Palatability and Bio-Functionality of Chalky Grains Generated by High-Temperature Ripening and Development of Formulae for Estimating the Degree of Damage Using a Rapid Visco Analyzer of Japonica Unpolished Rice.

Global warming inhibits grain filling in rice and leads to chalky grains, which are damaged in physical and cooking qualities. In the present paper, we evaluated 54 Japonica unpolished rice grains harvested in Japan in 2020, and these samples (original grains) were divided to two groups (whole grains and chalky grains). Using rice grains of 100% whole grains or those blended with 30% of chalky grains, we measured contents of sugars and amino acids, and textural properties of boiled rice grains. It was shown that the α-amylase activity and proteinase activity of raw chalky rice were significantly higher than those of whole rice grains, which led to the significant increase of low-molecular-weight sugars and free amino acids after boiling. Furthermore, hardness and toughness of the boiled rice grains were decreased markedly by blending chalky grains. The ratio of α-amylase activity of chalky grains to that of whole grains was shown to be a useful indicator for damage degree by high-temperature ripening. It became possible to estimate the degree of high-temperature damage of rice grains based on only the pasting properties of unpolished rice.

Evaluation of Hardness and Retrogradation of Cooked Rice Based on Its Pasting Properties Using a Novel RVA Testing.

With rice being one of the most important crops worldwide, rapid and objective quality evaluation methods based on physicochemical measurements of rice are necessary. We compared the pasting properties of various rice samples using three different heating and cooling programs (maximum temperatures were 93, 120, and 140 °C, respectively) in a newly developed high-temperature-type Rapid Visco Analyzer (RVA , RVA 4800). Furthermore, we investigated the relationship between the different pasting properties measured by the three programs, with starch microstructure measured by iodine scanning analysis, the physical properties of the cooked rice measured by a Tensipresser after 2 h at 25 °C or after 24 h at 6 °C, and prolamin ratio measured by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The consistency value (final viscosity-minimum viscosity) yielded by a new program of maintenance for 2 min at 120 °C using RVA 4800 had a higher positive correlation with retrograded surface hardness H1(R) (r = 0.92), retrograded overall hardness H2(R) (r = 0.90), and the absorbance at λmax (Aλmax) of cooked rice (r = 0.88) and resistant starch (r = 0.80) than those by the conventional program at 93 °C. We developed estimation formulae for H1(R) for various kinds of rice, of which the determination coefficient was 0.86. It led to an easy and rapid assay method for the cooking properties of the various rice samples.

Characterization of LGR5 expression in poorly differentiated colorectal carcinoma with mismatch repair protein deficiency.

BACKGROUND: Leucine-rich repeat-containing G-protein-coupled receptor 5 (LGR5) is a promising intestinal stem cell and carcinoma stem cell marker. We examined the relationship between mismatch repair (MMR) protein deficiency and LGR5 expression in poorly differentiated (PD) colorectal carcinoma (CRC). METHODS: In 29 cases of PD-CRC, deficiencies in MMR proteins (MLH1, PMS2, MSH2, MSH6) and ß-catenin expression were identified by immunohistochemistry (IHC). LGR5 expression was examined by the RNAscope assay in tissue microarrays. RESULTS: LGR5 H-scores in MMR-deficient (MMR-D) cases were significantly lower than those in MMR-proficient (MMR-P) cases (P = 0.0033). Nuclear ß-catenin IHC scores in MMR-D cases were significantly lower than those in MMR-P cases (P = 0.0024). In all cases, there was a positive correlation between LGR5 H-score and nuclear ß-catenin IHC score (r = 0.6796, P < 0.001). Even in MMR-D and MMR-P cases, there was a positive correlation between LGR5 H-score and nuclear ß-catenin IHC score (r = 0.7180, P < 0.0085 and r = 0.6574, P < 0.003, respectively). MMR-D CRC cases showed low expression of LGR5, which may be due to low activation of the Wnt/ß-catenin signaling pathway. CONCLUSIONS: Our results reveal the relationship between LGR5 expression and MMR protein profiles in PD-CRC. A further study is warranted to confirm these findings.

Leucine-rich repeat-containing G-protein-coupled receptor 5 expression and clinicopathological features of colorectal neuroendocrine neoplasms.

LGR5 is expressed in various tumors and has been identified as a putative intestinal stem cell marker. Here we investigated LGR5 expression in colorectal neuroendocrine neoplasms and analyzed the correlation with pathological characteristics. We evaluated the clinicopathological features of 8 neuroendocrine tumor (NET) grade 1 (NET G1), 4 NET Grade 2 (NET G2), and 8 NET Grade 3 (NET G3; also termed neuroendocrine carcinoma, or NEC) cases. We examined LGR5 expression using an RNAscope, a newly developed RNA in situ hybridization technique, with a tissue microarray of the neuroendocrine neoplasm samples. LGR5 staining in individual tumor cells was semi-quantitatively scored using an H-score scale. We also performed a combination of LGR5 RNA in situ hybridization and synaptophysin immunohistochemistry. All cases contained tumor cells with some LGR5-positive dots. For all cases, H-scores showed a positive correlation with nuclear beta-catenin expression. In the NEC group, there was a strong positive correlation between H-score and beta-catenin expression. Our findings suggest that LGR5 may serve as a stem cell marker in NEC, as is the case in colon adenocarcinoma. The positive correlation between H-score and beta-catenin expression suggests that LGR5 expression might be affected by beta-catenin expression in neuroendocrine neoplasms and especially in NEC.

Promoter hypomethylation of SKI in autoimmune pancreatitis.

The relationship between methylation abnormality and autoimmune pancreatitis (AIP)-a representative IgG4-related disease-has not yet been elucidated. We identified SKI might have a significant methylation abnormality in AIP through methylation array analysis using the Illumina Infinium Human Methylation 450K BeadChip array, and investigated the relationship of SKI with AIP clinicopathological features. The methylation rate of SKI was assessed by quantitative SYBR green methylation-specific PCR, and the degree of SKI expression in tissue specimens was assessed by immunohistochemistry in 10 AIP cases, 14 cases of obstructive pancreatitis area in pancreatic ductal adenocarcinoma (PDA) without a history of AIP, and 9 normal pancreas (NP) cases. The SKI methylation ratio was significantly lower in AIP than in PDA and NP. Additionally, the immunohistochemical staining-index (SI) score for SKI was significantly higher in AIP than NP, although there was no significant difference between AIP and PDA. There was a strong negative correlation between SI score and SKI methylation ratio, and between the serum concentrations of IgG4 and the SKI methylation ratio. There was a moderate positive correlation between the serum concentrations of IgG4 and SI. SKI is thought to be an oncogene indicating that SKI hypomethylation and carcinogenesis might be linked to AIP. Furthermore, the correlation between serum concentrations of IgG4 and SKI methylation levels suggest SKI might be involved in the pathogenesis of AIP. However, the role of SKI has not been clearly elucidated. Further studies are needed to understand further the function of SKI.

Relapsed and unresectable inflammatory myofibroblastic tumor responded to chemotherapy: A case report and review of the literature.

A 63-year-old female patient who had undergone cholecystectomy for inflammatory myofibroblastic tumor (IMT) in the gallbladder was referred to our hospital. The patient's disease relapsed, involving the pancreas, and was diagnosed as inoperable IMT 13 months after the cholecystectomy. The patient failed to respond to steroid and non-steroidal anti-inflammatory drug therapy, but subsequently exhibited a good response to vinorelbine and methotrexate combination chemotherapy. Little information is currently available on the efficacy of chemotherapy for adult-onset IMT. The present case suggests that chemotherapy with vinorelbine and methotrexate is a viable therapeutic option for adult patients with unresectable IMT.

Methylation of Tumor Suppressor Genes in Autoimmune Pancreatitis.

OBJECTIVES: Autoimmune pancreatitis (AIP) is a representative IgG4-related and inflammatory disease of unknown etiology. To clarify mechanisms of carcinogenesis resulting from AIP, we focused on methylation abnormalities and KRAS mutations in AIP. METHODS: Six tumor suppressor genes (NPTX2, Cyclin D2, FOXE1, TFPI2, ppENK, and p16) that exhibited hypermethylation in pancreatic carcinoma were selected for quantitative SYBR green methylation-specific polymerase chain reaction in 10 AIP specimens, 10 pancreatic adenocarcinoma cases without history of AIP containing carcinoma areas (CAs) and noncarcinoma areas (NCAs), and 11 normal pancreas (NP) samples. KRAS mutation in codons 12, 13, and 61 were also investigated using direct sequencing. RESULTS: Hypermethylation events (≥10%) were identified in NPTX2, Cyclin D2, FOXE1, TFPI2, ppENK, and p16 in 1, 2, 2, 0, 2, and 0 CA cases, respectively, but not in these 6 candidate genes in AIP, NCA, and NP. However, the TFPI2 methylation ratio was significantly higher in AIP than NCA and NP. Direct sequencing results for KRAS showed no single-point mutations in AIP. CONCLUSIONS: These are the first studies characterizing methylation abnormalities in AIP. AIP's inflammatory condition may be related to carcinogenesis. Further study will elucidate methylation abnormalities associated with carcinogenesis in AIP.

Distribution of Lgr5-positive cancer cells in intramucosal gastric signet-ring cell carcinoma.

Leucine-rich repeat-containing G-protein-coupled receptor 5 (Lgr5) is a putative intestinal stem cell marker that is also expressed in various tumors. To analyze its pathological characteristics in mucosal gastric signet-ring cell carcinoma (SRCC), we investigated Lgr5 expression in 35 intramucosal gastric SRCC patients using RNAscope, a newly developed RNA in situ hybridization technique. Lgr5 expression in individual tumor cells was scored semi-quantitatively from 0 to 400. Ki67 was also examined by immunohistochemistry, with a linear arrangement of Ki67-expressing cells present in 20 of 35 cases. This area of Ki67-expressing cells was topographically divided into upper, middle, and lower regions. All cases with linear Ki67 expression patterns also had Lgr5-positive cells arranged in a linear fashion in the lower area-which was distinct from the area of high Ki67 expression. The rate of Ki67 positivity in Lgr5-positive cells was significantly lower than that of Lgr5-negative cells in areas of high Ki67 expression. In intramucosal SRCC, the low mitotic activity of Lgr5-positive cells suggests that they may represent cancer stem cells as seen in other types of stomach carcinomas. Intramucosal SRCC may therefore contain stem cells expressing Lgr5 in the lower area of the lamina propria, akin to normal gastric pyloric mucosa.

Mucolipidosis IV: A milder form with novel mutations and serial MRI findings.

BACKGROUND: Mucolipidosis IV (MLIV; OMIM #252650) is an autosomal recessive lysosomal storage disorder, frequently observed in the Ashkenazi Jewish population. MLIV typically results in intellectual disability, corneal opacities, and delayed motor milestones during infancy, with a relatively static course. To date, reports of MLIV in other ethnic groups have been sparse. PATIENT: The present study is a case report of a 9-year-old Japanese boy, diagnosed via whole-exome sequencing, with compound heterozygous mutations of MCOLN1 (OMIM(*)605248): c.410T>C (p.Leu137Pro) and c.802_803delAG (p.Ser268Trpfs*17). Although his clinical course was mild (due to a lack of corneal clouding), other relevant features were present. These included strabismus, white matter signal abnormalities, and a hypoplastic corpus callosum at 2years of age. After a molecular diagnosis, a markedly elevated serum gastrin level (which is also common in MLIV) was confirmed. DISCUSSION: The present results suggest that MLIV could be added as a differential diagnosis for white matter disorders, regardless of ethnicity. Beyond neurological or ophthalmologic findings, serum gastrin could be a useful diagnostic marker for MLIV.

Clinicopathological characterisation of duodenal adenocarcinoma with high CD44 variant 9 expression.

CD44 has been identified as a cancer stem cell (CSC) biomarker in various malignancies. The aim of this study was to elucidate the clinicopathological features of CD44v9 positive cells in duodenal adenocarcinoma (DA).Twenty-nine DA patients were selected from medical archives at our hospital. CD44v9 expression was analysed together with that of MUC2, MUC5AC, and MUC6 by immunohistochemistry. High levels of CD44v9 expression weakly correlated with inflammatory cell infiltration (r = 0.431, p = 0.020) and MUC6 expression (r = 0.425, p = 0.022). Furthermore, double immunofluorescence staining of CD44v9 and Ki-67 or cleaved caspase 3 (CC3) was performed. High- and low-density areas of CD44v9 positive cells were designated as CD44v9 positive and negative areas, respectively. Within CD44v9 positive areas, the level of Ki-67 positivity among CD44v9 positive cells was significantly lower than that of CD44v9 negative cells (p = 0.002). Meanwhile, the level of CC3 positivity among CD44v9 positive cells was significantly lower than that of CD44v9 negative cells (p < 0.001).CD44v9 expression may be affected by mononuclear cell infiltration and MUC6 expression in DA. CD44v9 positive cells also have a low mitotic activity and resist apoptosis. These characteristics suggest that CD44v9 positive cells possess CSC-like properties in DA.

Two Cases of Thymic Carcinoma Initially Presenting as Bone Metastasis: A Clinical Report and the Usefulness of CD5 Immunohistochemistry for Assessing Bone Lesions.

Thymic carcinoma frequently spreads to the pleural space, regional lymph nodes, liver and lungs. However, an initial clinical presentation involving spinal or multiple bone metastases in patients with thymic carcinoma is extremely rare. We experienced two cases of thymic carcinoma that initially presented with spinal compression and severe pain due to multiple bone metastases, respectively. Both patients were histologically diagnosed with metastatic thymic squamous cell carcinoma based on the findings of specimens resected from the metastatic bone lesions. We herein describe the clinical courses of these cases and review the characteristics of bone metastasis of thymic carcinoma.

Insulin-Like Growth Factor II mRNA-Binding Protein 3 (IMP3) as a Useful Immunohistochemical Marker for the Diagnosis of Adenocarcinoma of Small Intestine.

The biological characteristics and roles of insulin-like growth factor II mRNA-binding protein 3 protein (IMP3) expression in small-intestinal adenocarcinoma were investigated. The value of IMP3 immunostaining in the diagnosis of small-intestinal epithelial lesions was also evaluated. Immunohistochemical expression of IMP3 in normal small-intestinal mucosa adjacent to adenoma and adenocarcinoma lesions, and inflamed duodenal and ileal mucosa was analyzed. Samples assessed were: duodenal ulcer (n=6), Crohn's disease (n=5), low-grade small-intestinal adenoma (n=10), high-grade small-intestinal adenoma (n=13), small-intestinal adenocarcinoma (n=23), lymph node metastases (LNM; n=7), and preoperative biopsies of small-intestinal adenocarcinoma (n=6). Immunohistochemical expression of Ki-67 and p53 was also analyzed in adenoma and adenocarcinoma samples. IMP3 was not expressed in normal epithelium, but weakly expressed in reparative epithelium. Meanwhile, increased IMP3 expression was associated with a higher degree of dysplasia in adenomas, higher T classification, LNM, Ki-67 positivity, histological differentiation, and lower 5-year disease-free survival, but not p53 expression in adenocarcinoma. IMP3 expression appears to be a late event in the small-intestinal carcinogenesis. Assessing the IMP3 staining pattern can be useful in the diagnosis of small-intestinal epithelial lesions when used in conjunction with other histological criteria.

Murine leukemia retrovirus integration induces the formation of transcription factor complexes on palindromic sequences in the signal transducer and activator of transcription factor 5a gene during the development of pre-B lymphomagenesis.

Murine leukemia retrovirus (MLV) vectors are highly effective tools for introducing a foreign gene into a target host genome. However, it remains unclear how integrated retroviral promoter activity is influenced by the upstream or downstream sequences and how the host cell phenotype is influenced by the integrated promoter activity. Herein, we analyzed a set of pre-B lymphoma clones in which the MLV genome was integrated into the signal transducer and activator of transcription factor 5a (Stat5a) gene. Among the clones, the lymphoma clones with a provirus integrating into the middle position of the palindromic target sequences showed significantly higher transcription of the Stat5a gene; and p300 and other transcriptional factors formed complexes, with binding to the proviral-host junctional DNA segment. By using a luciferase assay, the upstream and downstream sequences of the provirus contributed to the up-regulation of proviral promoter activity. In concomitance with the higher Stat5a transcription, the immunoglobulin gene recombination was arrested. Antiapoptotic activity was significantly higher, with an increase in Bcl-xL, one of the targets of STAT5A, when IL-7 was supplied. Thus, a minute difference between MLV integration sites can lead to large differences in the host phenotype through the formation of transcription factor complexes on the proviral-host junctional DNA segment, suggesting that caution is necessary in monitoring integration sites when working with MLV vectors.

Dual retrovirus integration tagging: identification of new signaling molecules Fiz1 and Hipk2 that are involved in the IL-7 signaling pathway in B lymphoblastic lymphomas.

IL-7R, FLT3, and CD43 are surface antigens expressed during the transition from pro-B to pre-B cells in BM. To understand interactions between their signaling pathways, we analyzed spontaneous mouse B-LBLs with dual MLV integration into Stat5a and Fiz1 or Stat5a and Hipk2. MLV integration resulted in up-regulation of these genes in lymphoma cells compared with normal pro-B cells from the BM. In lymphomas with both integrations into Stat5a and Fiz1, increases in phosphorylated STAT5A and expression of c-Myc, a target gene of STAT5A, were observed following stimulation of the FLT3. Clones with the dual integrations grew faster in IL-7 and FLT3L-supplemented medium than clones with Stat5a integration alone. On the other hand, in lymphomas with integrations into Stat5a and Hipk2, increases in phosphorylated STAT5A and expression of c-Myc were observed following cross-linking of CD43. In conclusion, FLT3 and CD43 signaling pathways involve STAT5A via Fiz1 and Hipk2 in B-LBLs. Identification of the dual MLV integration sites in B-LBLs, therefore, will provide an excellent tool for identification of the signaling pathways in B-LBLs.

Dependence on the electric power of the immersion-angle dependence of the resonant-frequency shift of a quartz crystal microbalance in a liquid.

We have investigated the immersion-angle dependence of the series resonant-frequency shift, DeltaF, of the quartz crystal microbalance, QCM, in a Newtonian liquid from the point of view of the supplied electric power level. In the low electric power levels, the immersion-angle dependence and the transition phenomenon of the DeltaF are observed. However, when the higher electric power levels are supplied to the QCM, the region of the transition phenomenon of the DeltaF decreases rapidly with an increase in the electric power level and disappears above 1.5 dBm. That is, above 1.5 dBm, the DeltaF values have only the values of 90 degrees immersion angle in all immersion angles. We suggest that the electric power is very important factor for the DeltaF in a liquid.

Kruppel-like factor 6 (KLF6) affects the promoter activity of the alpha1-proteinase inhibitor gene.

PURPOSE: Keratoconus is a progressive disease that thins and scars the cornea. In keratoconus corneas, levels of degradative enzymes, including lysosomal acid phosphatase (LAP) and cathepsin B, are elevated, and those of inhibitors alpha1-proteinase inhibitor (alpha1-PI) and alpha2-macroglobulin (alpha2-M) are reduced. The present study explored the possible involvement in keratoconus of Krüppel-like factor 6 (KLF6), a transcription factor previously described to be essential for the integrity of the corneal epithelium. The transcript and proteins level of KLF6 and its action in regulating the genes affected in keratoconus were examined in this study. METHODS: Semiquantitative RT-PCR, Western blot analysis, immunofluorescence and in situ hybridization were used to investigate the expression of KLF6 mRNA and protein in normal and keratoconus corneas. Modulation by KLF6 of the promoter activity of alpha1-PI, LAP, cathepsin B, and alpha2-M genes was studied after transient transfection of KLF6 expression plasmid into corneal epithelial cells using promoter-reporter gene assays. Chromatin immunoprecipitation (ChIP) assays were performed to confirm the interactions between KLF6 and promoters of the genes affected in keratoconus. RESULTS: A global increased expression of the transcription factor KLF6 in terms of mRNAs and proteins was observed in total cornea and/or the epithelium in a substantial number of the keratoconus specimens. The promoter activity of the human alpha1-PI gene was suppressed by expression of KLF6 in corneal epithelial cells. The ChIP assay confirmed a physical interaction between KLF6 and the alpha1-PI promoter. CONCLUSIONS: Transcription factor KLF6 downregulates the alpha1-PI gene in corneal epithelial cells and may thereby be involved in keratoconus.

Tomographic features of serous retinal detachment in Vogt-Koyanagi-Harada syndrome.

To clarify the nature of serous retinal detachment in Vogt-Koyanagi-Harada (VKH) syndrome, 42 consecutive eyes of 21 patients with acute phase VKH syndrome were examined using optical coherence tomography (OCT). OCT revealed two patterns of serous retinal detachment. Twenty-nine eyes (69%) had a true retinal detachment, 17 eyes (40%) had intraretinal fluid accumulation in the outer retina, and 4 eyes had both. Intraretinal fluid accumulation appeared as an oval space in the outer retina. On fluorescein angiography, the eyes with intraretinal fluid accumulation showed more severe dye leakage from the retinal pigment epithelium.

Mapping of Sp1 regulation sites in the promoter of the human alpha1-proteinase inhibitor gene.

Keratoconus is a potentially blinding disease that thins the central cornea. In afflicted corneas, the level of an inhibitor, alpha1-proteinase inhibitor (alpha1-PI), is found reduced. An increased expression of transcription factor Sp1 is also demonstrated. To examine the role of Sp1 in regulation of the human alpha1-PI gene, a 1.4-kb (-1397/+9) 5'-flanking promoter sequence that contains 10 Sp1 sites was cloned. Previous transient transfection experiments showed that Sp1 expression indeed repressed the alpha1-PI promoter activity. In this study, 12 DNA segments, a series of 5', 3', and internal deletions of the 1.4-kb alpha1-PI promoter sequence, were ligated into the SEAP (secreted alkaline phosphatase) reporter gene vector and transfected into human corneal stromal cells. Co-transfection with a Sp1 expression vector pPacSp1 was also performed in parallel. The SEAP enzyme activity was assayed. A fragment with 489 bp (-480/+9) of the 3' sequence, and three fragments with internal deletions, were found to confer a majority of the full promoter activity. Other deletions significantly abolished the promoter activity. Site-directed mutagenesis experiments further revealed that the most proximal Sp1 site (-100/-87) may be an essential element involved in the negative regulation of alpha1-PI promoter activity by Sp1. Interaction between the proximal and distal Sp1 sites also seemed to be important. These results provide the first in-depth characterization of the transcription mechanisms regulating the expression of alpha1-PI. Mapping of the Sp1 sites may help elucidate the molecular pathway leading to the alterations observed in keratoconus.

Reporter gene construct containing 1.4-kB alpha 1-proteinase inhibitor promoter confers expression in the cornea of transgenic mice.

A 1.4-kb human alpha1-proteinase inhibitor (alpha1-PI) 5'-flanking sequence fused to the E. coli LacZ gene was used to generate transgenic mice. The 1.4-kb alpha 1-PI fragment was found to target LacZ expression preferentially in the epithelium and stroma of the mouse cornea, and moderately or weakly in white blood cells and a few other tissues, such as the skin and brain. This finding implies that the alpha 1-PI promoter may offer an option for targeting foreign genes in both the epithelial and stromal layers of the cornea in future transgenic experiments.