Mutations of N-RAS, FLT3 and p53 genes are not involved in the development of acute leukemia transformed from myeloproliferative diseases with JAK2 mutation.
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Acute Disease↗
Biomedical subjects
Y Ito
Publications and source records attributed to Y Ito.
Expression profiles of melanogenesis-related genes and proteins in acquired melanocytic nevus.
UNLABELLED: We used three types of AMN to investigate the expression profiles of melanogenesis-related genes [tyrosinase, tyrosinase-related protein-1 (TRP1), dopachrome tautomerase (TRP2), Pmel-17/gp100, P-protein, and microphthalmia-associated transcription factor (MITF)], as well as tyrosinase, TRP1, Pmel-17/gp100, and MITF proteins. RESULTS: All melanogenesis-related genes examined in the junctional type were expressed in the basal epidermal layer. In the compound and intradermal types, mRNA for tyrosinase, TRP2, and MITF was expressed in all of the AMN cells. However, the expression of TRP1, P-protein, and Pmel-17/gp100 in the compound type and TRP1 in the intradermal type became weaker in accordance with the depth of the dermis layer, as compared to those levels in the basal to upper dermis layer. Although tyrosinase and Pmel-17/gp100 mRNA in the compound and intradermal types was expressed in the intraepidermal and dermal components, immunohistochemical staining showed that tyrosinase proteins were not detected in the lower dermis layer and Pmel-17/gp100 proteins were not detected in the dermis. CONCLUSIONS: Our results suggest that all nevus cells that constitute AMN tissue originate from melanocytes. Further, there may be differences in the transcription levels of melanogenesis-related genes as well as in their post-transcriptional regulation between nevus cells located in the basal epidermal to upper dermis layer and those in the lower dermis layer.
Biomarkers, Tumor↗
Identification of intestinal bacteria from Japanese flounder (Paralichthys olivaceus) and their ability to digest chitin.
AIMS: The aim of the present study was to clarify the taxonomic status of intestinal bacteria isolated from Japanese flounder (Paralichthys olivaceus) and describe their ability to digest chitin. METHODS AND RESULTS: Phylogenetic analysis based on 16S ribosomal DNA (rDNA) sequences showed that 82 representative isolates were closely related to three major species of marine vibrios, Vibrio scophthalmi-Vibrio ichthyoenteri group (41 isolates), Vibrio fischeri (39 isolates) and Vibrio harveyi (two isolates), with similarities of 97.2-99.8%, 96.4-100% and 98.6-99.5% respectively. These findings indicate that V. scophthalmi-V. ichthyoenteri group is indigenous to the intestinal tract of Japanese flounder. Moreover, the ability of 82 isolates to digest chitin was examined using the agar plate method and PCR amplification of the chiA gene. The two V. harveyi isolates and 36 of 41 V. scophthalmi-V. ichthyoenteri isolates digested chitin and were chiA PCR positive, whereas all 39 V. fischeri isolates digested chitin but were chiA PCR negative. CONCLUSIONS: Intestinal bacteria from Japanese flounder were mainly composed of Vibrio scophthalmi-V. ichthyoenteri group and V. fischeri. Taken together, the results showed that 81 of 82 isolates could digest chitin. However, only 38 of these isolates possessed a chiA homologue which could be identified by PCR. SIGNIFICANCE AND IMPACT OF THE STUDY: The present study shows that Japanese flounder harbours bacteria of the V. scophthalmi-V. ichthyoenteri group, and these results are similar to what has been found for turbot (Scophthalmus maximus).
Animals↗
Detection of Lactobacillus gasseri OLL2716 strain administered with yogurt drink in gastric mucus layer in humans.
In animal models and human trials, Lactobacillus gasseri OLL2716 (LG21) strain suppressed Helicobacter pylori colonization in the stomach. The aim of the present study was to clarify whether orally administered LG21 strain can enter the gastric mucus layer. Biopsy samples were taken from the gastric antrum and corpus of two healthy volunteers (H. pylori infected and non-infected) who drank yogurt supplemented with LG21 strains. DNA of LG21 and H. pylori in the mucus layer was detected using the laser-assisted microdissection and non-contact pressure catapulting (LMPC) method and the semi-nested PCR method with primer sets of RNA helicases of superfamily II gene-Insertion sequence for LG21 strain and those of ureA gene for H. pylori. In the volunteer with H. pylori infection, DNA fragments of LG21- and H. pylori-specific regions from both antrum and corpus were amplified, whereas in a non-infected volunteer, only the LG21 DNA from the antrum was amplified. The present study demonstrated that LG21 strains administered through a yogurt drink can enter into the gastric mucus layer. Our novel method may be useful in studying gastric probiotics for H. pylori infection.
Gastric Mucosa↗
Disorder of the saliva melatonin circadian rhythm in patients with Meniere's disease.
OBJECTIVES: Stress is involved in the development of symptoms of Meniere's disease (MD). Stress-related disease has been reported to be associated with disorders in the circadian rhythm of melatonin (MEL) which regulates that rhythm. We therefore investigated MEL circadian rhythm of patients with MD. PATIENTS AND METHODS: A comparison of 13 MD patients was made with age-matched controls. Saliva samples were collected every 3 h. A statistical analysis of the circadian rhythm of saliva MEL was performed for a circadian rhythm by the fit of a 24- and 12-h composite cosine model. In addition, we assessed the stress and depression status of the two groups. RESULTS: The rhythmic amplitude of MEL in the MD group was significantly lower than that in the control group (P < 0.05). The acrophase in the MD group was significantly earlier than that in the control group (P < 0.05). MD patients had significantly higher stress score and depression score than control subjects (P < 0.05). The amplitude in MD patients had no significant correlation with their otologic clinical data. CONCLUSIONS: These results suggest that MD patients have a MEL deficiency, which is related to their stress and depression status rather than the otologic pathological status of MD.
Adult↗
Expression and characterization of a Rana pipiens amelogenin protein.
Amelogenin, the major protein of developing enamel matrix, controls enamel crystal growth via unique supermolecular features. While much has been contributed to our understanding of mammalian amelogenin function, little is known about how amelogenin and its unique physico-chemical features have evolved among vertebrates. Here we report, for the first time, amphibian amelogenin recombinant protein expression and characterization in Rana pipiens. In order to characterize R. pipiens amelogenin, the newly discovered amelogenin coding sequence was amplified, subcloned, and expressed in Eshcerichia coli. Our newly generated R. pipiens amelogenin-specific antisera resolved a major 19-kDa band on western blots of frog tooth extracts and revealed an enamel organ tissue-specific localization pattern using immunohistochemistry. Using mass spectroscopy, a single major compound with a molecular weight of 21.6 kDa was detected, which corresponded to the amino acid sequence-based molecular weight prediction of the His fusion recombinant protein. Dynamic light scattering studies resolved 41-nm radius subunits compared with 14-nm radius subunits from mouse recombinant amelogenin controls. Transmission electron microscopy revealed defined spherical subunits in R. pipiens matrix self-assembly in contrast with a homogeneous 'stippled' matrix in mouse amelogenin matrix self-assembly. Our data suggest that R. pipiens amelogenin is distinguished from mammalian amelogenins by a number of unique physico-chemical properties which may be related to specific modes of crystal formation in frog enamel.
Amelogenin↗
Molecular cytogenetic characterization of chromosome 9-derived material in a human thyroid cancer cell line.
The incidence of papillary thyroid carcinoma (PTC) increases significantly after exposure of the head and neck region to ionizing radiation, yet we know neither the steps involved in malignant transformation of thyroid epithelium nor the specific carcinogenic mode of action of radiation. Such increased tumor frequency became most evident in children after the 1986 nuclear accident in Chernobyl, Ukraine. In the eight years following the accident, the average incidence of childhood PTCs (chPTC) increased 70-fold in Belarus, 200-fold in Gomel, 10-fold in the Ukraine and 50-fold in Tschnigov, Kiev, Rovno, Shitomyr and Tscherkassy compared to the rate of about 1 tumor incidence per 106 children per year prior to 1986 (Likhtarev et al., 1995; Sobolev et al., 1997; Jacob et al., 1998). To study the etiology of radiation-induced thyroid cancer, we formed an international consortium to investigate chromosomal changes and altered gene expression in cases of post-Chernobyl chPTC. Our approach is based on karyotyping of primary cultures established from chPTC specimens, establishment of cell lines and studies of genotype-phenotype relationships through high resolution chromosome analysis, DNA/cDNA micro-array studies, and mouse xenografts that test for tumorigenicity. Here, we report the application of fluorescence in situ hybridization (FISH)-based techniques for the molecular cytogenetic characterization of a highly tumorigenic chPTC cell line, S48TK, and its subclones. Using chromosome 9 rearrangements as an example, we describe a new approach termed 'BAC-FISH' to rapidly delineate chromosomal breakpoints, an important step towards a better understanding of the formation of translocations and their functional consequences.
Cell Line, Tumor↗
High sensitivity C-reactive protein in asthma.
Asthma is characterised by chronic inflammation of the airways, but the relevance of high-sensitivity assays for C-reactive protein (hs-CRP), which are known to be a sensitive marker of low-grade systemic inflammation, has not been fully studied in asthma. The objective was to examine serum hs-CRP levels in patients with asthma and their relationship to clinical characteristics and degree of airway inflammation. Serum hs-CRP levels were cross-sectionally examined in steroid-naive (n = 22) and steroid-inhaling (n = 23) adult patients with asthma and healthy controls (n = 14). All were nonsmokers. Serum hs-CRP levels were significantly increased in steroid-naive patients (mean+/-sd 1.33+/-1.48 mg.L(-1)) compared with controls (0.21+/-0.30 mg.L(-1)), but not in patients on inhaled corticosteroid. Among steroid-naive patients, serum hs-CRP levels significantly negatively correlated with indices of pulmonary function (forced expiratory volume in one second/forced vital capacity and forced mid-expiratory flow) and positively with sputum eosinophil count. Among patients on inhaled corticosteroid, hs-CRP levels did not correlate with any indices. In conclusion, an increase in serum C-reactive protein levels measured by high-sensitivity assays may be associated with airflow obstruction and airway inflammation, and may serve as a surrogate marker of airway inflammation in asthma.
Adult↗
Effects of tyrosine kinase inhibitors on voltage-dependent Ba(2+) currents in the guinea-pig gastric antrum.
We have investigated whether tyrosine kinases modify the activity of voltage-dependent Ba(2+) currents (I(Ba)) recorded from guinea-pig gastric myocytes by use of patch-clamp techniques. All experiments were carried on single smooth muscle cells, dispersed from the circular layer of the guinea-pig gastric antrum. Genistein ( > or = 10 microM), a specific tyrosine kinase inhibitor, reduced the peak amplitude of I(Ba) in a voltage- and concentration-dependent manner. Daidzein ( > or = 30 microM), an inactive analog of genistein, also inhibited I(Ba) in a concentration-dependent manner. Similarly, other types of tyrosine kinase inhibitors (lavendustin A and tyrphostin 23) suppressed the peak amplitude of I(Ba) in a concentration-dependent manner. These results indicate that tyrosine kinases may be essential to regulate Ca(2+) mobilization through voltage-dependent Ca(2+) channels in gastric myocytes.
Animals↗
Gastro-intestinal patch system for the delivery of erythropoietin.
The absorption of erythropoietin (EPO) from rat small intestine was studied using gastro-intestinal patches (GI-PS) in the presence of absorption enhancers. Surfactants such as a saturated polyglycolysed C8-C18 glyceride (Gelucire 44/14), PEG-8 capryl/caprylic acid glycerides (Labrasol), and polyoxyethylene hydrogenated castor oil derivative (HCO-60) were used as absorption enhancers at 143, 94 and 20 mg/kg, respectively. The absorption of EPO was studied by measuring serum EPO levels by an ELISA method after small intestinal administration of EPO-GI-PS preparation in rats at the EPO dose level of 100 IU/kg. Labrasol showed the highest absorption enhancing effect after intrajejunum administration with maximum serum EPO level of 84.1+/-11.4 mIU/ml while Gelucire 44/14 and HCO-60 showed 43.5+/-9.8 and 26.5+/-2.3 mIU/ml, respectively. The appropriate site for EPO absorption was also investigated. Jejunum was found to be the most efficient absorption site for the absorption of EPO from GI-PS. Using Labrasol as the absorption enhancer and jejunum as the absorption site, the effect of EPO dose on EPO absorption was studied by increasing the EPO dose from 50, to 100, 300 and 600 IU/kg. It was found that 100 IU/kg was the optimum dose with a serum EPO level of 84.1+/-11.4 mIU/ml while escalating doses showed decreases in serum EPO levels 48.3+/-5.6 for 300 IU/kg and 50.6+/-10.3 mIU/ml for 600 IU/kg. The percent bioavailability (BA) of EPO-GI-PS with Labrasol as absorption enhancer was 7.9 at 50 IU/kg, 12.1 at 100 IU/kg, 3.2 at 300 IU/kg and 1.2 at 600 IU/kg. Histological studies showed no adverse effect at the site of administration.
Administration, Oral↗
Presynaptic GABAA receptors facilitate spontaneous glutamate release from presynaptic terminals on mechanically dissociated rat CA3 pyramidal neurons.
Mossy fiber-derived giant spontaneous miniature excitatory postsynaptic currents have been suggested to be large enough to generate action potentials in postsynaptic CA3 pyramidal neurons. Here we report on the functional roles of presynaptic GABA(A) receptors on excitatory terminals in contributing to spontaneous glutamatergic transmission to CA3 neurons. In mechanically dissociated rat hippocampal CA3 neurons with adherent presynaptic nerve terminals, spontaneous excitatory postsynaptic currents were recorded using conventional whole-cell patch clamp recordings. In most recordings, unusually large spontaneous excitatory postsynaptic currents up to 500 pA were observed. These large spontaneous excitatory postsynaptic currents were highly sensitive to group II metabotropic glutamate receptor activation, and were still observed even after the blockade of voltage-dependent Na(+) or Ca(2+) channels. Exogenously applied muscimol (0.1-3 microM) significantly increased the frequency of spontaneous excitatory postsynaptic currents including the large ones. This facilitatory effect of muscimol was completely inhibited in the presence of 10 microM 6-imino-3-(4-methoxyphenyl)-1(6H)-pyridazinebutanoic acid HBr, a specific GABA(A) receptor antagonist. Pharmacological data suggest that activation of presynaptic GABA(A) receptors directly depolarizes glutamatergic terminals resulting in the facilitation of spontaneous glutamate release. In the current-clamp condition, a subset of large spontaneous excitatory postsynaptic potentials triggered action potentials, and muscimol greatly increased the frequency of spontaneous excitatory postsynaptic potential-triggered action potentials in postsynaptic CA3 pyramidal neurons. The results suggest that presynaptic GABA(A) receptors on glutamatergic terminals play an important role in the excitability of CA3 neurons as well as in the presynaptic modulation of glutamatergic transmission onto hippocampal CA3 neurons.
Action Potentials↗
Development of an in vitro skin sensitization test using human cell lines; human Cell Line Activation Test (h-CLAT). II. An inter-laboratory study of the h-CLAT.
Recent regulatory changes have placed a major emphasis on in vitro safety testing and alternative models. In regard to skin sensitization tests, dendritic cells (DCs) derived from human peripheral blood have been considered in the development of new in vitro alternatives. Human cell lines have been also reported recently. In our previous study, we suggested that measuring CD86 and/or CD54 expression on THP-1 cells (human monocytic leukemia cell line) could be used as an in vitro skin sensitization method. An inter-laboratory study among two laboratories was undertaken in Japan in order to further develop an in vitro skin sensitization model. In the present study, we used two human cell lines: THP-1 and U-937 (human histiocytic lymphoma cell line). First we optimized our test protocol (refer to the related paper entitled "optimization of the h-CLAT protocol" within this journal) and then we did an inter-laboratory validation with nine chemicals using the optimized protocol. We measured the expression of CD86 and CD54 on the above cells using flow cytometry after a 24h and 48h exposure to six known allergens (e.g., DNCB, pPD, NiSO(4)) and three non-allergens (e.g., SLS, tween 80). For the sample test concentration, four doses (0.1x, 0.5x, 1x, and 2x of the 50% inhibitory concentration (IC(50))) were evaluated. IC(50) was calculated using MTT assay. We found that allergens/non-allergens were better predicted using THP-1 cells compared to U-937 cells following a 24 h and a 48 h exposure. We also found that the 24h treatment time tended to have a better accuracy than the 48 h treatment time for THP-1 cells. Expression of CD86 and CD54 were good predictive markers for THP-1 cells, but for U-937 cells, expression of CD86 was a better predictor than CD54, at the 24h and the 48 h treatment time. The accuracy also improved when both markers (CD86 and CD54) were used as compared with a single marker for THP-1 cells. Both laboratories gave a good prediction of allergen/non-allergen, especially using THP-1 cells. These results suggest that our method, human Cell Line Activation Test (h-CLAT), using human cell lines THP-1 and U-937, but especially THP-1 cells at 24h treatment, may be a useful in vitro skin sensitization model to predict various contact allergens.
Allergens↗
Development of an in vitro skin sensitization test using human cell lines: the human Cell Line Activation Test (h-CLAT). I. Optimization of the h-CLAT protocol.
The aim of this study is to optimize the experimental conditions for an in vitro skin sensitization test using the human cell lines THP-1 and U-937. As regards pre-culturing time, the expression of CD86 on DNCB-treated THP-1 cells tended to be higher after 48h and 72h pre-culture compared with other time points evaluated. Next, we investigated the effect of chemical treatment time, and found that induction of CD86 expression on THP-1 cells by DNCB reached a plateau after 24h. Augmentation of CD86 expression is often observed when cells are treated with a subtoxic dose of allergens. To determine the appropriate dose of test samples, the cytotoxicity of test samples to THP-1 and U-937 cells was assessed with MTT assay, and the 50% inhibitory concentration (IC50) of each test sample was calculated. Based on the cytotoxicity assay data, four concentrations in the range between toxic and non-toxic were selected (0.1x, 0.5x, 1x and 2x IC50). Several kinds of antibodies were tested for staining THP-1 and U-937 cells treated with allergens/non-allergens (e.g., DNCB, Ni/SLS), and suitable antibodies for staining CD86 and CD54 were selected. We confirmed that the working dilutions of the selected CD86 and CD54 antibodies were appropriate for use in our method. The effect of an FcR blocking procedure was also evaluated. The mean fluorescence intensity (MFI value) was decreased by the FcR blocking procedure, which indicated that non-specific staining was blocked. Therefore, this procedure should be included in the method. Based on our findings, the protocol for this assay was optimized and the experimental conditions to be used in a future validation study were identified. We propose to call this kind of in vitro skin sensitization test h-CLAT, which is short for human Cell Line Activation Test.
Allergens↗
Feed-forward facilitation of glutamate release by presynaptic GABA(A) receptors.
Disynaptic GABAergic inputs from Schaffer collateral (SC) afferents on to the soma of glutamatergic CA1 pyramidal neurons are involved in feed-forward inhibition in the hippocampal neural circuits. Here we report the functional roles of presynaptic GABA(A) receptors on SC afferents projecting to CA1 pyramidal neurons. Muscimol (0.5 microM), a selective GABA(A) receptor agonist, increased SC-evoked EPSC amplitude and decreased paired-pulse ratio in the slice preparation, in addition, it facilitated spontaneous glutamate release on to mechanically dissociated CA1 pyramidal neurons in an external Ca2+-dependent manner. In field recordings, muscimol at low concentrations (< or = 0.5 microM) increased not only the excitability of SC afferents but glutamate release, however, it at high concentrations (> or = 1 microM) changed bidirectionally. These results suggest that the moderate activation of presynaptic GABA(A) receptors depolarizes SC afferents and enhances SC-mediated glutamatergic transmission. When endogenous GABA was disynaptically released by brief trains of stimulation of SC afferents, the axonal excitability in addition to glutamate release was increased. The effects of endogenous GABA on the excitability of SC afferents were blocked by either SR95531 or AMPA receptor blockers, which would be expected to block disynaptic feed-forward neural circuits. The present results provide a novel form of presynaptic modulation (feed-forward facilitation) of glutamatergic transmission by presynaptic GABA(A) receptors within the intrinsic hippocampal neural circuits.
Animals↗
Frequent loss of RUNX3 gene expression in remnant stomach cancer and adjacent mucosa with special reference to topography.
Our previous studies suggest that a lack of RUNX3 function is causally related to the genesis and progression of human gastric cancer. This study was conducted to determine whether alteration of RUNX3 gene expression could be detected in the normal-looking gastric remnant mucosa, and to ascertain any difference in the potential of gastric carcinogenesis between the anastomotic site and other areas in the remnant stomach after distal gastrectomy for peptic ulcer (RB group) or gastric cancer (RM group), by analysing RUNX3 expression with special reference to topography. A total of 89 patients underwent distal gastrectomy for gastric cancer from the intact stomach (GCI group) and 58 patients underwent resection of the remnant stomach for gastric cancer (RB group: 34 cases, RM group: 24 cases). We detected RUNX3 and gene promoter methylation by in situ hybridisation, quantitative reverse transcriptase-polymerase chain reaction (RT-PCR), and methylation-specific PCR. The interval between the initial surgery and surgery for remnant gastric cancer (interval time) was 10.4 years in the RM group, and 27.5 years in the RB group. Cancers in the RB group were significantly more predominant in the anastomosis area (P<0.05). Within the tumour, downregulation of RUNX3 expression ranged from 74.7 to 85.7% in the three groups. The rate of downregulation of RUNX3 of adjacent mucosa was 39.2% (11 in 28 cases) in RB and 47.6% (10 in 21 cases) in RM, which are significantly higher than that of the GCI group (19.5%, 17 in 87 cases). In noncancerous mucosa of the remnant stomach in the RB group, RUNX3 expression decreased more near the anastomosis area. In the RM group, however, there were no significant differences in RUNX3 expression by sampling location. Based on RUNX3 downregulation and clinical features, residual stomach mucosa of the RM group would have a higher potential of gastric carcinogenesis compared to the RB or GCI group. Gastric stump mucosa of the RB group has higher potential especially than other areas of residual stomach mucosa. Measurement of RUNX3 expression and detection of RUNX3 methylation in remnant gastric mucosa may estimate the forward risk of carcinogenesis in the remnant stomach.
Aged↗
Intracranial peripheral primitive neuroectodermal tumor manifesting as abducens nerve palsy.
INTRODUCTION: Peripheral primitive neuroectodermal tumor (PNET) occurring as an extra-axial lesion within the intracranial space and extending to the subarachnoid space is extremely rare. CASE REPORT: An 18-month-old girl presented with an intracranial peripheral PNET manifesting as abducens nerve palsy. Magnetic resonance imaging on admission revealed a lesion affecting the trigeminal and abducens nerves. The tumor was partially removed via the subtemporal approach. Histological examination showed a high-grade, undifferentiated neoplasm of small cell type with positive immunostaining for MIC2. The histological diagnosis was peripheral PNET. OUTCOME: Craniospinal radiotherapy reduced the tumor size, but adjuvant chemotherapy designed for Ewing's sarcomas and PNETs was not effective. She died 1 month after the last chemotherapy, despite whole craniospinal irradiation (total dose 53.2 Gy) and chemotherapy.
Abducens Nerve↗
Prenatal diagnosis of fetal hydrometrocolpos secondary to a cloacal anomaly by magnetic resonance imaging.
Fetal female urogenital anomalies are often difficult to evaluate by ultrasonography, especially in late gestation. We report a case of fetal hydrometrocolpos detected at 35 weeks of gestation. Ultrasonography revealed a large retrovesical septate hypoechogenic mass in the fetal abdomen, however the sonographic findings were inconclusive. Magnetic resonance imaging (MRI) confirmed that the abdominal mass was fluid-filled with a mid-plane septum in the midline posterior to the bladder, and showed a connection to the dilated uterus that was duplicated. These findings were consistent with a diagnosis of hydrometrocolpos with septate vagina and uterus didelphys. The neonate showed abdominal distension, ambiguous genitalia and anal atresia with a single perineal opening. Hydrometrocolpos was secondary to a urethral type of cloacal anomaly. Aspiration of the mass and a colostomy were performed on the first postnatal day, followed by anorectoplasty at 19 months of age. MRI is a useful complementary tool for assessing fetal urogenital anomalies when ultrasonography is inconclusive.
Adult↗
Nanowires and nanoribbons of charge-density-wave conductor NbSe3.
We report synthesis of nanowires and nanoribbons of the charge-density-wave conductor NbSe(3) through direct reaction of Nb and Se powders. The transverse dimension of the obtained nanostructures, as identified with scanning/transmission electron microscopy, ranges from 20 to 700 nm. X-ray and selected area electron diffraction analyses indicate that these nanowires and nanoribbons are single crystalline. Four-probe resistivity measurements confirm the expected charge-density-wave transitions, and furthermore, we find significant enhancement in the depinning threshold fields, which we attribute to a confinement effect.
Crystallization↗