Effect of Irradiance and Exposure Duration on Temperature and Degree of Conversion of Dual-Cure Resin Cement for Ceramic Restorations.

This study investigated the effects of irradiance and exposure duration on dual-cured resin cements irradiated through ceramic restorative materials. A single light-curing unit was calibrated to three different irradiances (500, 1000, and 1500 mW/cm2) and irradiated to three different attenuating materials (transparent acryl, lithium disilicate, zirconia) with 1-mm thicknesses for 20 or 60 seconds. The changes in irradiance and temperature were measured with a radiometer (or digital thermometer) under the attenuating materials. The degree of conversion (DC) of dual-cure resin cement after irradiation at different irradiances and exposure durations was measured with Fourier transform near infrared spectroscopy. Two-way analysis of variance revealed that irradiance ( p<0.001) and exposure duration ( p<0.001) significantly affected temperature and DC. All groups showed higher DCs with increased exposure times ( p<0.05), but there were no statistically significant differences between the groups irradiated with 1000 mW/cm2 and 1500 mW/cm2 ( p>0.05). Higher-intensity irradiances yielded higher temperatures ( p<0.05), but exposure time did not affect temperature when materials were irradiated at 500 mW/cm2 ( p>0.05).

The G-protein alpha-subunit gene CGA1 is involved in regulation of resistance to heat and osmotic stress in Chlamydomonas reinhardtii.

In eukaryotic cells, many important functions of specific G-proteins have been identified, but microalgal G-proteins are poorly studied. In this work, we characterized a gene (CGA1) encoding the G-protein α-subunit in Chlamydomonas reinhardtii. Independent knockdown mutants of CGA1 were generated via RNA interference (RNAi). CGA1 expression levels were consistently and significantly reduced in both independent CGA1 mutant cell lines (cga1). Both cga1 mutants had a higher survival rate at 35°C in comparison with the wild type. This stronger resistance of the cga1 mutants became more evident during simultaneous exposure to heat and osmotic stress. The stronger resistance of the CGA1 knockdown mutants to the two stressors was accompanied with significant morphological alterations-both cell size and cell wall thickness were different from those of the wild type. This finding supports the roles of CGA1 in C. reinhardtii morphology in response to stressors. To further understand biochemical mechanisms of the CGA1-mediated resistance, we thoroughly analyzed the level of reactive oxygen species (ROS) and the expression of several heat shock proteins or MAP kinase genes as possible downstream effectors of CGA1. Our data clearly indicated that CGA1 is implicated in the regulation of resistance to heat or osmotic stress in C. reinhardtii via HSP70A and MAPK6. Because the G-protein α-subunit is highly conserved across microalgal species, our results should facilitate future biotechnological applications of microalgae under extreme environmental conditions.

Preliminary evaluation of the pattern cutting and the ligating loop virtual laparoscopic trainers.

INTRODUCTION: The Fundamentals of Laparoscopic Surgery (FLS) trainer is currently the standard for training and evaluating basic laparoscopic skills. However, its manual scoring system is time-consuming and subjective. The Virtual Basic Laparoscopic Skill Trainer (VBLaST©) is the virtual version of the FLS trainer which allows automatic and real time assessment of skill performance, as well as force feedback. In this study, the VBLaST© pattern cutting (VBLaST-PC©) and ligating loop (VBLaST-LL©) tasks were evaluated as part of a validation study. We hypothesized that performance would be similar on the FLS and VBLaST© trainers, and that subjects with more experience would perform better than those with less experience on both trainers. METHODS: Fifty-five subjects with varying surgical experience were recruited at the Learning Center during the 2013 SAGES annual meeting and were divided into two groups: experts (PGY 5, surgical fellows and surgical attendings) and novices (PGY 1-4). They were asked to perform the PC or the ligating loop task on the FLS and the VBLaST© trainers. Their performance scores for each trainer were calculated and compared. RESULTS: There were no significant differences between the FLS and VBLaST© scores for either the PC or the ligating loop task. Experts' scores were significantly higher than the scores for novices on both trainers. CONCLUSION: This study showed that the subjects' performance on the VBLaST© trainer was similar to the FLS performance for both tasks. Both the VBLaST-PC© and the VBLaST-LL© tasks permitted discrimination between the novice and expert groups. Although concurrent and discriminant validity has been established, further studies to establish convergent and predictive validity are needed. Once validated as a training system for laparoscopic skills, the system is expected to overcome the current limitations of the FLS trainer.

Validation of the VBLaST peg transfer task: a first step toward an alternate training standard.

BACKGROUND: The FLS trainer lacks objective and automated assessments of laparoscopic performance and requires a large supply of relatively expensive consumables. Virtual reality simulation has a great potential as a training and assessment tool of laparoscopic skills and can overcome some limitations of the FLS trainer. This study was carried out to assess the value of our Virtual Basic Laparoscopic Surgical Trainer (VBLaST(©)) in the peg transfer task compared to the FLS trainer and its ability to differentiate performance between novice, intermediate, and expert groups. METHODS: Thirty subjects were divided into three groups: novices (PGY1-2, n = 10), intermediates (PGY3-4, n = 10), and experts (PGY5, surgical fellows and attendings, n = 10). All subjects performed ten trials of the peg transfer task on each simulator. Assessment of laparoscopic performance was based on FLS scoring while a questionnaire was used for subjective evaluation. RESULTS: The performance scores in the two simulators were correlated, though subjects performed significantly better in the FLS trainer. Experts performed better than novices only on the FLS trainer while no significant differences were observed between the other groups. Moreover, a significant learning effect was found on both trainers, with a greater improvement of performance on the VBLaST(©). Finally, 82.6% of the subjects preferred the FLS over the VBLaST(©) for surgical training which could be attributed to the novelty of the VR technology and existing deficiencies of the user interface for the VBLaST(©). CONCLUSION: This study demonstrated that the VBLaST(©) reproduced faithfully some aspects of the FLS peg transfer task (such as color, size, and shape of the peg board, etc.) while other aspects require additional development. Future improvement of the user interface and haptic feedback will enhance the value of the system as an alternative to the FLS as the standard training tool for laparoscopic surgery skills.

A laboratory study of the effects of processing blood through a cell salvage device and leucocyte depletion filter on levels of pro-inflammatory cytokines and bradykinin.

We investigated changes in concentrations of interleukin-1ß, interleukin-6, tumour necrosis factor-α and bradykinin in blood during passage through a cell salvage device and a leucocyte depletion filter, with or without application of subatmospheric pressure across the filter. Blood samples from 19 healthy women undergoing scheduled caesarean section showed concentrations of cytokines and bradykinin in blood filtered under gravity flow that were equal to or significantly lower than those of pre-operative venous blood samples. They were also significantly lower than that in postoperative orthopaedic shed blood, which is commonly reinfused after orthopaedic surgery. A minority of samples taken from blood that had been filtered using subatmospheric pressure showed raised interleukin-6 concentrations. We suggest that use of a leucocyte depletion filter for cell-salvaged blood with gravity flow is likely to be safe with regard to concentrations of cytokines and bradykinin. However, this may not hold true for the filter used with subatmospheric pressure. If transfusion of salvaged blood using a leucocyte depletion filter seems to induce hypotension, elevation of interleukin-6 should be suspected.

A Logarithmic Complexity Floating Frame of Reference Formulation with Interpolating Splines for Articulated Multi-Flexible-Body Dynamics.

An interpolating spline-based approach is presented for modeling multi-flexible-body systems in the divide-and-conquer (DCA) scheme. This algorithm uses the floating frame of reference formulation and piecewise spline functions to construct and solve the non-linear equations of motion of the multi-flexible-body system undergoing large rotations and translations. The new approach is compared with the flexible DCA (FDCA) that uses the assumed modes method [1]. The FDCA, in many cases, must resort to sub-structuring to accurately model the deformation of the system. We demonstrate, through numerical examples, that the interpolating spline-based approach is comparable in accuracy and superior in efficiency to the FDCA. The present approach is appropriate for modeling flexible mechanisms with thin 1D bodies undergoing large rotations and translations, including those with irregular shapes. As such, the present approach extends the current capability of the DCA to model deformable systems. The algorithm retains the theoretical logarithmic complexity inherent in the DCA when implemented in parallel.

The added value of virtual reality technology and force feedback for surgical training simulators.

Laparoscopic surgery requires more specialized training of the surgeons than traditional open surgery. The Virtual Basic Laparoscopic Surgical Trainer (VBLaST) is being developed as a virtual version of the Fundamentals of Laparoscopic Skills (FLS) trainer. This study assessed the current haptic and virtual reality (VR) technology of a virtual peg transfer task of the VBLaST, based on the subjective preference of surgeons and their objective task performance measures. Twenty-one surgical residents, fellows and attendings performed a peg-transfer task in the FLS and the VBLaST. Each subject performed 10 trials on each simulator. Results showed that subjects performed significantly better on the FLS than on the VBLaST. Subjects showed a significant learning effect on both simulators, but with an accelerated improvement on the VBLaST. Even so, 81% of the subjects preferred the FLS over the VBLaST for surgical training which could be attributed to the novelty of the VR technology and existing deficiencies of the haptic interface. Despite the subjective preference for the physical simulator, the performance results indicate an added value of VR and haptics in surgical training, which is expected to be demonstrated in more surgically relevant tasks such as suturing and knot-tying.

Staple line covering procedure after thoracoscopic bullectomy for the management of primary spontaneous pneumothorax.

BACKGROUND: Thoracoscopic bullectomy together with a pleural adhesive procedure is generally accepted as the standard for the definitive treatment of primary spontaneous pneumothorax (PSP). The purpose of this study was to evaluate whether the results of a thoracoscopic bullectomy followed by coverage of the staple line with cellulose mesh and fibrin glue could be comparable with those of adhesive procedures described in the literature. METHODS: Between May 2000 and February 2003, we performed 227 thoracoscopic surgeries on 219 patients with PSP using a single technique. After the bullectomy, the staple line was covered with cellulose mesh and fibrin glue. The postoperative status was evaluated with a mean follow-up of 46 months. RESULTS: The mean patient age was 24.3 years and 90.9 % of the 219 patients were male. Recurrent pneumothorax (37.4 %) was the most common operative indication, followed by persistent air leakage of more than 5 days (28.2 %). The mean duration of postoperative chest tube drainage was 1.6 days and the mean postoperative hospital stay was 3.8 days. Six patients experienced surgical complications (2.2 %); there was air leakage of more than 3 days in two cases, a small apical dead space in one case, a fever-associated wound problem in one case, and a reoperation due to air leakage of more than 7 days in two cases. Eleven patients (4.8 %) suffered a recurrence of pneumothorax during the follow-up period. Of these, nine cases required readmission and three (1.3 %) of these cases required a reoperation. CONCLUSIONS: Given the nature of a meticulous thoracoscopic bullectomy followed by coverage with cellulose mesh and fibrin glue, good surgical results can be expected without the need for a pleural adhesive procedure.

Gene expression profiles in squamous cell cervical carcinoma using array-based comparative genomic hybridization analysis.

Our aim was to identify novel genomic regions of interest and provide highly dynamic range information on correlation between squamous cell cervical carcinoma and its related gene expression patterns by a genome-wide array-based comparative genomic hybridization (array-CGH). We analyzed 15 cases of cervical cancer from KangNam St Mary's Hospital of the Catholic University of Korea. Microdissection assay was performed to obtain DNA samples from paraffin-embedded cervical tissues of cancer as well as of the adjacent normal tissues. The bacterial artificial chromosome (BAC) array used in this study consisted of 1440 human BACs and the space among the clones was 2.08 Mb. All the 15 cases of cervical cancer showed the differential changes of the cervical cancer-associated genetic alterations. The analysis limit of average gains and losses was 53%. A significant positive correlation was found in 8q24.3, 1p36.32, 3q27.1, 7p21.1, 11q13.1, and 3p14.2 changes through the cervical carcinogenesis. The regions of high level of gain were 1p36.33-1p36.32, 8q24.3, 16p13.3, 1p36.33, 3q27.1, and 7p21.1. And the regions of homozygous loss were 2q12.1, 22q11.21, 3p14.2, 6q24.3, 7p15.2, and 11q25. In the high level of gain regions, GSDMDC1, RECQL4, TP73, ABCF3, ALG3, HDAC9, ESRRA, and RPS6KA4 were significantly correlated with cervical cancer. The genes encoded by frequently lost clones were PTPRG, GRM7, ZDHHC3, EXOSC7, LRP1B, and NR3C2. Therefore, array-CGH analyses showed that specific genomic alterations were maintained in cervical cancer that were critical to the malignant phenotype and may give a chance to find out possible target genes present in the gained or lost clones.

Identification of differentially expressed genes using annealing control primer-based GeneFishing in human squamous cell cervical carcinoma.

AIMS: To compare different gene expression patterns between squamous cell cervical carcinoma (SCC) and normal cervical tissue in Korean women and to identify those genes that are specifically or predominantly expressed in SCC by employing annealing control primer (ACP)-based GeneFishing polymerase chain reaction (PCR). MATERIALS AND METHODS: Cervical cancer specimens were obtained from patients enrolled at the Department of Obstetrics and Gynecology, Kang Nam St. Mary's Hospital, Catholic University of Korea. We used a common reference that was mixed with an equal amount of RNA extracted from patients without cervical cancer. The profiles of expressed genes were compared between the SCC and normal cervix identified using GeneFishing differentially expressed gene kits, screened by a BLAST search, and confirmed by semi-quantitative reverse transcription-PCR (RT-PCR). RESULTS: Almost 100 differentially expressed genes were identified in the control and SCC samples. Using 60 arbitrary ACPs, 50 differentially expressed genes were identified, and 30 up-regulated and 20 down-regulated expressed genes were sequenced. Among 50 clones selected by ACP-based GeneFishing PCR, six genes with different expression patterns were determined and confirmed by semi-quantitative RT-PCR. The functional roles of two up-regulated genes, fibrillarin and calgranulin A, and one down-regulated gene, clusterin, were previously identified. However, the functional roles of two up-regulated genes and one down-regulated gene were not identified. CONCLUSION: We identified distinctive gene expression profiles in Korean women with SCC using ACP-based GeneFishing PCR.

Microscale Heat Transfer Transduced by Surface Plasmon Resonant Gold Nanoparticles.

Visible radiation at resonant frequencies is transduced to thermal energy by surface plasmons on gold nanoparticles. Temperature in

Distinctive cell cycle regulatory protein profiles by adenovirus delivery of p53 in human papillomavirus-associated cancer cells.

In this study, microarray analyses were performed to determine the time course of gene expression profiles in SiHa cells after infection with an adenovirus-expressing p53 (Adp53). We then investigated the consequences of Adp53 gene transfer on the expression level of six genes associated with cell cycle control and on apoptosis and cell cycle arrest in SiHa cells and compared these results with those from CaSki and HeLa cells. Gene expression profiling of the p53-targeted genes in SiHa cells revealed that p21, p53, and mdm2 protein expression was significantly upregulated at 24 and 48 h. Western blot results revealed that p21 and p53 expression levels had significantly increased after Adp53 infection. Cyclin-dependent kinase 4 levels were decreased 48 h after treatment in SiHa and CaSki cells. Proliferating cell nuclear antigen levels were unchanged after Adp53 infection. Only SiHa cells exhibited significant cell death. Cell cycle arrest at the G1 phase was induced in the SiHa and HeLa cells but was not induced at the G2/M and S phases in the CaSki cells. These data support the notion that the understanding of p53-dependent apoptosis and cell growth arrest could be applicable to advanced strategies in the development of preferential tumor cell-specific delivery.

Development of anticancer gene vaccine interact with human papillomavirus oncoprotein inhibition.

Adeno-associated virus (AAV) Rep 78 protein is known to inhibit the promoter site of several oncogenes and viral genes, including the human papillomavirus (HPV) type 16 E6 transforming genes. The biochemical studies of Rep 78 have been reported, but the effects of Rep 78 gene-mediated inhibition of HPV 16 E6 promoter activity on the various human cervical carcinoma cells have not been characterized. pEGFP-N1 vector, cloned by AAV-mediated Rep 78, is transfected into cervical carcinoma cells. Transfection efficiency of Rep 78 was approximately 30-60% different. Messenger RNA (mRNA) and protein expression of Rep 78 gene was significantly higher on day 1 of the transfection of Rep 78 DNA in CaSki cells, and DNA level of HPV 16 E6 was decreased on day 1 of the transfection. The growth of CaSki cervical cancer cells was only 10-15% inhibited by Rep 78, and the other cervical cells, HeLa, HeLaS3, HT3, and QGU, were unaffected by Rep 78 transfection. In spite of the high efficiency of Rep 78 gene transformation and expression rate, we could not show the significant growth inhibition in various cervical cancer cell lines. Taken together, long-term expression of Rep 78 strategy might be needed for cervical carcinoma gene therapy using AAV vector.

Diagnostic approach using the expression profiling of the P53 tumor suppressor gene and its related proteins in ovarian epithelial tumors.

The initial aim of this study was to examine the expression profiles of P53 and its upstream genes, downstream genes, and cell cycle regulators to determine whether these markers are useful for making a differential diagnosis among the benign, borderline, and malignant ovarian epithelial tumors. Between borderline and malignant tumors, the increased expression levels of P53, Bax, Cyclin E, and cyclin-dependent kinase-2 as well as the decreased expression levels of growth arrest and DNA damage (GADD45) and murine double minute-2 (MDM2) were significantly associated with malignancy (P < 0.01, each). Using the receiver operating curve (ROC), the most reliable cutoff value of the added-up staining scores of those markers was 4.5 with 79% sensitivity and 89% specificity for malignancy. Between benign and borderline tumors, the P21 and Bax expression levels were significantly higher in borderline tumors, whereas the Bcl-2 expression level was much higher in benign tumors (P < 0.01, each). Using the ROC, the cutoff value of the added-up staining scores used to discriminate between the two groups was 2.5 with 70% sensitivity and 74% specificity for borderline tumors. Thus, for the differential diagnosis between borderline and malignant tumors, the cutoff value 4.5 of the cumulative staining scores can be used. However, the cutoff value 2.5 for discrimination between benign and borderline tumors may not be useful because of its relatively low sensitivity and specificity. In addition, the P53, GADD45, Cyclin E, and MDM2 expression levels in malignant ovarian tumors might be useful for determining the histologic grade and type.

Cellular process classification of human papillomavirus-16-positive SiHa cervical carcinoma cell using Gene Ontology.

This study utilized mRNA differential display and the Gene Ontology (GO) analysis to characterize the multiple interactions of a number of genes involved in human papillomavirus (HPV)-16-induced cervical carcinogenesis. We used HPV-16-positive cervical cancer cell line (SiHa) and normal human keratinocyte cell line (HaCaT) as a control. Each gene has several biological functions in the GO, and hence, we chosen the several functions for each gene. and then, the specific functions were correlated with gene expression patterns. The results showed that 157 genes were up- or down-regulated above two-fold and organized into mutually dependent subfunction sets depending on the cervical cancer pathway, suggesting the potentially significant genes of unknown function. The GO analysis suggested that cervical cancer cells underwent repression of cancer-specific cell-adhesive properties. Also, genes belonging to DNA metabolism such as DNA repair and replication were strongly down-regulated, whereas significant increases were shown in protein degradation and in protein synthesis. The GO analysis can overcome the complexity of the gene expression profile of the HPV-16-associated pathway and identify several cancer-specific cellular processes as well as genes of unknown function. Also, it can become a major competing platform for the genome-wide characterization of carcinogenesis.

Natural killer cell activity and quality of life were improved by consumption of a mushroom extract, Agaricus blazei Murill Kyowa, in gynecological cancer patients undergoing chemotherapy.

A mushroom extract, Agaricus blazei Murill Kyowa (ABMK), has been reported to possess antimutagenic and antitumor effects. Here, we investigate the beneficial effects of ABMK consumption on immunological status and qualities of life in cancer patients undergoing chemotherapy. One hundred cervical, ovarian, and endometrial cancer patients were treated either with carboplatin (300 mg / m(2)) plus VP16 (etoposide, 100 mg / m(2)) or with carboplatin (300 mg / m(2)) plus taxol (175 mg / m(2)) every 3 weeks for at least three cycles with or without oral consumption of ABMK. We observed that natural killer cell activity was significantly higher in ABMK-treated group (ANOVA, n = 39, P < 0.002) as compared with nontreated placebo group (n = 61). However, no significant difference in lymphokine-activated killer and monocyte activities was observed in a manner similar to the count of specific immune cell populations between ABMK-treated and nontreated groups. However, chemotherapy-associated side effects such as appetite, alopecia, emotional stability, and general weakness were all improved by ABMK treatment. Taken together, this suggests that ABMK treatment might be beneficial for gynecological cancer patients undergoing chemotherapy.

Necrosis-like death with plasma membrane damage against cervical cancer cells by photodynamic therapy.

In order to elucidate the antitumor effect of photodynamic therapy (PDT) using the photosensitizing agent hematoporphyrin derivative (Photogem) and a diode laser, we evaluated the cell death of uterine cancer cell lines (CaSki, HT3, HeLa, and SKOV-3) and mice transplanted with TC-1 lung cancer cells. Morphological changes, MTT assay, flow cytometry, cytotoxicity, and tumor growth-inhibition study were evaluated at various time intervals after PDT. The results showed that the survival rates of each cell line decreased with time and dose-response after performing PDT. Also, PDT-induced damage of cancer cells was almost entirely confined to necrosis of the tumor cells in the early time courses. The irradiation of CaSki cells in the presence of Photogem induced plasma membrane disruption and cell shrinkage, indicating the plasma membrane as the main target for Photogem. In the experiment in vivo, the time courses of Photogem with irradiation showed significantly longer survival and a significantly smaller tumor size compared to those in the untreated control groups, and resorption of the tumor after PDT treatment was observed. Collectively, our results indicated that Photogem possesses tumor-specific affinity, and necrosis-like death with plasma membrane damage was postulated to be the principal mechanism of the antitumor effect of PDT using Photogem.

Anti-cancer effect of adenovirus p53 on human cervical cancer cell growth in vitro and in vivo.

To evaluate anti-tumor effects of recombinant adenovirus p53, time-course p53, E6 expression, and cell growth inhibition were investigated in vitro and in vivo using cervical cancer cell lines such as CaSki, SiHa, HeLa, HeLaS3, C33A, and HT3. The cell growth inhibition was studied via cell count assay, MTT assay and neutral red assay. After transfecting AdCMVp53 into SiHa cells-xenografted nude mice, the transduction efficiency and anti-tumor effect were investigated for a month. The results showed that adenoviral p53 expression induced significant growth suppression on the cancer cells, in which E6 transcript was strongly repressed, and that the expression of p53 and E6 were remarkably dependent on each cell type. The transduction efficiency was highly maintained in vivo as well as in vitro, and the size of tumor was remarkably decreased in comparison with AdCMVLacZ control. The results suggest that the adenovirus-mediated p53 gene transfection was done very effectively in vitro and in vivo experiment, and the cell growth was suppressed via p53-dependent apoptotic cell death, and that the anti-tumor effect could be related to E6 and p53 expression pattern.

Expression of cyclooxygenase 2, microsomal prostaglandin E synthase 1, and EP receptors is increased in rat oesophageal squamous cell dysplasia and Barrett's metaplasia induced by duodenal contents reflux.

BACKGROUND AND AIM: It is known that bile acids can induce mucosal injury, stimulate cell proliferation, and promote tumorigenesis. A large body of genetic and biochemical evidence indicate that the biosynthetic pathway of prostaglandin E2 (PGE2) may play an important role in human and rodent tumours. Therefore, we examined the expression pattern of cyclooxygenase 1 (COX-1), COX-2, and microsomal prostaglandin E synthase 1 (mPGES-1), as well as EP receptor subtypes in rat oesophageal lesions induced by duodenal contents reflux. METHODS: Oesophagoduodenal anastomosis was performed in rats to induce duodenal contents reflux. We examined histological changes and expression of COX-1, COX-2, mPGES-1, and EP receptor subtypes in the oesophagus by immunohistochemistry and reverse transcription-polymerase chain reaction. RESULTS: Normal control oesophageal tissues showed COX-1 expression in subepithelial stromal cells, including endothelial cells and muscular cells, and did not reveal expression of COX-2 or mPGES-1. In the case of squamous cell lesions, immunoreactivity of COX-1 was similar to that of normal lesions, and COX-2 was maximally expressed around the vascular papillae of tissues showing dysplasia and surrounding epithelial layer and basal layer. mPGES-1 was highly expressed in stromal cells with COX-2 expression. In the case of Barrett's oesophagus, COX-2 and mPGES-1 were predominantly in subepithelial stromal cells. mRNA levels of COX-2, mPGES-1, EP2, EP3, and EP4 were higher in the experimental groups than in controls. CONCLUSIONS: We suggest that the biosynthetic pathway of PGE2 may play an important role in oesophageal squamous cell dysplasia and glandular metaplasia induced by duodenal contents reflux.