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.

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).

Na(+)-dependent transporters mediate HCO(3)(-) salvage across the luminal membrane of the main pancreatic duct.

To study the roles of Na(+)-dependent H(+) transporters, we characterized H(+) efflux mechanisms in the pancreatic duct in wild-type, NHE2(-/-), and NHE3(-/-) mice. The pancreatic duct expresses NHE1 in the basolateral membrane, and NHE2 and NHE3 in the luminal membrane, but does not contain NHE4 or NHE5. Basolateral Na(+)-dependent H(+) efflux in the microperfused duct was inhibited by 1.5 microM of the amiloride analogue HOE 694, consistent with expression of NHE1, whereas the luminal activity required 50 microM HOE 694 for effective inhibition, suggesting that the efflux might be mediated by NHE2. However, disruption of NHE2 had no effect on luminal transport, while disruption of the NHE3 gene reduced luminal Na(+)-dependent H(+) efflux by approximately 45%. Notably, the remaining luminal Na(+)-dependent H(+) efflux in ducts from NHE3(-/-) mice was inhibited by 50 microM HOE 694. Hence, approximately 55% of luminal H(+) efflux (or HCO(3)(-) influx) in the pancreatic duct is mediated by a novel, HOE 694-sensitive, Na(+)-dependent mechanism. H(+) transport by NHE3 and the novel transporter is inhibited by cAMP, albeit to different extents. We propose that multiple Na(+)-dependent mechanisms in the luminal membrane of the pancreatic duct absorb Na(+) and HCO(3)(-) to produce a pancreatic juice that is poor in HCO(3)(-) and rich in Cl(-) during basal secretion. Inhibition of the transporters during stimulated secretion aids in producing the HCO(3)(-)-rich pancreatic juice.

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.

Why are different features central for natural kinds and artifacts?: the role of causal status in determining feature centrality.

Ahn and Lassaline [Ahn, W., Lassaline, M.E., 1995. Causal structure in categorization. Proceedings of the Seventeenth Annual Conference of the Cognitive Science Society, Pittsburgh, PA, pp. 521-526] recently proposed a causal status hypothesis which states that features that play a causal role in a relational structure are more central than their effects. This hypothesis can account for previous research demonstrating that compositional features are generally important for natural kinds but functional features are generally important for artifacts. The causal status hypothesis explains this category-feature interaction effect in terms of differences in the causal status of compositional and functional features between natural kinds and artifacts. Experiments 1 and 2 examined real-life categories used in previous studies, and found positive correlations between the causal status of the features and their centrality across natural and artifactual kinds. Experiments 3 and 4 manipulated the causal status of compositional and functional features in artificial categories, and showed that it was causal status rather than the interaction between the type of feature and the type of category per se that accounted for feature centrality. The implications of these results on the distinctions between natural kinds and artifacts are discussed.

Causal status effect in children's categorization.

The current study examined the causal status effect (weighing cause features more than effect features in categorization) in children. Adults (Study 1) and 7-9-year-old children (Study 2) learned descriptions of novel animals, in which one feature caused two other features. When asked to determine which transfer item was more likely to be an example of the animal they had learned, both adults and children preferred an animal with a cause feature and an effect feature rather than an animal with two effect features. This study is the first direct demonstration of the causal status effect in children.

The role of covariation versus mechanism information in causal attribution.

Traditional approaches to causal attribution propose that information about covariation of factors is used to identify causes of events. In contrast, we present a series of studies showing that people seek out and prefer information about causal mechanisms rather than information about covariation. Experiments 1, 2 and 3 asked subjects to indicate the kind of information they would need for causal attribution. The subjects tended to seek out information that would provide evidence for or against hypotheses about underlying mechanisms. When asked to provide causes, the subjects' descriptions were also based on causal mechanisms. In Experiment 4, subjects received pieces of conflicting evidence matching in covariation values but differing in whether the evidence included some statement of a mechanism. The influence of evidence was significantly stronger when it included mechanism information. We conclude that people do not treat the task of causal attribution as one of identifying a novel causal relationship between arbitrary factors by relying solely on covariation information. Rather, people attempt to seek out causal mechanisms in developing a causal explanation for a specific event.

A novel effect of rebamipide: generation of [Ca(2+)](i) oscillations through activation of CCK(1) receptors in rat pancreatic acinar cells.

The protective effect of 2-(4-chlorobenzoylamino)-3-[2(1H)-quinolinon-4-yl]-propionic acid (rebamipide) on gastric mucosa is well established. Here we demonstrate that rebamipide acts on pancreatic acinar cells to generate oscillations of intracellular Ca(2+) concentration ([Ca(2+)](i)) through the activation of cholecystokinin subtype 1 (CCK(1)) receptors. At concentrations higher than 5 microM, rebamipide induced [Ca(2+)](i) oscillations in individual fura-2-loaded pancreatic acinar cells. The frequency of oscillations increased with increasing concentrations of rebamipide, while the latency between stimulation of cells and initiation of [Ca(2+)](i) oscillations decreased with increasing concentration. The [Ca(2+)](i) oscillations evoked by rebamipide were inhibited by the CCK(1) receptor antagonist L-364,718 but not by atropine or the CCK(2) receptor antagonist L-365,260 indicating that rebamipide is a nonpeptide CCK(1) receptor agonist.

Protective effects of green tea extracts (polyphenon E and EGCG) on human cervical lesions.

We investigated clinical efficacy of green tea extracts (polyphenon E; poly E and (-)-epigallocatechin-3-gallate [EGCG]) delivered in a form of ointment or capsule in patients with human papilloma virus (HPV) infected cervical lesions. Fifty-one patients with cervical lesions (chronic cervicitis, mild dysplasia, moderate dysplasia and severe dysplasia) were divided into four groups, as compared with 39 untreated patients as a control. Poly E ointment was applied locally to 27 patients twice a week. For oral delivery, a 200 mg of poly E or EGCG capsule was taken orally every day for eight to 12 weeks. In the study, 20 out of 27 patients (74%) under poly E ointment therapy showed a response. Six out of eight patients under poly E ointment plus poly E capsule therapy (75%) showed a response, and three out of six patients (50%) under poly E capsule therapy showed a response. Six out of 10 patients (60%) under EGCG capsule therapy showed a response. Overall, a 69% response rate (35/51) was noted for treatment with green tea extracts, as compared with a 10% response rate (4/39) in untreated controls (P<0.05). Thus, the data collected here demonstrated that green tea extracts in a form of ointment and capsule are effective for treating cervical lesions, suggesting that green tea extracts can be a potential therapy regimen for patients with HPV infected cervical lesions.

Sterically stabilized anti-G(M3), anti-Le(x) immunoliposomes: targeting to B16BL6, HRT-18 cancer cells.

Various tumor-associated antigens have been identified as carbohydrates bound to lipids or to proteins expressed on tumor cell membranes. We prepared tumor-specific immunoliposomes by coupling anticarbohydrate antibodies, such as antiganglioside G(M3) antibody (DH2) or anti-Le(x) antibody (SH1), to polyethylene glycol (PEG)-coated liposomes. In vitro and in vivo targetability of anti-G(M3) and anti-Le(x) immunoliposomes to B16BL6 mouse melanoma cells and HRT-18 human colorectal adenocarcinoma cells were monitored with a fluorescence microscopy, and analyzed by biodistribution assay of the immunoliposome in mice bearing the tumor tissues. The antibody coupling to the PEG liposomes did not greatly diminish the circulation time of the liposome in the C57BL/6 mouse model. In vitro cytotoxicity of doxorubicin encapsulated in liposomes was enhanced by antibody coupling, but still behind free doxorubicin. However, in vivo antitumor therapeutic efficacy of doxorubicin encapsulated in the immunoliposomes was far greater than the free drug or in conventional liposomes. Doxorubicin encapsulated in anti-G(M3) immunoliposomes was able to reduce in vivo tumor growth and metastasis of B16BL6 mouse melanoma cells more greatly than any other formulations of the drug. This study suggests that tumor-associated antigens can be good target molecules for tumor-specific delivery of liposomal drugs or other synthetic drug delivery systems.

Pharmacodynamics of insulin in polyethylene glycol-coated liposomes.

To reduce the injection frequency and toxicity of intravenously administered protein drugs, it is necessary to develop safe and sustained injectable delivery systems. In this study, to evaluate liposomes as safe and sustained injectable delivery systems of proteins, we chose insulin as a model protein drug and tested its incorporation efficiency and pharmacodynamics in various liposomes with and without polyethylene glycol (PEG)-derivatized phospholipid. The liposomes coated with PEG showed 3-fold higher efficiency of insulin incorporation than did the liposomes without PEG. Moreover, among the liposomes coated with PEG, dipalmitoylphosphocholine (DPPC) liposomes showed higher incorporation efficiency than did dimyristoylphosphocholine (DMPC) liposomes. For pharmacodynamic study, insulin (2 IU/kg) was administered in various formulations, such as insulin alone in phosphate-buffered saline and insulin in the DPPC liposomes with and without PEG, to streptozotocin-treated diabetic rats. The pharmacodynamics of insulin alone, however, could not be measured due to the immediate death of rats caused by hypoglycemic shock. In contrast, all the rats treated with liposomal insulin survived, probably by the sustained release of insulin from liposomes. Pharmacodynamics of liposomal insulin showed that PEG-coated liposomes induced the lowest level of blood glucose-the nadir-1 h later than did the liposomes without PEG. These results indicate that PEG-coated liposomes could be developed as a relatively safe and sustained injectable delivery system for insulin with improved incorporation efficiency. Moreover, it is suggested that the liposomes coated with PEG might have a potential as safe injectable delivery systems for other protein and peptide drugs.

Comparison of the filtration characteristics between attached and suspended growth microorganisms in submerged membrane bioreactor.

An attached growth bioreactor was designed to minimize the effect of suspended microorganisms on membrane fouling in submerged membrane bioreactor. Comparison of mixed liquor from attached and suspended growth systems was made to elucidate major factors giving rise to different filtration characteristics. Unexpectedly, the rate of membrane fouling of the attached growth system was about 7 times higher than that of the suspended growth system despite similar characteristics of soluble fraction from the two reactors. Filtration performance proved to depend on the concentration of mixed liquor suspended solids (MLSS). Better filtration performance with suspended growth was explained by the formation of dynamic membranes with suspended solids. A series of analyses such as hydraulic resistance, specific cake resistance, scanning electron microscope, and atomic force microscope were carried out to elucidate the different filtration characteristics of the two systems.

Coordination of pancreatic HCO3- secretion by protein-protein interaction between membrane transporters.

Increasing evidence suggests that protein-protein interaction is essential in many biological processes including epithelial transport. In this report, we discuss the significance of protein interactions to HCO(3)(-) secretion in pancreatic duct cells. In pancreatic ducts HCO(3)(-) secretion is mediated by cystic fibrosis transmembrane conductance regulator (CFTR) activated luminal Cl(-)/HCO(3)(-) exchange activity and HCO(3)(-) absorption is achieved by Na(+)-dependent mechanisms including Na(+)/H(+) exchanger 3 (NHE3). We found biochemical and functional association between CFTR and NHE3. In addition, protein binding through PDZ modules is needed for this regulatory interaction. CFTR affected NHE3 activities in two ways. Acutely, CFTR augmented the cAMP-dependent inhibition of NHE3. In a chronic mechanism, CFTR increases the luminal expression of Na(+)/H(+) exchange in pancreatic duct cells. These findings reveal that protein complexes in the plasma membrane of pancreatic duct cells are highly organized for efficient HCO(3)(-) secretion.

An easy technique for long-term central venous catheterization and subcutaneous tunneling of the silastic catheter in neonates and infants.

Venous access is a common problem in the care of seriously ill infants and neonates on pediatric and pediatric surgical services, and recently there has been a high increase in indications for central venous lines. With the development of many types of silastic catheters, a new safe and fast method of catheter insertion, subcutaneous tunneling and fixation has been developed. Our method is based on Yoffa's (1965)1 description. In the past we used infraclavicular subclavian venipuncture,2 but now we prefer to use the supraclavicular subclavian venipuncture as the first choice in children. Thirty-one catheters were placed in 27 consecutive patients ranging in age from two days to ten years. Perioperative complications were nil, and those catheterized for a long period of time had relatively few complications.

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.