Artificial intelligence-based analysis of time-lapse images of sphere formation and process of plate adhesion and spread of pancreatic cancer cells.

Background: Most pancreatic cancers are pancreatic ductal adenocarcinomas (PDAC). Spherical morphology formed in three-dimensional (3D) cultures and the effects of anticancer drugs differ between epithelial and mesenchymal PDAC cell lines. In the human pancreas, cancer cells form 3D tumors, migrate to adjacent tissues, and metastasize to other organs. However, no effective methods exist to examine the ability of the tumor mass to migrate to surrounding tissues in vitro. We used spheres formed in 3D culture to investigate whether the migratory ability of tumors of PDAC cell lines, including epithelial and mesenchymal cell lines, varies. Methods: Sphere formation and adhesion and spread on culture plates were examined by artificial intelligence-based analysis of time-lapse imaging using five epithelial and three mesenchymal PDAC cell lines. Fused and non-fused areas of the sphere surface during sphere formation on low-attachment plates, the adhesion area to normal culture plates, and the sphere area maintaining its original form during adhesion to plates were measured. Results: Immunocytochemical staining confirmed that E-cadherin was highly expressed in epithelial PDAC spheres, as was vimentin in mesenchymal PDAC spheres, in 2D culture. When forming spheres using low-attachment plates, most epithelial PDAC cell lines initially showed decreased sphere area, and then the covering cells fused to form a smooth surface on the sphere. Mesenchymal PANC-1 and MIA PaCa-2 cells showed little reduction in sphere area and few areas of sphere surface fusion. When formed PDAC spheres were seeded onto normal culture plates, spheres of epithelial PK-8 cells-which have the highest E-cadherin expression, form numerous cysts, and have smooth sphere surfaces-did not adhere to normal plates even after 60 h, and epithelial PK45-P and T3M-4 spheres hardly adhered. Conversely, the area of adhesion and spread of mesenchymal PANC-1 and KP4 cell spheres on normal plates markedly increased from early on, forming large areas of attachment to plates. Conclusion: Seeding spheres formed in 3D culture onto culture plates can clarify differences in tumor migration potential to surrounding areas. The masses formed by each PDAC cell line varied in migratory ability, with mesenchymal PDAC masses being more migratory than epithelial PDAC masses.

Accelerated telomere shortening in adrenal zona reticularis in patients with prolonged critical illness.

Background: The number of patients with prolonged critical illness (PCI) has been increasing in many countries, and the adrenal gland plays an important role in maintaining homeostasis during PCI. Chronic disease burden is reportedly associated with shorter telomere lengths in human tissues. Telomere shortening in human somatic cells is largely dependent on cell divisions, and critically short telomeres lead to cellular dysfunction and aging. However, the association between PCI and telomere lengths in human adrenal cells is poorly understood. In this study, we investigated this association to assess whether the burden of PCI could accelerate the aging process in adrenal cells. Methods: Adrenocortical tissues from patients who died after PCI usually show a diffuse pattern of intracellular cholesterol ester depletion (i.e., lipid depletion). This study examined near-normal adrenal glands obtained from autopsied patients who died suddenly (control group) and lipid-depleted adrenal glands obtained from autopsied patients who died after PCI (PCI group). The control group included 7 men aged 80 to 94 years (mean age: 85.3 years) and 7 women aged 84 to 94 years (mean age: 87.7 years). The PCI group included 10 men aged 71 to 88 years (mean age: 78.8 years) and 8 women aged 77 to 95 years (mean age: 85.6 years). By using quantitative fluorescence in situ hybridization, relative telomere lengths (RTLs) were determined in the parenchymal cells of the three adrenocortical zones (zona glomerulosa, zona fasciculata, and zona reticularis [ZR]) and in the chromaffin cells of the medulla. The number of adrenal parenchymal cells was determined by immunohistochemistry and digital image analysis. Results: RTLs in ZR cells were significantly shorter in the PCI group than in the control group for both men and women (P = 0.0001 for men and P = 0.0012 for women). However, RTLs in the remaining three types of adrenal cells did not differ between the control and PCI groups for both men and women. The number of ZR cells was higher in the PCI group than in the control group for both men and women (P < 0.0001 for both men and women). The proportion of the number of ZR cells to the total number of adrenocortical parenchymal cells was also higher in the PCI group than in the control group (P < 0.0001 for both men and women). The Ki-67 proliferation index in ZR cells was higher in the PCI group than in the control group (P = 0.0039 for men and P = 0.0063 for women). Conclusions: This study demonstrated ZR cell-specific telomere shortening in patients with adrenal lipid depletion who died after PCI. Our results suggest that the reactive proliferation of ZR cells accelerates the telomere shortening and aging process in ZR cells in these patients. The results of our study may contribute to the understanding of adrenal aging during PCI.

Allelic haplotype combinations at the MS-P1 region, including P-class pentatricopeptide repeat family genes, influence wide phenotypic variation in pollen grain number through a cytoplasmic male sterility model in citrus.

In citrus breeding programs, male sterility is an important trait for developing seedless varieties. Sterility associated with the male sterile cytoplasm of Kishu mandarin (Kishu-cytoplasm) has been proposed to fit the cytoplasmic male sterility (CMS) model. However, it remains undetermined whether CMS in citrus is controlled by interactions between sterile cytoplasm and nuclear restorer-of-fertility (Rf) genes. Accordingly, mechanisms underlying the control of the wide phenotypic variation in pollen number for breeding germplasm should be elucidated. This study aimed to identify complete linkage DNA markers responsible for male sterility at the MS-P1 region based on fine mapping. Two P-class pentatricopeptide repeat (PPR) family genes were identified as candidates for Rf based on predicted mitochondrial localization and higher expression in a male fertile variety/selected strain than in a male sterile variety. Eleven haplotypes (HT1-HT11) at the MS-P1 region were defined based on genotyping of DNA markers. Association analysis of diplotypes at the MS-P1 region and the number of pollen grains per anther (NPG) in breeding germplasms harboring Kishu-cytoplasm revealed that the diplotypes in this region influenced NPG. Among these haplotypes, HT1 is a non-functional restorer-of-fertility (rf) haplotype; HT2, a less-functional Rf; HT3-HT5 are semi-functional Rfs; and HT6 and HT7 are functional Rfs. However, the rare haplotypes HT8-HT11 could not be characterized. Therefore, P-class PPR family genes in the MS-P1 region may constitute the nuclear Rf genes within the CMS model, and a combination of the seven haplotypes could contribute to phenotypic variation in the NPG of breeding germplasms. These findings reveal the genomic mechanisms of CMS in citrus and will contribute to seedless citrus breeding programs by selecting candidate seedless seedlings using the DNA markers at the MS-P1 region.

Clinicopathological and molecular characteristics of gastric papillary adenocarcinoma.

Papillary adenocarcinoma is defined as carcinoma with a well-defined papillary or villous structure. Despite sharing clinicopathological and morphological features with tubular adenocarcinomas, papillary adenocarcinomas frequently show microsatellite instability. The present study aimed to clarify the clinicopathological features, molecular classification, and programmed death-ligand 1 (PD-L1) expression characteristics of papillary adenocarcinoma, especially tumors with microsatellite instability. We examined the microsatellite status and expression of mucin core proteins and PD-L1 as well as the clinicopathological features in 40 gastric papillary adenocarcinomas. Surrogate immunohistochemical analysis of p53 and mismatch repair proteins along with Epstein-Barr virus-encoded RNA in situ hybridization were performed for molecular classification. Female predominance and frequent microsatellite instability were observed in papillary adenocarcinoma in comparison with tubular adenocarcinoma. The presence of microsatellite instability in papillary adenocarcinoma was significantly correlated with older age, tumor-infiltrating lymphocytes, and Crohn's-like lymphoid reactions. Surrogate examination demonstrated that the genomically stable type (17 cases, 42.5%) was the most common, followed by the microsatellite-unstable type (14 cases, 35%). Among the seven cases showing PD-L1-positive expression in tumor cells, four involved carcinomas with microsatellite instability. These results reveal the clinicopathological and molecular characteristics of gastric papillary adenocarcinoma.

Multiple cystic sphere formation from PK-8 cells in three-dimensional culture.

Three-dimensional (3D) culture of cancer cells mimics the in vivo environment. Recently, we reported that pancreatic ductal adenocarcinoma (PDAC) cell lines with epithelial and mesenchymal features formed differently shaped spheres in 3D culture. However, only PK-8 cells, the epithelial PDAC cell line with the highest E-cadherin expression among the eight PDAC cell lines, formed multiple cystic spheres in 3D culture. Optical coherence tomography revealed interconnected cysts inside the spheres. A weak inter-cellular adhesion, individual cell degeneration, necrosis, and secretory granules in the cytoplasm were observed in the PK-8 spheres using electron microscopy. The expression of MUC1, MUC5AC, and amylase was increased in PK-8 cells in the 3D culture compared with that in 2D culture. These findings suggest that highly E-cadherin-expressing epithelial PK-8 cells form multiple cystic spheres, which may be promoted by enhanced mucin and amylase synthesis in 3D culture.

Epithelial and Mesenchymal Features of Pancreatic Ductal Adenocarcinoma Cell Lines in Two- and Three-Dimensional Cultures.

Pancreatic ductal adenocarcinoma (PDAC) is an intractable cancer that is difficult to diagnose early, and there is no cure other than surgery. PDAC is classified as an adenocarcinoma that has limited effective anticancer drug and molecular-targeted therapies compared to adenocarcinoma found in other organs. A large number of cancer cell lines have been established from patients with PDAC that have different genetic abnormalities, including four driver genes; however, little is known about the differences in biological behaviors among these cell lines. Recent studies have shown that PDAC cell lines can be divided into epithelial and mesenchymal cell lines. In 3D cultures, morphological and functional differences between epithelial and mesenchymal PDAC cell lines were observed as well as the drug effects of different anticancer drugs. These effects included gemcitabine causing an increased growth inhibition of epithelial PDAC cells, while nab-paclitaxel caused greater mesenchymal PDAC cell inhibition. Thus, examining the characteristics of epithelial or mesenchymal PDAC cells with stromal cells using a 3D co-culture may lead to the development of new anticancer drugs.

[Fatal primary influenza virus pneumonia in a patient with secondary myelofibrosis during ruxolitinib therapy].

In recent years, fatal cases of primary influenza virus pneumonia have been rare. A 67-year-old woman with secondary myelofibrosis, who had been diagnosed with polycythemia vera 25 years prior, died of primary influenza virus pneumonia. She was immunocompromised due to the underlying disease and ruxolitinib therapy, but she was not vaccinated against influenza. She might have caught the flu from an infected family member. This case reminds us of the importance of infection control measures such as preventing familial infection during ruxolitinib therapy in severely immunocompromised patients.

Dissecting Breeders' Sense via Explainable Machine Learning Approach: Application to Fruit Peelability and Hardness in Citrus.

"Genomics-assisted breeding", which utilizes genomics-based methods, e.g., genome-wide association study (GWAS) and genomic selection (GS), has been attracting attention, especially in the field of fruit breeding. Low-cost genotyping technologies that support genome-assisted breeding have already been established. However, efficient collection of large amounts of high-quality phenotypic data is essential for the success of such breeding. Most of the fruit quality traits have been sensorily and visually evaluated by professional breeders. However, the fruit morphological features that serve as the basis for such sensory and visual judgments are unclear. This makes it difficult to collect efficient phenotypic data on fruit quality traits using image analysis. In this study, we developed a method to automatically measure the morphological features of citrus fruits by the image analysis of cross-sectional images of citrus fruits. We applied explainable machine learning methods and Bayesian networks to determine the relationship between fruit morphological features and two sensorily evaluated fruit quality traits: easiness of peeling (Peeling) and fruit hardness (FruH). In each of all the methods applied in this study, the degradation area of the central core of the fruit was significantly and directly associated with both Peeling and FruH, while the seed area was significantly and directly related to FruH alone. The degradation area of albedo and the area of flavedo were also significantly and directly related to Peeling and FruH, respectively, except in one or two methods. These results suggest that an approach that combines explainable machine learning methods, Bayesian networks, and image analysis can be effective in dissecting the experienced sense of a breeder. In breeding programs, collecting fruit images and efficiently measuring and documenting fruit morphological features that are related to fruit quality traits may increase the size of data for the analysis and improvement of the accuracy of GWAS and GS on the quality traits of the citrus fruits.

Fast calculation method based on hidden region continuity for computer-generated holograms with multiple cameras in a sports scene.

We propose, to the best of our knowledge, the world's first system capable of fast calculating computer-generated holograms (CGHs) from a large-scale outdoor sports scene captured with multiple RGB cameras. In the system, we introduce a fast calculation method focusing on hidden region continuity (HRC) that frequently appears in a point cloud of a 3D sports scene generated from free-viewpoint video technology. The experimental results show that the calculation time of the proposed HRC method is five to 10 times faster than that of the point-based method, which is one of the common CGH calculation methods.

Allelic composition of carotenoid metabolic genes in 13 founders influences carotenoid composition in juice sac tissues of fruits among Japanese citrus breeding population.

To enrich carotenoids, especially ß-cryptoxanthin, in juice sac tissues of fruits via molecular breeding in citrus, allele mining was utilized to dissect allelic variation of carotenoid metabolic genes and identify an optimum allele on the target loci characterized by expression quantitative trait (eQTL) analysis. SNPs of target carotenoid metabolic genes in 13 founders of the Japanese citrus breeding population were explored using the SureSelect target enrichment method. An independent allele was determined based on the presence or absence of reliable SNPs, using trio analysis to confirm inheritability between parent and offspring. Among the 13 founders, there were 7 PSY alleles, 7 HYb alleles, 11 ZEP alleles, 5 NCED alleles, and 4 alleles for the eQTL that control the transcription levels of PDS and ZDS among the ancestral species, indicating that some founders acquired those alleles from them. The carotenoid composition data of 263 breeding pedigrees in juice sac tissues revealed that the phenotypic variance of carotenoid composition was similar to that in the 13 founders, whereas the mean of total carotenoid content increased. This increase in total carotenoid content correlated with the increase in either or both ß-cryptoxanthin and violaxanthin in juice sac tissues. Bayesian statistical analysis between allelic composition of target genes and carotenoid composition in 263 breeding pedigrees indicated that PSY-a and ZEP-e alleles at PSY and ZEP loci had strong positive effects on increasing the total carotenoid content, including ß-cryptoxanthin and violaxanthin, in juice sac tissues. Moreover, the pyramiding of these alleles also increased the ß-cryptoxanthin content. Interestingly, the offset interaction between the alleles with increasing and decreasing effects on carotenoid content and the epistatic interaction among carotenoid metabolic genes were observed and these interactions complexed carotenoid profiles in breeding population. These results revealed that allele composition would highly influence the carotenoid composition in citrus fruits. The allelic genotype information for the examined carotenoid metabolic genes in major citrus varieties and the trio-tagged SNPs to discriminate the optimum alleles (PSY-a and ZEP-e) from the rest would promise citrus breeders carotenoid enrichment in fruit via molecular breeding.

Clinicopathological characteristics of gastric cancer with carbohydrate antigen 19-9 expression occurring in elderly individuals: An autopsy study.

The clinicopathological significance of carbohydrate antigen 19-9 (CA19-9) in gastric cancer (GC) remains obscure. Therefore, the current study aimed to clarify the clinicopathological value of CA19-9 in GC utilizing autopsy cases. We examined the expression of CA19-9 and mucin core proteins in GC immunohistochemically, and analyzed serum CA19-9 levels and clinicopathological variables or complications. We also investigated whether fucosyltransferases 2 and 3 (FUT2/3) allelic variants influence CA19-9 expression in GC. Compared to GC cases with negative CA19-9 expression (tCA19-9-N), those with positive CA19-9 expression (tCA19-9-P) demonstrated significant differences in characteristic features such as lymph node and distant organ metastases, lymphatic and venous permeation, and higher Tumor, Node, Metastasis (TNM) stages. Moreover, compared to GC cases with low serum CA19-9 levels (sCA19-9-L), those with high serum CA19-9 levels (sCA19-9-H) were related to venous permeation, higher proportion of lymph node and distant organ metastases, and higher TNM stages. Both tCA19-9-P GC and sCA19-9-H GC cases were significantly associated with coagulation abnormalities. sCA19-9-H GC cases correlated significantly with MUC1 and MUC5AC expression. FUT2/3 genotypes were not associated with CA19-9 expression in GC. These results suggest that CA19-9 can predict the risk of lymph node and distant metastases as well as of coagulation abnormalities.

Single-step genomic prediction of fruit-quality traits using phenotypic records of non-genotyped relatives in citrus.

The potential of genomic selection (GS) is currently being evaluated for fruit breeding. GS models are usually constructed based on information from both the genotype and phenotype of population. However, information from phenotyped but non-genotyped relatives can also be used to construct GS models, and this additional information can improve their accuracy. In the present study, we evaluated the utility of single-step genomic best linear unbiased prediction (ssGBLUP) in citrus breeding, which is a genomic prediction method that combines the kinship information from genotyped and non-genotyped relatives into a single relationship matrix for a mixed model to apply GS. Fruit weight, sugar content, and acid content of 1,935 citrus individuals, of which 483 had genotype data of 2,354 genome-wide single nucleotide polymorphisms, were evaluated from 2009-2012. The prediction accuracy of ssGBLUP for genotyped individuals was similar to or higher than that of usual genomic best linear unbiased prediction method using only genotyped individuals, especially for sugar content. Therefore, ssGBLUP could yield higher accuracy in genotyped individuals by adding information from non-genotyped relatives. The prediction accuracy of ssGBLUP for non-genotyped individuals was also slightly higher than that of conventional best linear unbiased prediction method using pedigree information. This indicates that ssGBLUP can enhance prediction accuracy of breeding values for non-genotyped individuals using genomic information of genotyped relatives. These results demonstrate the potential of ssGBLUP for fruit breeding, including citrus.

Correlation Between Differentiation of Adrenocortical Zones and Telomere Lengths Measured by Q-FISH.

CONTEXT: Adrenocortical zonation is associated with a markedly complex developmental process, and the pathogenesis and/or etiology of many disorders of adrenocortical zonal development have remained unknown. Cells from the three adrenocortical zones are morphologically and functionally differentiated, and the mature stage of cell development or senescence has been recently reported to be correlated with telomere length. However, the telomere length of each adrenocortical zonal cell has not yet been studied in human adrenal glands. OBJECTIVE: We aimed to study the telomere lengths of adrenocortical parenchymal cells from three different zones of the adrenal glands present during childhood, adolescence, and adulthood. METHODS: Adrenal glands of 30 autopsied subjects, aged between 0 and 68 years, were retrieved from pathology files. The normalized telomere to centromere ratio (NTCR), an index of telomere length, was determined in the parenchymal cells of the zona glomerulosa, zona fasciculata, and zona reticularis (ZR), using quantitative fluorescence in situ hybridization. RESULTS: NTCR of ZR cells was the longest, followed in decreasing order by that of zona glomerulosa and zona fasciculata cells in subjects aged 20 to 68 years, but no substantial differences in NTCR were detected among these three zones in the group <20 years of age. NTCR of ZR increased with age in subjects aged 20 to 68 years, whereas no important age-dependent changes in NTCR were detected in the group <20 years of age. CONCLUSION: The telomere lengths for three zones in adrenal cortex were correlated with their differentiation in adulthood but not in childhood and adolescence.

Acute Liver Failure Associated with Influenza A Virus Infection: an Autopsy Case Report.

An 84-year-old man with chronic renal failure, anemia, and diabetes was admitted for hemodialysis initiation. His vital signs were stable until the eighteenth hospital day, before acquiring an influenza A virus infection. Three days later, he died of septic shock with severe liver impairment. His leukocyte count, prothrombin time (PT-INR), and liver enzyme levels such as aspartate transaminase and alanine aminotransferase, were significantly increased. Hypercytokinemia was also observed. Autopsy revealed bilateral diffuse pneumonia with neutrophil infiltration. The liver showed extensive centrilobular hepatocyte necrosis. Immunohistochemistry for influenza A nucleoprotein revealed positivity in the ciliated columnar epithelium of the bronchi and negativity in the trachea, lungs, and liver. Hypoxic hepatitis is characterized by an abrupt and massive increase in aminotransferase levels (＞ 20 times upper normal limit) due to anoxic centrilobular hepatocyte necrosis. The occurrence of hypoxic hepatitis requires a pre-existing, chronic condition, such as anemia, causing reduced oxygen supply to the liver, followed by an acute decrease in hepatic oxygen supply, such as septic shock. Therefore, this report suggests that hypoxic hepatitis can be an important causative factor for acute liver failure associated with influenza virus infection.

Estimation of Outlet Temperature of a Flow Reactor Heated by Microwave Irradiation.

Flow reactors heated by microwave irradiation attract attention. The reactors are suitable for difficult synthesis processes due to rapid heating and cooling, and easy pressurization. In order to predict the quality of the product, it is appropriate to estimate the outlet conditions of the reactor. In this paper, the outlet temperature of the flow direction is estimated by using the flow condition and dynamic thermal energy balance of the reactor.

[An elderly case of squamous cell lung cancer treated continuously with pembrolizumab without any decline in the life function].

BACKGROUND: Whether or not immune checkpoint inhibitors are safe and effective for the elderly remains unclear, even though these drugs were approved two years ago for the treatment of advanced non-small cell lung cancer in Japan. Older cancer patients are vulnerable to chemotherapy, so their life function should be closely monitored before starting, continuing, or discontinuing cancer treatment. CASE: An 85-year-old man showed a wedge-shaped shadow in the apical portion of the left lung on chest computed tomography. Unfortunately, repeated bronchoscopy revealed no malignancy. The shadow progressed over about one year, so a third bronchoscopy was performed, leading to a diagnosis of squamous-cell carcinoma (cT3N2M1a, stage IVA). Because PD-L1 immunohistochemical staining was positive for 80%-90% of the tumor cells, the patient was treated with pembrolizumab as the first-line therapy, and the tumor dramatically regressed, notably without any decline in the patient's life functions. CONCLUSION: An elderly case of squamous-cell lung cancer was treated continuously with pembrolizumab without any decline in the patient's life function.