User-friendly cell patterning methods using a polydimethylsiloxane mold with microchannels.

Techniques for partitioning cell adhesion are useful tools in biological and medical experiments. However, conventional cell patterning methods require special apparatus, special materials or high-level skills. Therefore, we have developed a new cell patterning methodology which can be easily carried out in biological laboratories. Non-cell adhesive material including hydrogel or gas patterns to restrict cell adhesion on a culture dish or glass substrates can be constructed by exploiting a polydimethylsiloxane (PDMS) mold with microchannels. The PDMS molds suck non-adhesive materials into microchannels from the inlet of the microchannels and the materials are immobilized onto the substrates with a desired pattern. High resolution under a few micrometers and long-term stability can be realized. This method has been used for analysis of stem cells, muscle cells, neuron development and other cells in collaboration with many biological researchers. Several examples to use this technique are introduced in this review.

Thermoresponsive nanostructured surfaces generated by the Langmuir-Schaefer method are suitable for cell sheet fabrication.

Thermoresponsive poly(N-isopropylacrylamide) (PIPAAm)-immobilized surfaces for controlling cell adhesion and detachment were fabricated by the Langmuir-Schaefer method. Block copolymers composed of polystyrene and PIPAAm (St-IPAAms) having various chain lengths and compositions were synthesized by reversible addition-fragmentation chain transfer radical polymerization. The St-IPAAm Langmuir film at an air-water interface was horizontally transferred onto a hydrophobically modified glass substrate while regulating its density. Atomic force microscopy images clearly visualized nanoscaled sea-island structures on the surface. By adjusting both the composition of St-IPAAms and the density of immobilized PIPAAms, a series of thermoresponsive surfaces was prepared to control the strength, rate, and quality of cell adhesion and detachment through changes in temperature across the lower critical solution temperature range of PIPAAm molecules. In addition, a two-dimensional cell structure (cell sheet) was more rapidly recovered on the optimized surfaces than on conventional PIPAAm surfaces. These unique PIPAAm surfaces are suggested to be useful for controlling the strength of cell adhesion and detachment.

Cross-cultural comparison of nudging effects for environmental protection: A case-study of risk-averse attitudes toward disposable plastics.

Disposable plastics are drawing considerable attention as a source of environmental risk despite their benefits in daily life. Banning the use of disposable plastics could increase other types of risks, which may damage the public good in the long run. Considering the trade-off of the risks and benefits, one way to improve social welfare is to conduct proper recycling and to continue using plastics but limit them to essential use, avoiding an unnecessary ban. A potential barrier to such a policy might be risk-averse attitudes toward actions that are perceived to threaten future generations, which is a well-known phenomenon. We previously designed a framework for information provision using messages that remind individuals about familial support, which had significant effects in multiple countries on increasing positive attitudes toward air pollution caused by industrialization. We hypothesized that this information provision could also be effective for disposable plastic use. Thus, we conducted a randomized controlled trial via online surveys in Japan, Canada, and the US to identify the effects of our designed messages about recycling on increasing positive attitudes toward disposable plastics. The intervention effects were measured by the difference-in-difference method and panel analysis based on linear regression models using the respondents' attributes and personality traits. The effects were consistently correlated with a sense of familial support, with the effect sizes varying according to country (US > Japan > Canada). Attributes that positively contributed to the message being more effective were higher agreeableness, lower Machiavellianism, lower psychopathy, and being a woman. Although personal fear about COVID-19 moderated the message effects, concern about the threats to relatives and family boosted the effects. Although the effect sizes were influenced by external factors, the results suggested that our proposed framework for information provision has the potential to be applied to a wider variety of risk-related topics.

Mesh-based pneumostasis contributes to preserving gas exchange capacity and promoting rehabilitation after lung resection.

BACKGROUND: We recently introduced a technique of sutureless, mesh-based pneumostasis for preventing alveolar air leaks after lung resection. To verify the clinical usefulness of this technique, we examined if it can contribute to preserving gas exchange capacity and promoting postoperative rehabilitation. METHODS: We prospectively collected perioperative data, including arterial oxygen saturation on postoperative day (POD) 1 and the length of postoperative rehabilitation in 100 patients undergoing elective, video-assisted major lung resection for cancer. Before April, 2006, intraoperative air leaks were sealed with the conventional method (control group), and thereafter, with bioabsorbable mesh and glue, without suturing, (treated group). To reduce the bias in comparison of the nonrandomized control group, we paired the treated group with the control group using the nearest available matching method on the estimated propensity score. RESULTS: Thirty-five patients in the control group were matched to 35 patients in the treated group based on the estimated propensity score. The length of both chest tube drainage and postoperative rehabilitation were significantly shorter in the treated group than in the control group (median, 1 versus 1 d, P = 0.03; 2 versus 3 d, P = 0.01, respectively). The arterial oxygen saturation on POD 1 was significantly higher in the treated group than in the control group (median, 94.0 versus 92.5 %, P = 0.03). CONCLUSION: Mesh-based pneumostasis during video-assisted major lung resection enabled early chest tube removal, preserved postoperative oxygenation capacity, and promoted postoperative rehabilitation, which may facilitate fast-track surgery for patients undergoing video-assisted major lung resection for cancer.

Salivary secretion and occlusal force in patients with unilateral cerebral stroke.

The purpose of this study was to investigate salivary secretion in unilateral cerebral stroke patients, including the effects of masticatory forces. We compared the volumes of unstimulated and acid-stimulated saliva between 30 patients with unilateral cerebral stroke and 30 age-matched healthy subjects. The volume of whole-mouth salivary secretion was measured by the modified cotton swab method. Occlusal forces were measured with an occlusal force meter in patients/subjects, in groups with normal teeth and dentures, respectively. The volume of unstimulated saliva in stroke patients was significantly lower than that in controls. For subjects with normal teeth, the volume of acid-stimulated saliva and the occlusal force on the hemiplegic side in stroke patients were significantly lower than those in control subjects. For those with dentures, while the volume of unstimulated saliva in stroke patients was significantly lower than that in controls, there were no significant differences in occlusal forces between the two groups. These results suggest that salivary secretion might be reduced in cerebral stroke patients, which might be caused partly by an inability to generate sufficient masticatory force, and which, in turn, might lead to a reduced masticatory-parotid reflex and consequent disuse atrophy of the salivary glands.

Flow analysis on microcasting with degassed polydimethylsiloxane micro-channels for cell patterning with cross-linked albumin.

Patterned cell culturing is one of the most useful techniques for understanding the interaction between geometric conditions surrounding cells and their behaviors. The authors previously proposed a simple method for cell patterning with an agarose gel microstructure fabricated by microcasting with a degassed polydimethylsiloxane (PDMS) mold. Although the vacuum pressure produced from the degassed PDMS can drive a highly viscous agarose solution, the influence of solution viscosity on the casting process is unknown. This study investigated the influences of micro-channel dimensions or solution viscosity on the flow of the solution in a micro-channel of a PDMS mold by both experiments and numerical simulation. It was found experimentally that the degassed PDMS mold was able to drive a solution with a viscosity under 575 mPa·s. A simulation model was developed which can well estimate the flow rate in various dimensions of micro-channels. Cross-linked albumin has low viscosity (1 mPa·s) in aqueous solution and can undergo a one-way dehydration process from solution to solid that produces cellular repellency after dehydration. A microstructure of cross-linked albumin was fabricated on a cell culture dish by the microcasting method. After cells were seeded and cultivated on the cell culture dish with the microstructure for 7 days, the cellular pattern of mouse skeletal myoblast cell line C2C12 was observed. The microcasting with cross-linked albumin solution enables preparation of patterned cell culture systems more quickly in comparison with the previous agarose gel casting, which requires a gelation process before the dehydration process.

Vacuum microcasting of 2-methacryloyloxyethyl phosphorylcholine polymer for stable cell patterning.

This study demonstrates the rapid fabrication and utility of 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer film for cell patterning. The film was obtained on a cell culture surface by microcasting MPC polymer ethanol solution into a degassed polydimethylsiloxane mold with a desired pattern. After removal of the mold, 293AD cells were cultured on the surface of the polymer film with the patterned microstructures. Patterned cell adhesion restricted by the film was successfully maintained during at least a 168-h cultivation. The microcast MPC polymer film can be prepared rapidly and used for efficient long-term cell confinement.

Analysis of Long-term Morphological Changes of Micro-patterned Molecules and Cells on PDMS and Glass Surfaces.

We demonstrated that our previously developed gas-phase fluoroalkylsilane patterning method was applicable to polydimethylsiloxane (PDMS) and we compared the stability of patterned proteins and cultured cells between PDMS and glass surfaces. The shapes of the protein patterns were stable on both glass and PDMS surfaces for more than 1 week. The cell patterns were stable on glass surfaces for 1 week, while those on PDMS collapsed within a few days. These results indicated that our method was applicable to PDMS, although, compared with glass, PDMS has an unsolved issue for its application to long-term patterning of cells.

Enhanced methanogenic degradation of cellulose-containing sewage via fungi-methanogens syntrophic association in an anaerobic membrane bioreactor.

An anaerobic membrane bioreactor was configured for methanogenic degradation of cellulose-containing sewage. The degradation performance and microbial changes were evaluated under five hydraulic retention times (HRTs). The results indicated the methane production was largely enhanced with 92.6% efficiency of chemical oxygen demand (COD) converting to methane and 80% proportion of methane in produced biogas, meanwhile the biomass yield presented the fewest at the shortest HRT 8h. Enhanced methane production with decreased biomass yield was attributed to an association between fungi and methanogens. Microbial analysis showed fungi Basidiomycota and methanogen Methanoregula apparently established the association, especially Basidiomycota reaching 93% relative abundance at HRT 8h. Specific methanogenic activity (SMA) and biochemical methane potential (BMP) tests suggested the association was derived from H2 production by fungi and H2 consumption by methanogens, during the process of cellulose degradation. The methanogenic degradation of cellulose-containing sewage was markedly promoted via the fungi-methanogens syntrophic association.

Development of novel sustained-release system, disintegration-controlled matrix tablet (DCMT) with solid dispersion granules of nilvadipine (II): in vivo evaluation.

A novel sustained-release (SR) system, disintegration-controlled matrix tablet (DCMT), was developed for poorly water-soluble drugs. DCMT, consisting of wax and solid dispersion (SD) granules containing a disintegrant, could control the release of nilvadipine (NiD), a model compound, by its disintegration. In the present study, two DCMTs (DCMT-1 and DCMT-2) with different release rates of NiD were orally administered to beagle dogs, and in vivo absorption of NiD from DCMTs was compared with that from immediate-release (IR) tablets. DCMTs successfully sustained the absorption of NiD longer than IR tablets, while they did not decrease the bioavailability of NiD. DCMT-2, providing the slower release of NiD than DCMT-1, prolonged the absorption longer than DCMT-1. In vivo absorption profiles of NiD from DCMTs were significantly correlated with in vitro release profiles, suggesting that the release property from DCMTs would maintain regardless of the change in physiological condition through the gastrointestinal tract. Furthermore, the food intake did not affect the absorption of NiD after oral administration of DCMT-2. The present results strongly indicate that the DCMT system would be a promising SR system, which could improve the solubility and sustain the absorption of poorly water-soluble drugs.

Response of microalgae to elevated CO2 and temperature: impact of climate change on freshwater ecosystems.

To estimate the combined effects of elevated CO2 and temperature on microalgae, three typical and worldwide freshwater species, the green alga Scenedesmus acuminatus, the diatom Cyclotella meneghiniana, and the cyanobacterium Microcystis aeruginosa, as well as mixes of these three species were continuously cultured in controlled environment chambers with CO2 at 390 and 1000 ppm and temperatures of 20, 25, and 30 °C. CO2 and temperature significantly affected the production of microalgae. The cell productivity increased under elevated CO2 and temperature. Although the green alga dominated in the mixed culture within all CO2 and temperature conditions, rising temperature and CO2 intensified the competition of the cyanobacterium with other microalgae. CO2 affected the extracellular polymeric substances (EPS) characteristics of the green alga and the cyanobacterium. Elevated CO2 induced the generation of humic substances in the EPS fractions of the green alga, the cyanobacterium, and the mixed culture. The extracellular carbohydrates of the diatom and the extracellular proteins of the cyanobacterium increased with elevated CO2 and temperature, while the extracellular carbohydrates and proteins of the green alga and the mixes increased under elevated CO2 and temperature. There were synergistic effects of CO2 and temperature on the productivity and the EPS of microalgae. Climate change related CO2 and temperature increases will promote autochthonous organic carbon production in aquatic ecosystems and facilitate the proliferation of cyanobacteria, which potentially changes the carbon cycling and undermines the functioning of ecosystems.

Preparation of Thermoresponsive Nanostructured Surfaces for Tissue Engineering.

Thermoresponsive poly(N-isopropylacrylamide) (PIPAAm)-immobilized surfaces for controlling cell adhesion and detachment were fabricated by the Langmuir-Schaefer method. Amphiphilic block copolymers composed of polystyrene and PIPAAm (St-IPAAms) were synthesized by reversible addition-fragmentation chain transfer (RAFT) radical polymerization. A chloroform solution of St-IPAAm molecules was gently dropped into a Langmuir-trough apparatus, and both barriers of the apparatus were moved horizontally to compress the film to regulate its density. Then, the St-IPAAm Langmuir film was horizontally transferred onto a hydrophobically modified glass substrate by a surface-fixed device. Atomic force microscopy images clearly revealed nanoscale sea-island structures on the surface. The strength, rate, and quality of cell adhesion and detachment on the prepared surface were modulated by changes in temperature across the lower critical solution temperature range of PIPAAm molecules. In addition, a two-dimensional cell structure (cell sheet) was successfully recovered on the optimized surfaces. These unique PIPAAm surfaces may be useful for controlling the strength of cell adhesion and detachment.

Aberrant methylation of multiple genes and clinicopathological features in oral squamous cell carcinoma.

The purpose of this study was to examine the methylation profile of various oral squamous cell carcinomas and to correlate the methylation of particular chromosomal loci with the clinicopathological features of the tumors. A semiquantitative analysis of the methylation status of 12 loci in 96 primary tumors and 13 cell lines was carried out. Methylation frequency was calculated as the percentage of methylated alleles detected by bisulfate-PCR. Of the 12 loci examined, 9 (p16INK4A, p15INK4B, p14ARF, DCC, DAP kinase, MINT1, MINT2, MINT27, and MINT31) exhibited aberrant methylation at various frequencies, whereas 3 (hMLH1, HRK, and CACNA1G) showed no methylation. Dense methylation of the 5' CpG island of DAP kinase and MINT1 was well correlated with loss of gene expression. In addition, methylation of DCC was correlated with bone invasion by gingival tumors (P = 0.036), with aggressive invasiveness of tumors of the tongue (P = 0.046), and with reduced survival (P = 0.050). Methylation of MINT1 and MINT31 also correlated with poor prognoses (P = 0.058 and 0.041), whereas methylation of p14ARF correlated with a good prognosis (P = 0.021). Cox regression analysis showed methylation of MINT31 to be an independent predictor of outcome (hazard ratio, 3.79; 95% confidence interval, 1.58-9.10) and to be associated with the T4 disease group (hazard ratio, 5.71; 95% confidence interval, 1.25-26.07). Analysis of DNA methylation is a useful approach to evaluation of the biological characteristics of oral cancers and may be a useful diagnostic indicator of patient prognosis.

ESR study of camphorquinone/amine photoinitiator systems using blue light-emitting diodes.

New light-activation units equipped with high-illuminant blue light-emitting diodes (LEDs) have recently been proposed as a replacement for the halogen units that are widely used in dentistry to polymerize light-cured resins. The photoinitiators in light-cured dental resins, typified by the camphorquinone (CQ)/amine photoinitiator system, generate primary radicals with light irradiation that attack the double bonds of resin monomers. The physical properties of the cured resins are affected by the generation of primary radicals during the initial stage of polymerization. This study examined two types of photoinitiator systems, CQ/DMPT and CQ/DMAEMA, and three types of curing units, a new LED unit and two conventional halogen units. The primary radicals generated by irradiation were quantified using electron spin resonance (ESR) spectroscopy with a trapping method, using phenyl-tert-butyl nitrone as the trapping agent. The energy efficiencies of the LED and halogen units were compared by quantifying the generated radicals and emitted light energy (J/cm(2)). The energy required to generate a given amount of radicals using the LED unit was smaller than that using the halogen units (p<0.05). These results suggest that the new LED unit performs better than conventional halogen units with respect to light energy.

Usefulness of bioabsorbable thread pins after resection arthroplasty for rheumatoid forefoot reconstruction.

BACKGROUND: Bioabsorbable thread pin has been used for internal fixation of bone. The results of resection arthroplasty of the lesser metatarsophalangeal (MTP) joints using internal intramedullary fixation with bioabsorbable pins have not been reported. METHODS: Resection arthroplasty of the MTP joints of the lesser toes with poly-L-lactic acid (PLLA) thread pins or Kirschner wires was performed at random in reconstruction of the 87 rheumatoid forefeet (62 patients) with a grommet-protected silicone-rubber implant insertion of the first MTP joint. Clinical symptoms, the state of radiographic changes, and complications were assessed 5-10 years (average, 7.7 years) postoperatively. RESULTS: The mean American Orthopaedic Foot and Ankle Society clinical scores at the preoperative and latest points were 31 and 91, respectively, in the operated patients with PLLA pins, while the mean scores were 32 and 82, respectively, in the operated patients with Kirschner wires. The lesser toes treated by bioabsorbable pins did not become rigid, although they were stable. Recurrent dorsal subluxation of the lesser MTP joints was visible on radiographs in three of the 46 feet with PLLA pins, while two feet had three dislocated MTP joints and one subluxated MTP joint postoperatively and recurrent dorsal subluxation of the lesser MTP joints was visible in four of the 41 feet with Kirschner wires. The postoperative hallux valgus did not progress to the preoperative level during the follow-up period in both groups. Two of the 46 feet with PLLA pins and two of the 41 feet with Kirschner wires had radiographic evidence of silicone synovitis without pathological fracture. Three patients with Kirschner wires had wire-track infection, and one patient had severe circulation disturbance of the corrected lesser toes necessitating wire removal. CONCLUSIONS: A new trial of internal fixation with bioabsorbable pins may lead to the establishment of a safe method for enhancing stability of the lesser toes after resection arthroplasty of the lesser MTP joints.

Micro-patterned cell-sheets fabricated with stamping-force-controlled micro-contact printing.

Cell-sheet-engineering based regenerative medicine is successfully applied to clinical studies, though cell sheets contain uniformly distributed cells. For the further application to complex tissues/organs, cell sheets with a multi-cellular pattern were highly demanded. Micro-contact printing is a quite useful technique for patterning proteins contained in extracellular matrix (ECM). Because ECM is a kind of cellular adherent molecules, ECM-patterned cell culture surface is capable of aligning cells on the pattern of ECM. However, a manual printing is difficult, because a stamp made from polydimethylsiloxane (PDMS) is easily deformed, and a printed pattern is also crushed. This study focused on the deformability of PDMS stamp and discussed an appropriate stamping force in micro-contact printing. Considering in availability in a medical or biological laboratory, a method for assessing the stamp deformability was developed by using stiffness measurement with a general microscope. An automated stamping system composed of a load cell and an automated actuator was prepared and allowed to improve the quality of stamped pattern by controlling an appropriate stamping force of 0.1 N. Using the system and the control of appropriate stamping force, the pattern of 8-mm-diameter 80-µm-stripe fibronectin was fabricated on the surface of temperature-responsive cell culture dish. After cell-seeding and cell culture, a co-culture system with the micro-pattern of both fibroblasts and endothelial cells was completed. Furthermore, by reducing temperature to 20 °C, the co-cultured cell sheet with the micro-pattern was successfully harvested. As a result, the method would not only provide a high-quality ECM pattern but also a breakthrough technique to fabricate multi-cellular-patterned cell sheets for the next generation of regenerative medicine and tissue engineering.

Control of cell adhesion and detachment on Langmuir-Schaefer surface composed of dodecyl-terminated thermo-responsive polymers.

This study used Langmuir-Schaefer (LS) method to produce thermo-responsive poly(N-isopropylacrylamide) (PIPAAm) modified surface. Dodecyl terminated-PIPAAm (PIPAAm-C12) was synthesized by reversible addition-fragmentation chain transfer radical polymerization. PIPAAm-C12 was dropped on an air-water interface and formed Langmuir film by compressing. A surface pressure measurement revealed that PIPAAm-C12 was floated and Langmuir films were formed on the interface. And the Langmuir film was transferred on a hydrophobic substrate to produce PIPAAm-C12 transferred surface (PIPAAm-LS surface). In the results of atomic force microscope, attenuated total reflection Fourier transform infrared spectroscope, and X-ray photoelectron spectroscope measurement, the transference of Langmuir films was demonstrated and densities could be precisely controlled. Ellipsometric measurements of PIPAAm-LS surfaces showed that the thicknesses of the surfaces were less than 10 nm. Cell adhesion and detachment were observed on the PIPAAm-LS surfaces. The amount of adhered cells on all LS surfaces was found to be similar on the control hydrophobic substrate at 37 °C. In regard to cell detachment, adhering cells rapidly detached themselves with higher densities and shorter PIPAAm-C12 molecules. In this method, the effect of densities and molecular weights on cell adhesion and detachment were observed. Our method should be proved novel insights for investigating cell adhesion and detachment on thermo-responsive surfaces.

Cell sheet technology for cardiac tissue engineering.

In this chapter, we describe the methods for the fabrication and transfer/transplantation of 3D tissues by using cell sheet technology for cardiac tissue regeneration. A temperature-responsive culture surface can be fabricated by grafting a temperature-responsive polymer, poly(N-isopropylacrylamide), onto a polystyrene cell culture surface. Cells cultured confluently on such a culture surface can be recovered as an intact cell sheet, and functional three-dimensional (3D) tissues can then be easily fabricated by layering the recovered cell sheets without any scaffolds or complicated manipulation. Cardiac cell sheets, myoblast sheets, mesenchymal stem cell sheets, cardiac progenitor cell sheets, etc., which are prepared from temperature-responsive culture surfaces, can be easily transplanted onto heart tissues of animal models, and those cell sheet constructs enhance the cell transplant efficiency, resulting in the induction of effective therapy.

Rate control of cell sheet recovery by incorporating hydrophilic pattern in thermoresponsive cell culture dish.

Thready stripe-polyacrylamide (PAAm) pattern was fabricated on a thermoresponsive poly(N-isopropylacrylamide) (PIPAAm) surface, and their surface properties were characterized. A PIPAAm surface spin-coated with positive photoresist was irradiated through a 5 µm/5 µm or a 10 µm/10-µm black and white striped photomask, resulting in the radical polymerization of AAm on the photoirradiated area. After staining with Alexa488 bovine serum albumin, the stripe-patterned surface was clearly observed and the patterned surface was also observed by a phase contrast image of an atomic force microscope. NIH-3T3 (3T3) single cells were able to be cultured at 37°C on the patterned surfaces as well as on a PIPAAm surface without pattern, and the detachment of adhered cells was more rapidly from the patterned surface after reducing temperature. Furthermore, the rate of detachment of 3T3 confluent cell sheet on the patterned surface was accelerated, compared with on a conventional PIPAAm surface under the static condition. The rate control of cell sheet recovery should contribute the preservations of cell phenotype and biological functions of cell sheet for applying to clinical trials.

Sutureless pneumostasis using bioabsorbable mesh and glue during major lung resection for cancer: who are the best candidates?

OBJECTIVE: Preventing air leaks after major lung resection for cancer is mandatory for successful fast-track surgical intervention. We reported our preliminary results with performance of pneumostasis by combining polyglycolic acid mesh and fibrin glue; however, the advantages of this combination over the conventional method have not been clarified. METHODS: We controlled air leaks detected during an intraoperative water-seal test by using sutures and fibrin glue before April 2006 and by combining polyglycolic acid mesh and fibrin glue without sutures thereafter. We removed the chest tube the day after the air leaks stopped. For bias reduction in comparison with the 2 historical cohorts, we used the nearest available matching method with the estimated propensity score. RESULTS: The durations of chest tube drainage and postoperative hospital stay were significantly shorter in the mesh-and-glue group (n = 61) than in the glue-alone group (n = 61). The incidence of postoperative pulmonary complications was lower in the mesh-and-glue group than in the glue-alone group (0% vs 7%, P = .042). According to a stratification analysis, the benefit of combining mesh and glue to reduce the duration of chest tube drainage was limited in patients undergoing upper lobe resection and in patients with severe emphysema undergoing other types of resection. CONCLUSION: Combining bioabsorbable mesh and glue for pneumostasis can reduce the duration of chest tube drainage, postoperative hospital stay, and pulmonary complications after major lung resection for cancer. Patients undergoing upper lobe resection and those with severe emphysema might be the best candidates for this technique.