs. The second type of method is the combination of program slicing and program spectrum methods for fault localization. For example, After Spectrum-based tuning is one of the most popular tuning techniques in the automated tuning process. The spectral information of program execution is used to calculate the suspiciousness of each statement , Ochiai In SBFL, ranking is based solely on the suspiciousness score of each element. Elements with high scores are placed at the beginning of the list, and elements with low scores are placed at the bottom. To enhance the impact of failed test cases on fault localization, In this article, we have proposed the improved dynamic slices to improve the effectiveness of fault localization. In details, we collect the dynamic slice information of program execution. Secondly, we use dynamic slices and execution results to build a hybrid spectrum. Last, a fault localization report is finally obtained by computing the suspiciousness of each statement in the hybrid slice spectrum. In the empirical study, our approach is compared with three fault localization approaches. Experimental results indicate that our approach outperforms other compared approaches. Furthermore, it can reduce approximately 1% to 17.27% of the average cost of examined code significantly. In the future, we will continue to verify the performance of our proposed technique on more subjects and further improve the accuracy of fault localization.In the future, we want to further consider the following issues. Firstly, we plan to apply our approach to locate multiple faults. Secondly, we want to use the methods like deep learning to deal with the massive information of slice computing, so that we can improve our method to locate faults in more large-scale and real-world applications. Last but not least, we want to apply our approach to more subjects written by other programming languages such as C++, Python and conduct more detailed empirical studies.10.7717/peerj-cs.1071/supp-1Supplemental Information 1Click here for additional data file.10.7717/peerj-cs.1071/supp-2Supplemental Information 2Click here for additional data file.10.7717/peerj-cs.1071/supp-3Supplemental Information 3Click here for additional data file.


Web-based advance care planning (ACP) programs may support patients in thinking about and discussing their preferences for future treatment and care. However, they are not widely available, and only a limited number of programs are evidence based.We aimed to develop and evaluate an evidence-based, interactive web-based ACP program that guides users through the process of thinking about, discussing, and recording of preferences for treatment and care.The program “Explore your preferences for treatment and care” was developed, pilot-tested on feasibility, and subsequently evaluated; engagement in ACP was assessed before program completion and 2 months after program completion using the ACP Engagement Survey (score 1-5) among 147 persons with chronic disease. Usability (score 0-100) and user satisfaction (score 1-5) were also assessed.P<.001); contemplation about ACP increased from 2.6 to 2.8 (P=.003), and readiness for ACP increased from 2.2 to 2.5 (P<.001). No changes were found for knowledge about ACP  and self-efficacy for ACP . The program was perceived as usable , attractive , and comprehensible .ACP engagement increased from 2.8 before program completion to 3.0 two months after program completion (We developed an evidence-based, interactive web-based ACP program in cocreation with patients, relatives, and health care professionals. Before-and-after evaluation showed that the program can support people in taking first steps in ACP and in reflecting on preferences for treatment and care, by guiding them through the process of ACP using a stepwise approach. Participants perceived the program as usable and understandable, and they were satisfied with the program and with the amount of information. Health care professionals may use the program as a tool to start ACP discussions with their patients. The program may increase awareness of ACP. Reflecting on future treatment and care can be relevant for everyone, but especially for older persons and people with chronic diseases . AdvanceACP is typically conducted through face-to-face conversations between patients, their relatives, and health care professionals . HoweverInteractive web-based programs may support patients in the first steps of ACP, for instance, in communicating their preferences and in recording these preferences . Web-basPrepare For Your Care program [Making Your Wishes Known program [Examples of web-based ACP programs that have been shown to support people in ACP include the  program  and the  program . A scopi program . Further program . EvaluatThe aim of this study was to develop and evaluate an evidence-based, interactive web-based program for ACP in the Netherlands.The developmental process of the web-based ACP program was based on the main principles of the Medical Research Council guidance for developing and evaluating complex interventions, which consists of four phases: development or identification of the intervention, feasibility, evaluation, and implementation; while in each phase considering context, developing and refining program theory, engaging stakeholders, identifying key uncertainties, refining the intervention, and economic considerations . From thThe objectives of the program were to develop a web-based program that (1) informs about ACP and its possibilities and impossibilities; (2) invites patients to think about preferences and goals for future treatment and care; (3) invites patients and relatives to share preferences and goals for future treatment and care with each other and with health care professionals; (4) invites patients to record preferences and goals for treatment and care; and (5) invites patients to appoint a health care representative. Users could choose the steps of ACP they are ready to engage in and are not required to complete the entire program.Input for the program’s content and structure came from several sources :In a scoping review, we examined the content, feasibility, and effectiveness of evidence-based, interactive web-based ACP programs . We idenIn an interview study, we identified information needs of patients with chronic diseases and their relatives regarding web-based ACP, such as the need for information about ACP and its relevance, the need for reliable information about their disease and (arranging) care, and the need for peer support, as well as search terms for finding ACP related information .Thuisarts.nl [A stakeholder group was formed to include the perspectives of different stakeholders during the development, evaluation, and implementation of the web-based ACP program. The stakeholder group included 1 patient (co-author FRMDVH), 2 relatives, and representatives of the Dutch College of General Practitioners (Nederlands Huisartsen Genootschap), the Dutch Association for Kidney Patients (Nierpatiënten Vereniging Nederland), the Dutch Association for Patients (de Nederlandse Patiëntenvereniging), and Agora . Furthermore, the stakeholder group included 1 expert in health communication of the Nivel , 1 expert in eHealth of the University of Twente, 1 representative of Vital Innovators, an organization that conducts Social Return of Investment analyses, and the researchers  with expertise in shared decision-making, care at the end of life, and eHealth. During the entire 4-year project, the stakeholder group met approximately 2 to 3 times per year for 2-hour meetings. During these meetings, the progress of the project, preliminary results, and planned next steps were discussed. The stakeholders provided their feedback, which was processed by the researchers. The members of the group also assisted in the implementation of the program, for example, by disseminating the program, and participating in media interviews and in seminars about the program. The program was embedded in the general practitioners’ platform sarts.nl  (Englishsarts.nl ). Repressarts.nl . It is vsarts.nl ,14.The program’s feasibility was evaluated in a pilot study. On the basis of the definition of feasibility of Bowen et al ,16, we eThe pilot study and evaluation study were approved by the Medical Research Ethics Committee of the Erasmus MC, University Medical Center Rotterdam on October 21, 2019 (MEC-2019-0590). Participants provided written informed consent. The data for the pilot study were collected in February 2020, and the data for the evaluation study were collected from April 2020 to June 2020.Participants were recruited via an internet-based Dutch research portal . In thisHaving a chronic disease, defined as a disease that lasts 3 months or longer, is not (completely) curable, and which reoccurs regularly. Examples are chronic obstructive pulmonary disease, multiple sclerosis, and cancer. Participants with a psychological disorder or dementia were not invited to the study.Participants aged ≥18 years were included.We used purposive sampling by inviting comparable numbers of men and women, with diverse educational backgrounds, living across the Netherlands.Members of the research portal were asked to (1) complete the baseline survey on ACP engagement, health literacy, and demographics; (2) complete the web-based ACP program and a survey on usability and user satisfaction; and (3) complete a survey on ACP engagement after 2 months. Reminders were sent if participants had not completed the measurement within 1 to 2 weeks. All participants had completed the baseline survey before the launch of the web-based ACP program.We assessed participants’ age, level of education, country of birth , and theACP has evolved from focusing on completing advance directives to an ongoing behavior change process of considering, discussing, and recording goals, values, and preferences for treatment and care ,15. The Participants completed the validated Dutch ACP Engagement Survey (34 items) -24 beforWe assessed the program’s usability with the System Usability Scale  ,27. A toWe statistically analyzed participants’ responses on the ACP Engagement Survey using the software IBM SPSS Statistics. As the data were not entirely normally distributed, we conducted nonparametric testing with Wilcoxon Signed Rank statistical tests to compare participants’ responses on the ACP Engagement Survey before and 2 months after program completion. To see whether the readiness items indicated a behavior change since baseline, for instance, whether participants moved from the precontemplation behavior change stage to contemplation, action, or maintenance , we conducted McNemar tests . To asseThis study was approved by the Medical Research Ethics Committee of the Erasmus MC, University Medical Center Rotterdam on October 21, 2019 (MEC-2019-0590). All methods were performed in accordance with the relevant guidelines and regulations (Declaration of Helsinki). The study conforms with the International Committee of Medical Journal Editors (ICMJE) recommendations for the conduct, reporting, editing, and publication and for the protection of research participants. Participants were recruited via an internet-based Dutch research portal  and provExplore your preferences for treatment and care [The web-based ACP program and care  consistsThinking about preferences for future medical treatment and careDiscussing preferences for treatment and care with relatives and health care professionals and appointing a personal representativeRecording preferences for treatment and care; instructions are provided on how to record preferences in an advance directive and to review preferences (it is not possible to create an advance directive in the program itself)Users can choose the steps of ACP they are ready for to engage in and are not required to complete the entire program. The program contains videos, questions, and links to information on health and disease. The user can print or save a document in PDF with the responses to the questions in the program. Content of the programMain topic (every bullet point is described on a separate page):Home pageAbout the programAbout ACPFor whom and whenUseful websitesDisclaimerStep 1: Thinking about your treatment and care preferencesInformation about what is important in life and thinking about preferences (with video)Question: what is important to you?Question: what does this mean for your treatment and care preferences?Question: which care would you like to receive or not?Your preferences when being severely ill and when you will not recover anymoreQuestion: what have you learned from previous experiences?Statements:I want to live as long as possible, even when my quality of life is not good.I want to try various treatments, but I want to stop when my quality of life is no longer good.I want to live as comfortable and free from pain as possible, even if this would mean my life would be shorter.Step 2: Discussing your treatment and care preferencesThe health care representative (with video)Question: have you already thought about a health care representative?Question: who would you choose as your health care representative?The role of your health care representativeQuestion: are there additional things you would want your health care representative to address?Discussing your preferences with your health care representativeQuestion: what do you need to start the conversation with your health care representative?Discussing your preferences with your doctorQuestion: what do you want to discuss with your doctor?Step 3: Recording your treatment and care preferencesHow to make an advance directive (with video)Question: have you already recorded your preferences in an advance directive?Question: what topics would you want to record in an advance directive?Discussing your advance directive and sharing itQuestion: when would you review your advance directive?End of programYour answers as given while completing this program (user sees answers and can save these in PDF or print these)Functionalities of the programUsers can generate a document with the questions and their answers, this document can be printed and saved in PDFUsers can navigate in the program: they can skip steps or can go back and forward in the programUsers can track their progress in the programUsers can answer open and closed questions, and statementsThe program is interactive: users can answer questions, click on links for additional informationUsers can watch videos with patient experiences with ACPThe program refers to useful information about disease, treatment and care, information for relatives, patient associations, and peer support, partly within the website “Thuisarts.nl.”Audio can be used, text-to-speech option: text can be read out loudThe program can be accessed by phone, computer, and tabletClear and understandable languageClear structure and layoutIn the pilot study, patients mentioned that the program made them think about their treatment and care preferences and they understood the questions in the program well. The participating physicians thought the program would be valuable for patients to support them in ACP. All interviewees were able to complete the program without problems. Some minor suggestions were given to the web design team. For example, sometimes interviewees clicked on a hyperlink to an external website without noticing they left the program website. Subsequently, the web design team inserted a notification, and they also applied small language improvements based on the interviewees’ feedback. All participants thought the program was interesting. Some interviewees mentioned that they would recommend the program to others.The baseline questionnaire was sent to 550 members of the research portal with chronic disease. The baseline measurement was completed by 70.9% (390/550) of the participants, the second measurement  was completed by 40.8% (159/390) of the participants, and the measurement after 2 months was completed by 92.5% (147/159) of the participants .Participants who completed all questionnaires (n=147) were included in this study. They were 60.5 years of age on average , and of the 147 participants, 82 (55.8%) were male and 65 (44.2%) were female; this was largely representative for the general Dutch population of people aged ≥18 years  . Of the P<.001). Contemplation about ACP increased from 2.6 to 2.8 (P=.003). Readiness increased from 2.2 to 2.5 (P<.001). No changes were found for knowledge about ACP  and for self-efficacy for ACP . Comparing baseline and the measurement after 2 months, we found significant increases for three of four domains: (1) surrogate decision makers increased from 2.8 to 3.0 (P<.001); (2) what matters most in life: health situations increased from 3.0 to 3.2 (P=.003) and care at the end of life increased from 2.9 to 3.0 (P=.02); and (3) flexibility in medical decision-making increased from 2.6 to 2.9 (P<.001). We found no significant differences for domain 4, ask your doctors questions . The total ACP engagement score increased from 2.8 before program completion to 3.0 after 2 months (P=.11), except for the domain “flexibility in medical decision-making”; participants moved from precontemplation to higher stages of behavior change ; this difference was significant (P=.04).The McNemar test showed no significant difference in participants’ stages of behavior change after 2 months compared with baseline  and neither for the domains .The mixed ANOVAs with a post hoc Bonferroni test indicated no significant differences on level of education  for the subscales (knowledge: P=.19), education level (P=.29), and levels of ACP engagement  and the domains: surrogate decision makers, P=.98; what matters most in life: health situations, P=.74; and care at the end of life, P=.99; flexibility in medical decision-making, P=.43; and ask your doctors questions, P=.33), suggesting there was no selection bias owing to the dropout.The incomplete responses or dropout in the measurement in which the program needed to be completed was 53.2% (181/340) of participants; they started the questionnaire, which contained the  link to the web-based ACP program, but did not complete the postprogram questionnaire. As user data are not recorded in the program, we were not able to see whether these participants completed the program. When comparing the 147 participants who completed all measurements with the 390 participants who completed only the baseline, we found no significant differences among the groups for age  participants indicated they would use the program frequently, 25 (17%) participants would not and 72 (49%) participants were neutral. Of the 147 participants, 115 (78.2%) thought the program was easy to use and 26 (17.7%) participants were neutral. In total, of the 147 participants, 96 (65.3%) participants thought the functions were well-integrated and 115 (78.2%) participants felt they did not need to learn a lot before they could use the program. The mean total SUS score was 70 . On average, participants rated the attractiveness of the program as 3.8 , its comprehensibility as 4.2 , and the emotional support it provided as 3.4 . Of the 147 participants, 96 (65.3%) would recommend the program to others . ParticiParticipants thought the amount of information in the program was enough . Of 147 participants total of 54 (36.7%) participants added an explanation, of which 47 (87%) were positive, mentioning the content of the program was enough and the information was clear; 6 (11.1%) participants found the information quite a lot to complete at once.Explore your preferences for treatment and care [The web-based ACP program and care  was consThe program was evidence-based and developed in cocreation with patients, relatives, and health care professionals; their input ensured that it would meet the needs of its potential users. We had a varied sample of participants with chronic diseases with different ages and levels of education.As user data are not recorded in the program, we were not able to see whether participants completed the program and were unable to analyze their responses. Numbers of incomplete responses or dropout were quite high in the measurement immediately following the completion of the ACP program. Filling in the measurement required participants to return to the questionnaire after completing the program on a separate website or web page. It may be the case that this was not clear to participants or, alternatively, that they thought the program was too long or too difficult. However, the response rates in the measurement 2 months after completion of the program were sufficient and rather high according to the research portal: 92.5% (147/159); and we found no significant differences in participant characteristics in our baseline measurement that suggested we had no selection bias owing to the dropout.Thuisarts.nl. We used the ACP Engagement Survey to evaluate the program’s effects and found that it could support patients in ACP engagement. This confirmed the findings considering the web-based ACP program Prepare For Your Care from the United States [Most evidence-based, web-based ACP programs have been developed in the United States and only a few have been thoroughly evaluated . We deved States -35. We fd States .The availability of the program on the web may improve access to ACP information at any preferred time and place; this can be important as ACP is considered a process over time. The web-based ACP program may be an addition to the traditional ACP process as facilitated by health care professionals, as it includes information, questions to be answered, and videos. We believe web-based programs should not replace discussions with relatives or health care professionals, but the program may support patients in preparing for ACP discussions . Health The program was launched in April 2020, and it has been frequently used .As most participants are born in the Netherlands, we recommend to evaluate the program in persons with other countries of birth as well. In addition, since the participants were members of an internet-based research portal, their level of computer skills may be above the average skill of the Dutch population. As readiness for ACP can differ across patients , we recoExplore your preferences for treatment and care in cocreation with patients, relatives, and health care professionals. The before-and-after evaluation showed that the program can support people in taking first steps in ACP and in reflecting on preferences for treatment and care, by guiding them through the process of ACP using a stepwise approach. Participants perceived the program as usable and understandable, and they were satisfied with the program and the amount of information. Health care professionals may use the program as a tool to start ACP discussions with their patients. The program may increase awareness of ACP.We developed an evidence-based, web-based ACP program


Physicians must increasingly lead change for improvement in the value of health care for individuals and populations. Leadership, stewardship, and population health competencies are not explicitly part of the Accreditation Council for Graduate Medical Education (ACGME) requirements and are best appreciated in the context of Health Systems Science (HSS).HSS education is best approached at the institutional level, yet almost all graduate medical education (GME) curriculum is at the program level. We describe the process of designing and implementing an institutional HSS GME curriculum in a hospital-based sponsoring institution.A group of diverse stakeholders drafted a curriculum to build competencies in leadership, stewardship, and population health, which was further refined by our Graduate Medical Education Committee (GMEC) and Resident Forum in the academic years 2015–2017. The refined curriculum was implemented at the institutional level of a large urban teaching hospital with over 80 ACGME accredited programs in the 2017–2018 academic year, participation was tracked and impact surveys were conducted.All programs participate in at least parts of the curriculum with sustained use. Annual surveys show a progression in assessment of our target competencies and/or opportunities to reflect and provide feedback. The annual program review meeting and GMEC meetings are used to troubleshoot and identify new curricular opportunities.This innovative institutional curriculum has been sustained for over four years and we believe that other training institutions with similar goals will find our experience implementing an institutional curriculum translatable to their clinical learning environment.The online version contains supplementary material available at 10.1186/s12909-022-03706-y. Future physicians will need competencies beyond those currently measured by the Accreditation Council for Graduate Medical Education (ACGME) milestones . PhysiciThe current competencies largely focus on the role of the physician in individual patient care, while the additional competencies focus on the role of the physician within an integrated health care delivery system focused on population health. Physicians will require leadership competencies to implement these changes and will increasingly be expected to collaborate with advanced practice providers and other members of the health care team, functioning as master clinicians and leading process improvement .Internal Medicine was the first discipline to consider additional competencies that future physicians will need  and otheTable These additional competencies are best appreciated in the context of health systems science (HSS) . HSS is Our GME leaders established meetings and worked collaboratively with senior institutional leaders, medical educators, and trainees, to create an experiential HSS curriculum utilized by all GME training programs in our large teaching hospital. We wanted to integrate this curriculum seamlessly with our health system’s clinical transformation and quality and safety initiatives to improve value of care for patients (stewardship) and for communities we serve . Our desire is to empower our graduates to utilize their acquired knowledge, skills, and attitudes not only during training but throughout their career. This requires intrinsic motivation gained by satisfaction with learning experiences in a supportive environment .Create a unified set of experiences shared across disciplines to build competencies in leadership, stewardship, and population health.Build upon existing undergraduate curricula in Health Systems Science (HSS) that have recently evolved.Provide our graduates with the relevant knowledge, skills, and attitudes that impact their future practice.Our overarching goal was to create an institutional HSS curriculum. Our curricular goals were:We are a large urban teaching hospital sponsoring over 80 ACGME accredited programs enrolling over 800 residents and fellows. We wanted to ensure incorporation of this training in every program and in an inter-disciplinary fashion whenever possible. We believe that curriculum transcending disciplines is very important for Health Systems Science as we train future physicians who will contribute to team and population well-being, as well as to the health of individual patients. Aside from onboarding orientation modules, we know of few other large institutions that have an institutional curriculum. A suggested framework for Health Systems Science Education was recently published . We descWe began the initiative in 2015. Many stakeholders (approximately 60) took part in review and discussion of the curriculum during the subsequent year. A GME Advisory task force comprised of program directors  and department chairs met to discuss the HSS curriculum monthly during the 2016–2017 academic year. Our Resident Forum, comprised of peer-selected residents from each training program, and the GMEC, comprised of program directors from a variety of disciplines, also included this as an agenda item during regularly scheduled monthly meetings. Hospital leaders, including the Chief Operating Officer and affiliated medical school leaders including the Assistant Dean for Health Systems Science and the Vice Dean for Education, also offered guidance in individual meetings.We chose Leadership, Stewardship, and Population Health to categorize our target competencies. Leadership includes professionalism, patient-centered communication, and teamwork. Stewardship includes quality improvement for high value care. Population health includes stewardship of quality improvement projects and stewardship for groups of patients. Once the desired competencies were articulated, we began to design curriculum.The first draft of the curriculum was detailed and included some course work similar to a Master’s in Public Health degree. We iteratively simplified the curriculum and made it less didactic and more experiential. We included perspectives on feasibility from multiple programs including medical, procedural, and hospital-based disciplines. Consensus was reached after one year, when no further changes were proposed.The curriculum is divided into 3 competencies, with learning opportunities for program directors/faculty and residents/fellows. Table Our primary goal was to create an institutional curriculum. The institutional, rather than program level, implementation, helped underscore the importance of our work. Obtaining buy-in and participation from over 80 different programs was the primary success metric.The annual internal GME faculty and resident surveys were modified in the 2016–2017 (baseline) academic year to include questions about frequency of assessment or personal reflection on competencies included in the curriculum. These questions were subsequently included in each annual survey after the HSS curriculum was implemented in 2017–2018 to assess trends in utilization and impact on clinical work. The results are discussed in the next section and the data is in supplementary tables.We included resources from our health system Leadership Institute, Quality Institute and GME office to encourage experiential learning in the areas of desired competencies. All residents are required to participate each year, and program directors participate to role model lifelong learning.Learning activities are differentiated for program directors/ faculty and for residents/fellows. Program director participation is mandatory, but faculty participation is optional at the discretion of the program. The program director and faculty expectations are set to promote role-modeling of life-long learning over an interval of many years, while the resident/fellow expectations are set to acquire competencies in the shorter interval of training.Learning objectives for Leadership include incorporating reflection in self-improvement of communication. We allowed program directors flexibility in implementation for either individual or group reflection. The online curriculum includes a link to an Association of Professors of Dermatology resource with suggested topics for reflection on communication in the clinical teaching environment. This link also includes a scoring rubric for assessing written essays that may be utilized by the Clinical Competency Committee (CCC) charged with assessing communication skills in ACGME accredited programs. We suggest that residents and fellows frame reflections in a way that will make them useful as preparation for future job or fellowship interviews when asked to respond to behavioral questions. For example, residents might write about caring for a patient whose values conflicted with their own and discuss this as a growth experience.Reflective essays induced anxiety, and the Graduate Medical Education Committee had concerns about requiring reflective essays in terms of evidence for educational value, time required for program leaders to read the essays, and risk of discovery in the event of a medical malpractice claim. We addressed these by providing literature demonstrating benefit . We alsoLearning objectives for Stewardship included knowledge of resources such as Choosing Wisely and experience with understanding patient perspective of value in their care. All programs are required to have a patient participate in one didactic session each year where the patient can explain their perception of value in each step of their disease journey. This is intended to develop the skill of listening for value from a patient perspective.Learning objectives for Population Health included knowledge of the populations we serve, specifically available local resources and challenges. Presented at institutional orientation as a bus  tour of inner ring neighborhoods near our flagship campus, we include instruction on structural racism and health disparities. Local experts explain the health care challenges as well as the health care and cultural resources in each neighborhood.There are many similar challenges between design and implementation of the more established undergraduate curricula in HSS and the GME HSS curriculum that we designed and implemented. Program directors strongly preferred learning experiences that did not interfere with scheduled didactic sessions, as they worried about interference with teaching time for basic and clinical sciences necessary to pass American Board of Medical Specialties examinations.Likewise, residents disliked activities away from clinical learning sites. We began with a requirement for live leadership training because of our belief that group learning is best for this competency. However, residents were concerned about potential time away from clinical education experience and interference with studying. This is similar to reported medical student resistance to undergraduate Health Systems Science curricula because of concern about interference with United States Medical Licensing Exams (USMLE) preparation . Some ReHospital leaders preferred a less experiential, more didactic curriculum with particular emphasis on adherence to coding and documentation and other policies. Undergraduate medical school leaders were also more familiar with classroom rather than experiential approaches, as didactic approaches are more uniform and measurable.We allowed programs to modify learning experiences if they presented their alternative as a “best practice’ at our Graduate Medical Education Committee meeting for approval, and if they opened the learning activity to residents/fellows from other disciplines. This has resulted in sporadic increases in inter-disciplinary learning, usually at the time of an external guest facilitated workshop. The idea of sharing common curricular experiences across disciplines was new to our program directors, but we felt that it was important to learn health systems science as a health system. This difficulty has also been documented in the literature in medical schools instituting their own HSS curricula .The list of alternative learning opportunities and contact information is posted on our GME intranet site in the section devoted to the Health Systems Science Curriculum for all programs to view. Examples of alternatives include a group discussion of a system issue followed by quality improvement project rather than individual reflective essays; this was appealing to our surgical specialists who felt that reflection alone felt too passive. Our psychiatry program offered their existing activity to trainees from other disciplines as a Population Health experience; they watch the 2017 documentary “Knife Skills” and then have dinner at the local restaurant that hires ex-convicts and was the focus of the movie.There was significant anxiety upon introduction of the curriculum about detracting from opportunities to acquire medical knowledge, and concern that each discipline had unique educational needs such that an institutional curriculum might be harmful . The anxResults of our annual GME internal surveys of faculty and residents show gradual progression in reflection and assessment of knowledge, skills, and attitudes that constitute our target competencies .Trainees were also surveyed annually 11 months after orientation and asked about whether they applied the knowledge gained from the orientation “See the City You Serve” tour in a patient care setting. In 2019 40% answered “yes”. Even residents and fellows who have lived in our city for many years commented that they had never visited these neighborhoods and found the experience very worthwhile.The ultimate measure of graduate outcomes is what physicians do in practice. Many of our programs have incorporated competencies in leadership, stewardship, and population health into their program aims. Success at meeting program aims is measured with periodic program level alumni surveys. We will encourage our programs to include questions in their graduate surveys that measure the graduates’ perception of how much these learning experiences benefited their professional lives and how marketable these experiences were in securing their first job. In 2015 as part of our GME strategic plan we articulated institutional aims for graduate performance including: Routinely seek perspective of other disciplines and professions for complex cases, practice high-value care, contribute to health policy, and work effectively within teams to ensure patient safety is the top priority.A limitation of our process is that the HSS curriculum is part of ongoing improvement in our health system, and it is not possible to define the exact contribution of the curriculum as opposed to other internal or external influences on our clinical learning environment. It is difficult to measure the impact on competencies of a curriculum that was modified as needed for a variety of disciplines.We have found the establishment of an institutional curriculum invaluable to increase visibility and use of resources from our Leadership Institute, Quality Institute, and GME professional development intranet toward meeting aims for graduate outcomes. We continuously improve this curriculum as an improvement plan overseen by our GMEC Annual Institutional Review Committee. For example, our Palliative Care team has recently added a communication workshop on shared end of life goals of care to the Leadership offerings; our Clinical Transformation Team is developing mini-rotations as part of our Population Health offerings to sites such as rehabilitation hospitals, home health, and other sites that many trainees never experience.Program aims are shared with applicants during recruitment. We expect that the HSS curricular aims will increasingly serve as a marketing tool despite only 7% of U.S. Seniors endorsing “opportunity for training in systems-based practice” as an important aspect of choosing a GME training program in the 2019 National Residency Matching Program survey, increasing to 7.5% in the 2021 survey .Use of our Health Systems Science Curriculum has been sustained for over four years, and the curriculum continues to evolve in response to user feedback. The curriculum was designed and implemented over approximately 18 months with only the costs of educators’ time, meaning that no dedicated budget for physical resources (other than buses for our orientation tour) was required.Expansion of assessments for impact on clinical practice of our graduates after they have completed GME training and are engaged in independent practice may lead to further changes in our curriculum to better meet our goals. We require program directors to survey their graduates to assess program and institutional aims. We will have more data available to analyze in the coming years.We believe that our experiences are translatable to other teaching hospitals. Each institution will need to consider the perspectives of their own stakeholders and the best approach to change management to create or improve the process of building new physician workforce competencies. These competencies should support institutional and program aims for graduate performance, and the degree to which graduates endorse behaviors consistent with these aims is the ultimate GME outcome metric.Additional file : Supplementary Table 1. Legend: Survey of Faculty  the year prior and the first two years after implementation of the curriculum. 1/3 respondents were program directors’. Note that all program directors, but not all faculty, participate in the curriculum.Supplementary Table 2:Legend:Survey of Residents and Fellows  the year prior and the first two years after implementation of the curriculum.


Tourism destinations constitute a conglomerate of attractions, service providers, and retailers that make up the overall offerings and experiences that attract visitors. However, given the severe consequences that the COVID-19 pandemic has had on the tourism industry, it is crucial to appraise consumer loyalty towards destinations in the context of the coronavirus disruptions. An increasing number of academic works examining the factors that influence destination loyalty have been carried out since the pandemic breakout, but no evaluation of their cumulative results and findings has been offered in the literature. Therefore, this research conducts a review of studies that have empirically investigated the drivers of destination loyalty during the pandemic in diverse geographical settings. By analyzing 24 journal articles selected from the Web of Science (WoS) database, this work adds to the literature by providing an assessment of the state-of-the-art body of knowledge about the explanation and prediction of loyalty for tourism destinations in the context of COVID-19. Building consumer loyalty is an essential objective for managers and is among the core relational constructs that have drawn greater attention in marketing and consumer behavior . For insDestination loyalty can be defined as the “behavioral consistency of repeated visits to a destination fueled by a psychological desire to visit the destination “, p. 870.By conducting a review of empirical results from studies elaborated during the pandemic, this work contributes to identify the drivers of destination loyalty as the focal construct, supporting the advancement of marketing theory based on inductive-statistical explanations . UnderstThe review of the literature was conducted through the Web of Science (WoS) platform , as in r2) ranging from 0.234 to 0.942. The outcome variables employed scale items corresponding to conative, affective, and/or cognitive loyalty to elicit revisit intentions or likelihood to return to the same destination. The operationalization of destination loyalty typically included scale items referring to recommendation or positive word-of-mouth (WOM) about the destinations, combined with other revisit intentions items within the same loyalty construct. However, some investigations specified and operationalized tourists’ recommendations or endorsements as additional, separate constructs in their models , with samples ranging from 123 to 774 (average sample size = 382). As indicated in Journal of Travel Research), rated “A” , or rated “B” . Three journals published more than one of the works reviewed: Sustainability (five articles), Journal of Destination Marketing & Management (three articles), and Current Issues in Tourism (two articles). The fact that two studies were published in Spanish language journals by Revista Universidad & Empresa, and by El Periplo Sustentable reflects the growing interest of academics based in Latin America to disseminate their research to broader audiences through journals indexed in international databases  tourist’s destination loyalty in the research of Three of the reviewed works addressed technological innovations used by consumers and also as part of the destination’s offerings. Due to the lack of a synthesis about research on tourism destination loyalty in COVID-19 settings, a literature review was conducted by examining 24 journal articles published in 2021 and 2022. Although the search for studies encompassed the time period since the coronavirus appeared, none of the 24 studies was published with an assigned volume/issue during 2020. It seems logical that when the pandemic crisis first broke out early in 2020 (first epidemic wave), many authors devoted their work to design, execute, write, and submit their research for peer-review, which ultimately led to final journal publication in the following years. In this respect, it should be noted that the effects of the constructs in the estimated loyalty models could have been influenced by the distinct time periods in which survey-data was obtained for each study. The perceived threat of infection according to fluctuations in coronavirus waves (upward or downward trends) has an impact on tourists’ risk assessments and intentions to travel . ConsideWhile some of the articles studied destination loyalty drawing from existing models in the marketing and tourism literature framed within the context of COVID-19 impacts, others works hypothesized moderation and/or mediation effects with new constructs specifically relevant to the pandemic disruptions e.g., . The oveNonetheless, it appears that loyalty towards destinations in some parts of the world could have been additionally influenced by the restrictions imposed on overseas travel to certain regions during the pandemic. This is suggested by the large variance explained in the loyalty outcomes reported for highly populated countries with significant domestic markets such as China , India , or someThe literature review was performed based on the records provided by the Web of Science (WoS) database derived from key term queries in English. Therefore, studies that could have been obtained by searching key terms in other languages were not included in the review. Further, the 24 works analyzed correspond to studies published in the form of journal articles, so other types of research in books, chapters, conference proceedings, or dissertations/theses were out of the scope of the review. The findings of the studies were based on convenience samples, which calls for caution in inferring generalizations given the limitations of non-probabilistic sampling . In termThe review of journal articles found that only one of the examined models specified cognitive and affective dimensions as drivers of destination loyalty . Hence, OC-M conceived, designed the concept, collected the data, wrote the manuscript, and read and agreed to the published version of the manuscript.This publication was supported by the TAMU-CC Open Access Publication Fund.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.