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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.

BackgroundThe 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.MethodsAdrenocortical 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.ResultsRTLs 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).ConclusionsThis 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.

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.

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.

“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.

Mucin-producing urothelial-type adenocarcinoma of the prostate (MPUAP) is a rare, aggressive neoplasm thought to originate from the prostatic urethral urothelium. Our case is the first reported to show significantly increased serum levels of carcinoembryonic antigen (CEA) and carbohydrate antigen (CA) 19-9 in MPUAP. An autopsy revealed occult MPUAP with prominent seminal vesicle, perineural, lymphatic vessel, and vascular invasion and extensive bone metastases. Duration of survival in this case was far shorter than average, and extensive bone metastases were rare in previously reported cases. This suggests serum CEA and CA19-9 levels can be an important prognostic factor in MPUAP. Keywords: Mucin-producing urothelial-type adenocarcinoma of prostate, Carcinoembryonic antigen, Carbohydrate antigen 19-9, Prostate-specific antigen

To enrich carotenoids, especially β-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 β-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 β-cryptoxanthin and violaxanthin, in juice sac tissues. Moreover, the pyramiding of these alleles also increased the β-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.

This paper proposes a new parallel approach to solve connected components on a 2-D binary image. The following strategies are employed to accelerate neighborhood exploration after dividing an input image into independent blocks: 1) in the local labeling stage, a coarse-labeling algorithm, including row-column connection and unification, is applied first to reduce the complexity of an initialized local label map; a refinement algorithm is then introduced to merge separated sub-regions from a single component; and 2) in the block merge stage, we scan the pixels on the block boundary instead of solving the connectivity of all the pixels. With the proposed method, the length of label-equivalence lists in both the local labeling stage and global labeling stage are compressed and the number of memory accesses is reduced. Thus, the efficiency of connected component labeling is improved. The proposed strategies are illustrated using 4-neighbor connectivity, and the case of 8-neighbor connectivity is also discussed. The YACCLAB data sets, including both synthetic and real images, are used to evaluate the new algorithm and compare it to existing algorithms. The comparative results show that the proposed new algorithm outperforms the other approaches in both the 4-neighbor connectivity and 8-neighbor connectivity cases.

Carbohydrate antigen 19-9 (CA19-9) is a well-known tumor marker for pancreatobiliary cancer, and several studies have shown that an elevated serum CA19-9 level is associated with more aggressive biological behavior in gastric cancer (GC). However, the clinicopathological characteristics of CA19-9-positive GC remain unclear. We herein report an autopsy case of CA19-9-positive GC in an 84-year-old man who was admitted to our hospital because of paralysis and anemia. Autopsy revealed an ulcerative-invasive tumor measuring 72 × 60 mm in the anterior wall of the gastric body. The tumor had invaded beyond the muscularis propria, and metastasized to the lung, liver, and regional lymph nodes. Histologically, the tumor cell had oval nuclei with abundant clear cytoplasm, and tubular and/or papillary features with prominent lymphovascular permeation and perineural invasion, mimicking pancreatobiliary carcinoma. Immunohistochemically, the tumor cells showed diffuse immunopositivity for CA19-9 and carcinoembryonic antigen. According to a review of cases reported in the literature, CA19-9-positive GCs show clinicopathological characteristics such as antral location, ulcerative-infiltrating gross feature, differentiated histology, prominent lymphatic and venous invasion, higher proportion of metastasis, and higher clinical stage. These results suggest that CA19-9-positive GC is pathologically a distinctive type of tumor with aggressive biological behavior.

Abstract Background Gangliocytic paraganglioma (GP) is a rare neuroendocrine neoplasm, which occurs mostly in the periampullary portion of the duodenum; the majority of the reported cases of duodenal GP has been of benign nature with a low incidence of regional lymph node metastasis. GP arising from the pancreas is extremely rare. To date, only three cases have been reported and its clinical characteristics are largely unknown. Case presentation A nodule located in the pancreatic head was incidentally detected in an asymptomatic 68-year-old woman. Computed tomography revealed 18-, 8-, and 12-mm masses in the pancreatic head, the pancreatic tail, and the left adrenal gland, respectively. Subsequent genetic examination revealed an absence of mutations in the MEN1 and VHL genes. Macroscopically, the tumor located in the pancreatic head was 22 mm in size and displayed an ill-circumscribed margin along with yellowish-white color. Microscopically, it was composed of three cell components: epithelioid cells, ganglion-like cells, and spindle cells, which led to the diagnosis of GP. The tumor was accompanied by a peripancreatic lymph node metastasis. The tumor in the pancreatic tail was histologically classified as a neuroendocrine tumor (NET) G1 (grade 1, WHO 2010), whereas the tumor in the left adrenal gland was identified as an adrenocortical adenoma. The patient was disease-free at the 12-month follow-up examination. Conclusions Pancreatic GP is associated with a higher incidence of metastasis and larger tumor size than duodenal GPs, suggesting that the primary organ of GP is an important prognostic factor.

Abstract Novel genomics-based approaches such as genome-wide association studies (GWAS) and genomic selection (GS) are expected to be useful in fruit tree breeding, which requires much time from the cross to the release of a cultivar because of the long generation time. In this study, a citrus parental population (111 varieties) and a breeding population (676 individuals from 35 full-sib families) were genotyped for 1,841 single nucleotide polymorphisms (SNPs) and phenotyped for 17 fruit quality traits. GWAS power and prediction accuracy were increased by combining the parental and breeding populations. A multi-kernel model considering both additive and dominance effects improved prediction accuracy for acidity and juiciness, implying that the effects of both types are important for these traits. Genomic best linear unbiased prediction (GBLUP) with linear ridge kernel regression (RR) was more robust and accurate than GBLUP with non-linear Gaussian kernel regression (GAUSS) in the tails of the phenotypic distribution. The results of this study suggest that both GWAS and GS are effective for genetic improvement of citrus fruit traits. Furthermore, the data collected from breeding populations are beneficial for increasing the detection power of GWAS and the prediction accuracy of GS.

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.

In the breeding of citrus (Citrus spp.), suitable fruit quality is essential for consumer acceptance of new cultivars. To identify parental combinations producing F1 progeny with fruit-quality traits exceeding certain selection criteria, we developed a simple and practical method for predicting multiple-trait segregation in an F1 progeny population. This method uses breeding values of parental genotypes and an additive genetic (co)variance matrix calculated by the best linear unbiased prediction method to construct a model for trait segregation in F1 progeny. To confirm the validity of our proposed method, we calculated the breeding values and additive genetic (co)variances based on phenotypic records on nine fruit-quality traits in 2122 genotypes, and constructed a trait segregation model. Subsequently, we applied the trait segregation model to all pairs of the 2122 genotypes (i.e., 2,252,503 combinations), and predicted the most promising combinations and evaluated their probabilities of producing superior genotypes exceeding the nine fruit-quality traits of satsuma mandarin (Citrus unshiu Marcow.) or 'Shiranuhi' ('Kiyomi' × 'Nakano No. 3' ponkan), two popular citrus cultivars in Japan. We consider these results to be useful not only for selecting good parental combinations for fruit quality or other important traits but also for determining the scale of breeding programs required to achieve specific breeding goals.

Most indigenous citrus varieties are assumed to be natural hybrids, but their parentage has so far been determined in only a few cases because of their wide genetic diversity and the low transferability of DNA markers. Here we infer the parentage of indigenous citrus varieties using simple sequence repeat and indel markers developed from various citrus genome sequence resources. Parentage tests with 122 known hybrids using the selected DNA markers certify their transferability among those hybrids. Identity tests confirm that most variant strains are selected mutants, but we find four types of kunenbo (Citrus nobilis) and three types of tachibana (Citrus tachibana) for which we suggest different origins. Structure analysis with DNA markers that are in Hardy-Weinberg equilibrium deduce three basic taxa coinciding with the current understanding of citrus ancestors. Genotyping analysis of 101 indigenous citrus varieties with 123 selected DNA markers infers the parentages of 22 indigenous citrus varieties including Satsuma, Temple, and iyo, and single parents of 45 indigenous citrus varieties, including kunenbo, C. ichangensis, and Ichang lemon by allele-sharing and parentage tests. Genotyping analysis of chloroplast and mitochondrial genomes using 11 DNA markers classifies their cytoplasmic genotypes into 18 categories and deduces the combination of seed and pollen parents. Likelihood ratio analysis verifies the inferred parentages with significant scores. The reconstructed genealogy identifies 12 types of varieties consisting of Kishu, kunenbo, yuzu, koji, sour orange, dancy, kobeni mikan, sweet orange, tachibana, Cleopatra, willowleaf mandarin, and pummelo, which have played pivotal roles in the occurrence of these indigenous varieties. The inferred parentage of the indigenous varieties confirms their hybrid origins, as found by recent studies.

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-offertility (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. [ABSTRACT FROM AUTHOR]