Joint unloading inhibits articular cartilage degeneration in knee joints of a monosodium iodoacetate-induced rat model of osteoarthritis.

OBJECTIVE: The aim of the study was to examine how mechanical unloading affects articular cartilage degeneration in the patellofemoral (PF) and tibiofemoral (TF) joints of a monosodium iodoacetate (MIA)-induced rat model of osteoarthritis (OA). DESIGN: The study involved 60 male rats. OA was induced by intra-articular injecting MIA into both knee joints. All animals were equally divided into two groups: sedentary (SE) and hindlimb unloading (HU) groups. Histopathological changes in the articular cartilage of the PF and TF joints were evaluated using the Osteoarthritis Research Society International (OARSI) score and modified Mankin score at 2 and 4 weeks after MIA injection. RESULTS: In the SE and HU groups, representative histopathological changes in OA were detected in the PF and TF joints. The OARSI and modified Mankin scores for the PF and TF joints tended to increase over time after the injection of 0.2 mg or 1.0 mg of MIA in the SE and HU groups. Both the scores for the HU group were significantly lower than those for the SE group [OARSI score: P < 0.0001 (1.0-mg injection at 4 weeks); modified Mankin score: P = 0.0116 (0.2-mg injection at 4 weeks); P = 0.0004 and < 0.0001 (1.0-mg injection at 2 and 4 weeks, respectively)]. CONCLUSION: This study revealed new histological evidence that indicates that unloading condition suppresses articular cartilage degeneration and is beneficial in many areas of basal and clinical research involving OA.

A tuberculosis outbreak at an insecure, temporary housing facility, manga café, Tokyo, Japan, 2016-2017.

In November 2016, a woman in her 30s who stayed at an insecure, temporary housing facility, a manga café in Tokyo, Japan, for a year was diagnosed with sputum smear-positive tuberculosis (TB). Since the café had 31 staff members and provided with accommodation to many people, the local health office initiated a contact investigation. This study aims to characterise the cases found in the outbreak. A TB case was defined as a person tested bacteriologically positive for TB, or was determined to have TB by a physician. A latent TB infection case was defined as a person tested positive by interferon-γ release assay. From January 2016 through November 2017, there were 31 staff members at the manga café, of which, six developed TB disease (one smear-negative, culture-positive and five smear- and culture-negative) in addition to seven LTBI. Another long-term customer was found having sputum smear-positive TB. Variable numbers tandem repeat (VNTR) test revealed that the index patient and the long-term customer had the identical type of VNTR; however, one staff member had a different VNTR. Local health authorities should intensify screening long-term customers of such facilities for TB regularly as well as once a TB outbreak occurs.

Ir(iii) complex-based oxygen imaging of living cells and ocular fundus with a gated ICCD camera.

Phosphorescence lifetime imaging methods using oxygen-sensitive probes are very useful for visualizing the oxygen status of living cells and tissues with high spatial resolution. We aim to develop a useful oxygen detection technique combining a phosphorescent oxygen probe and an optimal detection method. Herein we present a biological oxygen imaging method using a microscope equipped with a gated intensified charge-coupled device (ICCD) camera as a detector and an Ir(iii) complex as a phosphorescent oxygen probe. Microscopic luminescence images of monolayer HT-29 cells (human colorectal adenocarcinoma cells) obtained using the cell-penetrating Ir(iii) complex BTPDM1 and an inverted microscope demonstrated that this method allowed visualization of the oxygen gradient produced in a monolayer of cultured cells when the monolayer is covered with a thin coverslip. Furthermore, combining the IR-emitting Ir(iii) complex DTTPH-PEG24 with a macrozoom microscope equipped with a gated ICCD camera enabled both the visualization of retinal vessels near the optic disc and the monitoring of oxygen level changes in a rabbit retina upon changing the inhaled oxygen content.

Preoperative prediction for regaining ambulatory ability in paretic non-ambulatory patients with metastatic spinal cord compression.

STUDY DESIGN: Retrospective multicenter study. OBJECTIVES: To analyze the predictive factors for postoperative ambulatory recovery in paretic non-ambulatory patients with metastatic spinal cord compression (MSCC). SETTING: Japan. METHODS: Eighty-two consecutive patients (74.4% men; mean age, 66.2 years) who could not walk before surgery due to cervical or thoracic MSCC and underwent posterior decompressive surgery between 2003 and 2014 were included. Patients were divided into two groups according to ambulatory status at 6 weeks after surgery: recovery (group R) and non-recovery (group NR). To evaluate the speed of progression of motor deficits, we assessed the period from onset of neurological symptoms to gait inability (T1). RESULTS: Fifty patients (61.0%) regained the ability to walk (group R). The period of T1 demonstrated a positive correlation with probability of ambulatory recovery (P=0.00; Kendall's tau-b=0.38), and a receiver operating characteristic curve analysis showed that the cutoff value of T1 was 5 days (area under the curve=0.72; P=0.001). In multivariate analysis, <6 days of T1 was one of the independent risk factors for failing to regain ambulatory ability (odds ratio, 8.74; P=0.00). CONCLUSIONS: The speed of progression of motor deficits can independently and powerfully predict the chance of postoperative ambulatory recovery as well as previously identified predictors. Since information about the speed of progression can be obtained easily by interviewing patients or family members, even if the patient is in an urgent state, our results will be helpful in clinical decision-making.

Growth of bulk ß-Ga2O3 crystals from melt without precious-metal crucible by pulling from a cold container.

We report the growth of bulk ß-Ga2O3 crystals based on crystal pulling from a melt using a cold container without employing a precious-metal crucible. Our approach, named oxide crystal growth from cold crucible (OCCC), is a fusion between the skull-melting and Czochralski methods. The absence of an expensive precious-metal crucible makes this a cost-effective crystal growth method, which is a critical factor in the semiconductor industry. An original construction 0.4-0.5 MHz SiC MOSFET transistor generator with power up to 35 kW was used to successfully grow bulk ß-Ga2O3 crystals with diameters up to 46 mm. Also, an original diameter control system by generator frequency change was applied. In this preliminary study, the full width at half maximum of the X-ray rocking curve from the obtained ß-Ga2O3 crystals with diameters ≤ 46 mm was comparable to those of ß-Ga2O3 produced by edge-defined film fed growth. Moreover, as expected, the purity of the obtained crystals was high because only raw material-derived impurities were detected, and contamination from the process, such as insulation and noble metals, was below the detection limit. Our results indicate that the OCCC technique can be used to produce high-purity bulk ß-Ga2O3 single crystalline substrate.

HIF-2α Inhibits Ameloblast Differentiation via Hey2 in Tooth Development.

Enamel is the highly mineralized outer layer of teeth; the cells responsible for enamel formation are ameloblasts. Local hypoxia and hypoxia inducible factor (HIF) in embryonic tissues are important to promote normal organogenesis. However, hypoxic state in tooth germs and the roles of HIF in ameloblast differentiation have not been understood. The aim of this study is to clarify the role of HIF in ameloblast differentiation during tooth germ development. We found that tooth germs were under hypoxia and HIF-1α and HIF-2α were expressed in tooth germs in embryonic mice. Then, we used HIF inhibitors to evaluate the function of HIF during tooth germ development. The HIF-2α inhibitor significantly decreased the size of tooth germs in organ culture, while the HIF-1α inhibitor did not apparently affect the size of tooth germs. The HIF-2α inhibitor enhanced the expression of amelogenin, a marker of ameloblast differentiation, in the tooth germs in organ culture and rat dental epithelial SF2 cells. Moreover, we found that the HIF-2α inhibitor-stimulating amelogenin expression was regulated by hes-related family basic helix-loop-helix transcription factor with YRPW motif 2(Hey2) in SF2 cells. These findings suggest that the HIF-2α-Hey2 axis plays an important role in ameloblast differentiation during tooth germ development.

Relationship between the combination of polyunsaturated fatty acids intake and psychological distress during pregnancy: The Tohoku Medical Megabank Project Birth and Three-Generation Cohort Study.

No studies have examined the association of the combination of n-3 polyunsaturated fatty acids (PUFAs) and n-6 PUFAs intake with psychological distress during pregnancy. To examine these associations, we divided Japanese pregnant women into 25 groups based on combining quintiles of n-3 PUFAs intake and quintiles of n-6 PUFAs intake. We conducted multivariable logistic regression analyses to assess the risk of psychological distress during pregnancy (Kessler Psychological Distress Scale ≥ 5 or 13). Compared to the third quintile of both n-3 PUFAs and n-6 PUFAs intake, the groups with unbalanced intake, high intake of both, and low intake of both were associated with a higher risk of both Kessler Psychological Distress Scale ≥ 5 and 13 in early and mid-pregnancy. Further research is needed to identify the precise combination of n-3 PUFAs and n-6 PUFAs intake associated with the lowest psychological distress during pregnancy.

Knee osteoarthritis, knee joint pain and aging in relation to increasing serum hyaluronan level in the Japanese population.

OBJECTIVE: To investigate relationship between serum hyaluronan (HA) level and the presence and severity of radiographic knee osteoarthritis (OA) as well as degree of knee pain in Japanese population. DESIGN: A total of 616 volunteers participated in this study. Based on the Kellgren-Lawrence (K-L) grade, participants were radiographically classified into three groups: Normal (K-L grade 0 or 1), Moderate (grade 2) and Severe (grade 3 or 4). The degree of knee pain was quantified by visual analogue scale (VAS) and Knee injury and Osteoarthritis Outcome Score (KOOS) Pain. Serum HA levels were compared among the Normal, Moderate and Severe groups, and the relationship between serum HA level and the severity of knee OA was analyzed after age, sex and body mass index (BMI) were adjusted. In addition, the correlation between serum HA level and the degree of knee pain was analyzed in each group. RESULTS: Regarding relationship between serum HA level and the severity of radiographic knee OA, serum HA levels of the Moderate and Severe groups were significantly higher than in the Normal group (P<0.001). Furthermore, serum HA level correlated with the severity of radiographic knee OA (r=0.289, P<0.001) after adjusting for age, sex and BMI. Serum HA level correlated with VAS of knee pain and/or KOOS Pain in the Normal and Moderate groups. CONCLUSION: Serum HA level has the potential to be useful for the diagnosis of the presence and severity of knee OA.

Limitin: An interferon-like cytokine that preferentially influences B-lymphocyte precursors.

We have identified an interferon-like cytokine, limitin, on the basis of its ability to arrest the growth of or kill lympho-hematopoietic cells. Limitin strongly inhibited B lymphopoiesis in vitro and in vivo but had little influence on either myelopoiesis or erythropoiesis. Because limitin uses the interferon alpha/beta receptors and induces interferon regulatory factor-1, it may represent a previously unknown type I interferon prototype. However, preferential B-lineage growth inhibition and activation of Janus kinase 2 in a myelomonocytic leukemia line have not been described for previously known interferons.

Adult orbital xanthogranulomatous disease: adult-onset xanthogranuloma of periorbital location.

Adult orbital xanthogranulomatous disease (AOXGD) is a rare granulomatous disorder, which has four subtypes: adult-onset xanthogranuloma (AOX), adult-onset asthma with periocular xanthogranuloma, necrobiotic xanthogranuloma and Erdheim-Chester disease. We report a 42-year-old woman who presented with yellowish nonulcerative nodules on her eyelids. On histopathological examination of a nodule, mild degeneration of collagen fibres was seen, with surrounding infiltration of numerous foam cells and Touton giant cells in the deep dermis. Lymphoid follicles were seen in the reticular dermis. There was no apparent necrobiosis of collagen fibres. There were no clinical symptoms of asthma and no laboratory signs of paraproteinaemia during a follow-up of more than 5 years. We diagnosed this case as AOX, but further long-term follow-up would be required for the differentiation from the other AOXGDs. Dermatologists should be aware of these rare granulomatous disease conditions with ocular/orbital location, because they may cause ophthalmological complications.

Alteration of interleukin 4 production results in the inhibition of T helper type 2 cell-dominated inflammatory bowel disease in T cell receptor alpha chain-deficient mice.

T cell receptor alpha chain-deficient (TCR-alpha(-/-)) mice are known to spontaneously develop inflammatory bowel disease (IBD). The colitis that develops in these mice is associated with increased numbers of T helper cell (Th)2-type CD4(+)TCR-betabeta (CD4(+)betabeta) T cells producing predominantly interleukin (IL)-4. To investigate the role of these Th2-type CD4(+)betabeta T cells, we treated TCR-alpha(-/-) mice with anti-IL-4 monoclonal antibody (mAb). Approximately 60% of TCR-alpha(-/-) mice, including those treated with mock Ab and those left untreated, spontaneously developed IBD. However, anti-IL-4 mAb-treated mice exhibited no clinical or histological signs of IBD, and their levels of mucosal and systemic Ab responses were lower than those of mock Ab-treated mice. Although TCR-alpha(-/-) mice treated with either specific or mock Ab developed CD4(+)betabeta T cells, only those treated with anti-IL-4 mAb showed a decrease in Th2-type cytokine production at the level of mRNA and protein and an increase in interferon gamma-specific expression. These findings suggest that IL-4-producing Th2-type CD4(+)betabeta T cells play a major immunopathological role in the induction of IBD in TCR-alpha(-/-) mice, a role that anti-IL-4 mAb inhibits by causing Th2-type CD4(+)betabeta T cells to shift to the Th1 type.

gamma/delta T cell-deficient mice have impaired mucosal immunoglobulin A responses.

Mucosal tissues of mice are enriched in T cells that express the gamma/delta T cell receptor. Since the function of these cells remains unclear, we have compared mucosal immune responses in gamma/delta T cell receptor-deficient (TCRdelta-/-) mice versus control mice of the same genetic background. The frequency of intestinal immunoglobulin (Ig) A plasma cells as well as IgA levels in serum, bile, saliva, and fecal samples were markedly reduced in TCRdelta-/- mice. The TCRdelta-/- mice produced much lower levels of IgA antibodies when immunized orally with a vaccine of tetanus toxoid plus cholera toxin as adjuvant. Conversely, the antigen-specific IgM and IgG antibody responses were comparable to orally immunized control mice. Direct assessment of the cells forming antibodies against the tetanus toxoid and cholera toxin antigens indicated that significantly lower numbers of IgA antibody-producing cells were present in the intestinal lamina propria and Peyer's patches of TCRdelta-/- mice compared with the orally immunized control mice. The selective reduction of IgA responses to ingested antigens in the absence of gamma/delta T cells suggests a specialized role for gamma/delta cells in mucosal immunity.

Induction of MMP-2 at the interface between epithelial cells and fibroblasts from human periodontal ligament.

BACKGROUND AND OBJECTIVE: MMP-2 can degrade type IV collagen and MMP-14 can activate pro MMP-2. The present study was undertaken to examine the expression of MMP-2 and MMP-14 with respect to interaction between the cells of the epithelial rests of Malassez and fibroblasts from human periodontal ligament. MATERIAL AND METHODS: Explants of human periodontal ligament tissues produced outgrowths containing both putative epithelial rests of Malassez cells and human periodontal ligament fibroblasts after incubation in a modified serum-free medium. The distribution and expression of MMP-2 and MMP-14 were analysed using immunohistochemistry, in situ hybridization and RT-PCR analysis. The conditioned media and cell extracts were collected for western blot analysis for MMP-2. RESULTS: Putative epithelial rests of Malassez cells at the interface between the cells of the epithelial rests of Malassez and fibroblasts expressed MMP-2 and MMP-14 strongly. However, in situ hybridization analysis revealed that human periodontal ligament fibroblasts expressed MMP-2 mRNA while putative epithelial rests of Malassez cells expressed MMP-14 mRNA at the interface. The RT-PCR analysis showed that the expression of MMP-2 mRNA was significantly higher when putative epithelial rests of Malassez cells and human periodontal ligament fibroblasts were cultured together than when cultured alone. Western blot analysis showed that the active form of MMP-2 was detected at higher levels in the conditioned medium of the co-cultured cells. CONCLUSION: These findings indicate that putative epithelial rests of Malassez cells stimulate the production of MMP-2 in human periodontal ligament fibroblasts. Up-regulated proMMP-2 bound by MMP-14 expressed in epithelial rests of Malassez cells can degrade matrix molecules, such as type IV collagen, in the basal membrane between putative epithelial rests of Malassez cells and human periodontal ligament fibroblasts.

Analysis of hypoxia-associated dendritic cells in colitic mice and effects of probiotics on IL-10 production in inflammatory dendritic-cells under hypoxia.

The aim of this study was to analyse hypoxia-associated dendritic cells (DCs) in colitic mice and the effects of probiotics on interleukin (IL)-10 production in inflammatory DCs under hypoxic conditions. Extensive hypoxia was observed in the colonic mucosa of dextran sodium sulphate-induced colitic mice. Flow cytometric analysis demonstrated that hypoxia-inducible factor-1α+ DCs in colonic lamina propria (CLP) lymphocytes and mesenteric lymph nodes (MLN) were more abundant in colitic mice than those in controls. Among three subsets of DCs, i.e. plasmacytoid DCs, conventional DCs (cDCs), and monocyte-derived DCs (mDCs), cDCs and mDCs were more abundant in CLP of colitic mice. Bone marrow-derived Flt-3L-induced DCs (Flt-DCs) but not bone marrow-derived GM-CSF-induced DCs (GM-DCs), incubated with 1% O2 exhibited an inflammatory phenotype, with higher CD86, IL-6, and tumour necrosis factor-α expression, and lower IL-10 levels than those in Flt-DCs incubated with 21% O2. The hypoxia-induced decrease in IL-10 expression in Flt-DCs was restored by Bifidobacterium bifidum JCM 1255T promoted IL-10 expression through the p38 pathway under normoxic conditions. The anti-inflammatory effects of B. bifidum JCM 1255T in Flt-DCs were mediated through different cellular mechanisms under hypoxic and normoxic conditions. B. bifidum JCM 1255T could be used therapeutically for its anti-inflammatory effects.

Prevalence of adult-onset multifactorial disease among offspring of atomic bomb survivors.

The first study to examine whether parental radiation exposure leads to increased heritable risk of common adult-onset multifactorial diseases (i.e., hypertension, diabetes mellitus, hypercholesterolemia, ischemic heart disease, and stroke) was conducted among 11,951 participants in the clinical examination program out of a potential of 24,673 mail survey subjects who were offspring of survivors born from May 1946 through December 1984. Logistic regression analyses demonstrated no evidence of an association between the prevalence of multifactorial diseases in the offspring and parental radiation exposure, after adjusting for age, city, gender and various risk factors. The odds ratio (OR) for a paternal dose of 1 Gy was 0.91 [95% confidence interval (CI) 0.81-1.01, P = 0.08], and that for a maternal dose of 1 Gy was 0.98 (95% CI 0.86-1.10, P = 0.71). There was no apparent effect of parental age at exposure or of elapsed time between parental exposure and birth, but male offspring had a low odds ratio (OR = 0.76 at 1 Gy) for paternal exposure, but cautious interpretation is needed for this finding. The clinical assessment of nearly 12,000 offspring of A-bomb survivors who have reached a median age of about 50 years provided no evidence for an increased prevalence of adult-onset multifactorial diseases in relation to parental radiation exposure.

Effects of long-term training on neutrophil function in male university judoists.

OBJECTIVES: To clarify the effects of high-intensity and high-frequency long-term/chronic training on neutrophil function and serum levels of myogenic enzymes in male university judoists. METHODS: The subjects were 24 male judoists who had stopped judo training for 6 months and then restarted their training. The following parameters were examined before and after a 2 h unified exercise loading (UEL) at the beginning of the restarted quotidian training (pre-training) and at 2 months, 4 months and 6 months thereafter: myogenic enzymes, neutrophil and leucocyte counts, and neutrophil phagocytic activity (PA) and oxidative burst activity as a measure of reactive oxygen species (ROS) production capability. RESULTS: Myogenic enzymes that were measured after UEL at all four points significantly increased except for creatine kinase at the 2-month point (p<0.01 in each) and neutrophil counts significantly increased after UEL at the pre-training, 2-month and 4-month points (p<0.01 in each), but these changes became smaller from the 2-month point. PA significantly decreased after UEL at the pre-training and 2-month points (p<0.01 in each), but no change was seen at the 4-month and 6-month points. On the other hand, no change in ROS production per cell after UEL was seen at the pre-training point, but it significantly increased after UEL at the 2-month, 4-month and 6-month points (p<0.01 in each). CONCLUSION: The changing rate of the levels of UEL-mediated myogenic enzymes, neutrophil mobilisation and neutrophil function was seen to decrease at the 2-month, 4-month and 6-month assessments, compared with the pre-training point: these may comprise at least some of the long-term training effects.

Systemically derived large intestinal CD4(+) Th2 cells play a central role in STAT6-mediated allergic diarrhea.

Systemically primed BALB/c mice developed severe diarrhea after repeated oral administration of ovalbumin (OVA). Histological analysis demonstrated that dramatic infiltration of eosinophils and mast cells occurred in the large intestine but not in the small intestine of mice with diarrhea. Interestingly, CD4(+) alphabeta T cells of the large intestine secreted IL-4 and IL-13 at high levels. Identically treated STAT6 gene-disrupted mice failed to develop OVA-induced diarrhea. Further, treatment of BALB/c mice with monoclonal anti-IL-4 antibody prevented the development of allergic diarrhea. An adoptive transfer study showed that systemically primed splenic CD4(+) T cells were preferentially recruited into the large intestine upon exposure to oral OVA. These results strongly suggest that systemically derived CD4(+) alphabeta T cells of the large intestine play a critical role in the onset of Th2-mediated intestinal allergic disorders via STAT6 signal transduction.

Mucosal immunity to infection with implications for vaccine development.

The induction of effective mucosal immunity that also provides systemic immunity is a considerable challenge. Over the past two years, efforts to develop novel mucosal vaccine delivery systems to induce mucosal immunity against bacterial and viral diseases, including HIV, have dramatically increased. Here we cite novel vaccines and delivery systems being used to establish effective mucosal immunity.

Effects of rugby sevens matches on human neutrophil-related non-specific immunity.

AIMS: To evaluate the influences of the accumulative effect of two consecutive rugby sevens matches (Sevens) on aspects of human neutrophil-related non-specific immunity. METHODS: In seven players participating in the Japan Sevens, neutrophil reactive oxygen species (ROS) production capability and phagocytic activity were measured using flow cytometry, and serum opsonic activity (SOA) was assessed by measuring neutrophil ROS using the peak height of lucigenin-dependent chemiluminescence before and after two consecutive matches. RESULTS: ROS showed no change immediately after the first match, and had significantly (P<0.05) increased 4 h later, but showed a decrease after the second match. Phagocytic activity showed no change immediately after the first match, but had significantly (P<0.01) decreased 4 h later, and showed a further decrease after the second match, although it was not significant. SOA significantly (P<0.01) increased after the first match, and still maintained its high 4 h later, but decreased after the second match. ROS production capability, phagocytic activity and SOA significantly (P<0.01) decreased after the second match. CONCLUSIONS: When rugby players play two consecutive Sevens matches, the exercise loading is thought to be hard, similar to that experienced during a marathon race and intensive or long training in a training camp, although the expected changes were not seen after the first match. Differences between after the first and the second matches may be due to the "cumulative effect".

Stim1 Regulates Enamel Mineralization and Ameloblast Modulation.

Loss-of-function mutations in the Ca2+ release-activated Ca2+ channel genes ORAI1 and STIM1 abolish store-operated Ca2+ entry (SOCE) and result in ectodermal dysplasia with amelogenesis imperfecta. However, because of the limited availability of patient tissue, analyses of enamel mineralization or possible changes in ameloblast function or morphology have not been possible. Here, we generated mice with ectodermal tissue-specific deletion of Stim1 ( Stim1 cKO [conditional knockout]), Stim2 ( Stim2 cKO), and Stim1 and Stim2 ( Stim1/2 cKO) and analyzed their enamel phenotypes as compared with those of control ( Stim1/2fl/fl) animals. Ablation of Stim1 and Stim1/2 but not Stim2 expression resulted in chalky enamel and severe attrition at the incisor tips and molar cusps. Stim1 and Stim1/2 cKO, but not Stim2 cKO, demonstrated inferior enamel mineralization with impaired structural integrity, whereas the shape of the teeth and enamel thickness appeared to be normal in all animals. The gene expression levels of the enamel matrix proteins Amelx and Ambn and the enamel matrix proteases Mmp20 and Klk4 were not altered by the abrogation of SOCE in Stim1/2 cKO mice. The morphology of ameloblasts during the secretory and maturation stages was not significantly altered in either the incisors or molars of the cKO animals. However, in Stim1 and Stim1/2 cKO incisors, the alternating modulation of maturation-stage ameloblasts between the smooth- and ruffle-ended cell types continued beyond the regular cycle and extended to the areas corresponding to the zone of postmodulation ameloblasts in the teeth of control animals. These results indicate that SOCE is essential for proper enamel mineralization, in which Stim1 plays a critical role during the maturation process.