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Understanding of the genetic control of female reproductive performance in pigs would offer the opportunity to utilize natural variation and improve selective breeding programs through marker-assisted selection. The Chinese Meishan is one of the most prolific pig breeds known, farrowing 3 to 5 more viable piglets per litter than Western breeds. This difference in prolificacy is attributed to the Meishan's superior prenatal survival. Our study utilized a 3-generation resource population, in which the founder grandparental animals were purebred Meishan and Duroc pigs, in a genome scan for QTL. Grandparent, [F.sub.1], and [F.sub.2] animals were genotyped for 180 microsatellite markers. Reproductive traits, including number of corpora lutea (number of animals = 234), number of fetuses per animal (n = 226), number of teats (n = 801), and total number born (n = 288), were recorded for F2 females. Genome-wide significance level thresholds of 1, 5, and 10% were calculated using a permutation approach. We identified 9 QTL for 3 traits at a 10% genome-wise significance level. Parametric interval mapping analysis indicated evidence of a 1% genome-wise significant QTL for corpora lutea on SSC 3. Nonparametric interval mapping for number of teats found 4 significant QTL on chromosomes SSC3 (P < 0.01), SSC7 (P < 0.01), SSC8 (P < 0.01), and SSC12 (P < 0.05). Partial imprinting of a QTL affecting teat number (P < 0.10) was detected on SSC8. Using the likelihood-ratio test for a categorical trait, 2 QTL for pin nipples were detected on SSC2 and SSC16 (P < 0.01). Fine mapping of the QTL regions will be required for their application to introgression programs and gene cloning. Key words: corpora lutea, imprinting, number of teats, pig, quantitative trait loci

We constructed a pig F2 resource population by crossing a Meishan sow and a Duroc boar to locate economically important trait loci. The F2 generation was composed of 865 animals (450 males and 415 females) from four F1 males and 24 F1 females and was genotyped for 180 informative microsatellite markers spanning 2,263.6 cM of the whole pig genome. Results of the genome scan showed evidence for significant quantitative trait loci ( Key Words: Growth, Linkage Analysis, Meat Quality, Pigs, Quantitative Trait Loci

Understanding of the genetic control of female reproductive performance in pigs would offer the opportunity to utilize natural variation and improve selective breeding programs through marker-assisted selection. The Chinese Meishan is one of the most prolific pig breeds known, farrowing 3 to 5 more viable piglets per litter than Western breeds. This difference in prolificacy is attributed to the Meishan's superior prenatal survival. Our study utilized a 3-generation resource population, in which the founder grandparental animals were purebred Meishan and Duroc pigs, in a genome scan for QTL. Grandparent, F1, and F2 animals were genotyped for 180 microsatellite markers. Reproductive traits, including number of corpora lutea (number of animals = 234), number of fetuses per animal (n = 226), number of teats (n = 801), and total number born (n = 288), were recorded for F2 females. Genome- wide significance level thresholds of 1, 5, and 10% were calculated using a permutation approach. We identified 9 QTL for 3 traits at a 10% genome-wise significance level. Parametric interval mapping analysis indicated evidence of a 1% genome-wise significant QTL for corpora lutea on SSC 3. Nonparametric interval mapping for number of teats found 4 significant QTL on chromosomes SSC3 (P < 0.01), SSC7 (P < 0.01), SSC8 (P < 0.01), and SSC12 (P < 0.05). Partial imprinting of a QTL affecting teat number (P < 0.10) was detected on SSC8. Using the likelihood-ratio test for a categorical trait, 2 QTL for pin nipples were detected on SSC2 and SSC16 (P < 0.01). Fine mapping of the QTL regions will be required for their application to introgression programs and gene cloning. [ABSTRACT FROM AUTHOR]

Spaceflight induces hepatic damage, partially owing to oxidative stress caused by the space environment such as microgravity and space radiation. We examined the roles of anti-oxidative sulfur-containing compounds on hepatic damage after spaceflight. We analyzed the livers of mice on board the International Space Station for 30 days. During spaceflight, half of the mice were exposed to artificial earth gravity (1 g) using centrifugation cages. Sulfur-metabolomics of the livers of mice after spaceflight revealed a decrease in sulfur antioxidants (ergothioneine, glutathione, cysteine, taurine, thiamine, etc.) and their intermediates (cysteine sulfonic acid, hercynine, N-acethylserine, serine, etc.) compared to the controls on the ground. Furthermore, RNA-sequencing showed upregulation of gene sets related to oxidative stress and sulfur metabolism, and downregulation of gene sets related to glutathione reducibility in the livers of mice after spaceflight, compared to controls on the ground. These changes were partially mitigated by exposure to 1 g centrifugation. For the first time, we observed a decrease in sulfur antioxidants based on a comprehensive analysis of the livers of mice after spaceflight. Our data suggest that a decrease in sulfur-containing compounds owing to both microgravity and other spaceflight environments (radiation and stressors) contributes to liver damage after spaceflight., (© 2021. The Author(s).)

Euglena gracilis is a green photosynthetic microalga that swims using its flagellum. This species has been used as a model organism for over half a century to study its metabolism and the mechanisms of its behavior. The development of mass-cultivation technology has led to E. gracilis application as a feedstock in various products such as foods. Therefore, breeding of E. gracilis has been attempted to improve the productivity of this feedstock for potential industrial applications. For this purpose, a characteristic that preserves the microalgal energy e.g., reduces motility, should be added to the cultivars. The objective of this study was to verify our hypothesis that E. gracilis locomotion-defective mutants are suitable for industrial applications because they save the energy required for locomotion. To test this hypothesis, we screened for E. gracilis mutants from Fe-ion-irradiated cell suspensions and established a mutant strain, M 3 - ZFeL, which shows defects in flagellum formation and locomotion. The mutant strain exhibits a growth rate comparable to that of the wild type when cultured under autotrophic conditions, but had a slightly slower growth under heterotrophic conditions. It also stores 1.6 times the amount of paramylon, a crystal of β -1,3-glucan, under autotrophic culture conditions, and shows a faster sedimentation compared with that of the wild type, because of the deficiency in mobility and probably the high amount of paramylon accumulation. Such characteristics make E. gracilis mutant cells suitable for cost-effective mass cultivation and harvesting., Competing Interests: Shuki Muramatsu, Kohei Atsuji, Koji Yamada, Hideyuki Suzuki, Takuto Takeuchi, Yuka Hashimoto-Marukawa, Kengo Suzuki, and Osamu Iwata are employees of euglena Co., Ltd. which is a private company selling E. gracilis products. All other authors declare that they have no conflict of interest., (©2020 Muramatsu et al.)

A haptonema is an elongated microtubule-based motile organelle uniquely present in haptophytes. The most notable and rapid movement of a haptonema is 'coiling', which occurs within a few milliseconds following mechanical stimulation in an unknown motor-independent mechanism. Here, we analyzed the coiling process in detail by high-speed filming and showed that haptonema coiling was initiated by left-handed twisting of the haptonema, followed by writhing to form a helix from the distal tip. On recovery from a mechanical stimulus, the helix slowly uncoiled from the proximal region. Electron microscopy showed that the seven microtubules in a haptonema were arranged mostly in parallel but that one of the microtubules often wound around the others in the extended state. A microtubule stabilizer, paclitaxel, inhibited coiling and induced right-handed twisting of the haptonema in the absence of Ca 2+ , suggesting changes in the mechanical properties of microtubules. Addition of Ca 2+ resulted in the conversion of haptonematal twist into the planar bends near the proximal region. These results indicate that switching microtubule conformation, possibly with the aid of Ca 2+ -binding microtubule-associated proteins is responsible for rapid haptonematal coiling., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

Euglena gracilis is a microalga, which has been used as a model organism for decades. Recent technological advances have enabled mass cultivation of this species for industrial applications such as feedstock in nutritional foods and cosmetics. E. gracilis degrades its storage polysaccharide (paramylon) under hypoxic conditions for energy acquisition by an oxygen-independent process and accumulates high amount of wax-ester as a by-product. Using this sequence of reactions referred to as wax-ester fermentation, E. gracilis is studied for its application in biofuel production. Although the wax-ester production pathway is well characterized, little is known regarding the biochemical reactions underlying the main metabolic route, especially, the existence of an unknown sulfur-compound metabolism implied by the nasty odor generation accompanying the wax-ester fermentation. In this study, we show sulfur-metabolomics of E. gracilis in aerobic and hypoxic conditions, to reveal the biochemical reactions that occur during wax-ester synthesis. Our results helped us in identifying hydrogen sulfide (H 2 S) as the nasty odor-producing component in wax-ester fermentation. In addition, the results indicate that glutathione and protein degrades during hypoxia, whereas cysteine, methionine, and their metabolites increase in the cells. This indicates that this shift of abundance in sulfur compounds is the cause of H 2 S synthesis.

With the aging of the population of Japan and Westernization of the dietary life, the number of cases in which cardiovascular diseases are merged in non-cardiac surgery is increasing year by year.Many of the abdominal aortic aneurysms are asymptomatic and it is not uncommon to be discovered accidentally in preoperative examination of non-cardiac surgery.When gastrointestinal surgery involves malignant diseases of the gastrointestinal tract and abdominal aortic aneurysm, the two life prognosis-related diseases are merged, depending on the severity and urgency of the disease for each case, its treatment to determine the priority order.Abdominal aortic aneurysm occurred at the time of malignant disease surgery in 14 cases of gastrointestinal cancer patients who underwent surgery at the department during the 5 years from 2012 to 2016.T he actual condition of treatment for these cases was investigated.

The clinical condition of oncologic emergency associated with colorectal cancer includes hemorrhage, perforation and obstruction. Obstructive colorectal cancer is an oncologic emergency commonly observed in our daily clinical practice. Colonic stent placement for obstructive colorectal cancer is relatively easy and safe and may be considered as an effective treatment method that enables favorable intestinal decompression preoperatively and one-stage resection. Colonic stent use can be a bridge to surgery, enabling shorter duration of hospitalization, and reduced postoperative complications, and colostomy rates, as compared to emergency surgery. From January 2009 to December 2016, this study was designed to evaluate the clinical outcomes of 68 patients who underwent surgery for obstructive colorectal cancer. The patients were divided into 2 groups: 32 cases receiving colonic stent placement(the S group), 36 cases receiving ileus tube and emergency surgery(the NS group). There was no significant difference in terms of morbidity or survival rate between the 2 groups. For the S group, 31 out of 32 could one-stage resection(94%). The colostomy rate in the S group was significantly lower than that in the NS group(3% vs 33%). In the S group, number of dissected lymph nodes was significantly larger and the duration of postoperative stay was shorter than that in the NS Group.

A 74-year-old man was referred to our hospital for further investigation of a cystic lesion in the pancreatic body, which had been detected by ultrasonography at a local hospital. He was diagnosed as intraductal papillary mucinous neoplasm(IPMN) and further preoperative examinations were conducted. Upper gastrointestinal endoscopy demonstrated a type 0-II c tumor of the greater curvature in the upper third of the stomach. Endoscopic ultrasonography showed no sign of submucosal invasion. Endoscopic submucosal dissection(ESD)was carried out and pathological examination of a specimen revealed well differentiated adenocarcinoma with submucosal invasion, which fulfilled the indication for additional gastrectomy with lymph node dissection. Laparoscopy-assisted proxymal gastrectomy with D1 plus lymph node dissection and distal pancreatectomy with splenectomy was performed. Pathological examination demonstrated intraductal papillary mucious adenoma(IPMA)in the pancreatic body and no residual gastric cancer in a specimen, however 7lymph node metastases from gastric cancer was confirmed(pN3a), including 3 metastatic lymph nodes incidentally-detected adjacent to the pancreatic parenchyma. We report a rare case of early gastric cancer with N3 lymph node metastases, with a brief literature review.

Euglena gracilis Z (Euglena) is a unicellular, photosynthesizing, microscopic green alga. It contains several nutrients such as vitamins, minerals, and unsaturated fatty acids. In this study, to verify the potential role of Euglena consumption on human health and obesity, we evaluated the effect of Euglena on human adipose-derived stem cells. We prepared a Euglena extract and evaluated its effect on cell growth and lipid accumulation, and found that cell growth was promoted by the addition of the Euglena extract. Interestingly, intracellular lipid accumulation was inhibited in a concentration-dependent manner. Quantitative real-time PCR analysis and western blotting analysis indicated that the Euglena extract suppressed adipocyte differentiation by inhibiting the gene expression of the master regulators peroxisome proliferator-activated receptor-γ (PPARγ) and one of three CCAAT-enhancer-binding proteins (C/EBPα). Further Oil Red O staining experiments indicated that the Euglena extract inhibited the early stage of adipocyte-differentiation. Consistent with these results, we observed that down-regulation of gene expression was involved in the early stage of adipogenesis represented by the sterol regulatory element binding protein 1 c (SREBP1c), two of three CCAAT-enhancer-binding proteins (C/EBPβ, C/EBPδ), and the cAMP regulatory element-binding protein (CREB). Taken together, these data suggest that Euglena extract is a promising candidate for the development of a new therapeutic treatment for obesity.

β-Glucans are a class of polysaccharides consisting of D-glucose units that are polymerized primarily via the β-1,3 glycosidic bonds, in addition to the β-1,4 and/or β-1,6 bonds. They are present in various food products such as cereals, mushrooms, and seaweeds and are known for their numerous effects on the human body, depending on their structures, which are diverse. The major physicochemical properties of β-glucans include their antioxidant property, which is responsible for the scavenging of reactive oxygen species, and their role as dietary fiber for preventing the absorption of cholesterol, for promoting egestion, and for producing short-chain fatty acids in the intestine. Dietary β-glucans also exert immunostimulatory and antitumor effects by activation of cells of the mucosal immune system via β-glucan receptors, such as dectin-1. In this review, we elaborate upon the diversity of the structures and functions of β-glucans present in food, along with discussing their proposed mechanisms of action. In addition to the traditional β-glucan-containing foods, recent progress in the commercial mass cultivation and supply of an algal species, Euglena gracilis, as a food material is briefly described. Mass production has enabled consumption of paramylon, a Euglena-specific novel β-glucan source. The biological effects of paramylon are discussed and compared with those of other β-glucans.

In recent years, many have focused on the use of oncolytic virus capable of lysing cancer cells by a cancer-selective replication for a new treatment modality of cancer. In the present study, we used oncolytic adenovirus, AdCEAp/Rep, genetically modified to selectively replicate in CEA-expressing cancer cells, and investigated whether AdCEAp/Rep could induce selective cytotoxicity to CEA expressing cancer cells, and where the AdCEAp/Rep induced cytotoxic effect could be enhanced by 5 FU in using human gastric cancer cell lines, MKN45 (CEA-positive) and MKN74 (CEA-negative). The results showed that AdCEAp/Rep showed cytotoxicity against MKN45 in a dose-dependent manner, while it did not against MKN74. Furthermore, 5-FU remarkably enhanced AdCEAp/Rep-induced cytotoxicity against MKN45 by being added 3 days after adenovirus infection. These findings strongly suggest that AdCEAp/Rep may be applicable as a new therapeutic agent for clinical cancers expressing CEA in combination with chemotherapeutic agents such as 5-FU.

In adoptive immunotherapy, in vivo trafficking of adoptively transferred cells, including their accumulation at tumor sites, remains to be further investigated. Tracking of these cells by visualization is useful to clarify antitumor mechanisms and develop new modalities to enhance antitumor capacities. In the present study, an in vivo tracking study was performed using an adoptive transfer model of lymphokine-activated killer (LAK) cells induced from green mice into C57/BL6 mice with B16 melanoma metastases. Green mice are green fluorescent protein (GFP) transgenic mice originating from C57/BL6 mice. All of the tissues, except for erythrocytes and hair, express green fluorescence under excitation light. Although LAK cells in combination with IL-2 potently suppressed pulmonary metastases with survival prolongation, very few LAK cells accumulated in tumor tissues compared to those localized in the spleen, as visualized by fluorescent microscopy and quantitated by flow cytometry. The present method using transfer of green mice-derived cells into parental tumor-bearing mice is simple because there is no need for in vitro labeling and is feasible for the in vivo tracking of effector cells in an adoptive immunotherapy model.

Milky spots (MS), peritoneal lymphoid tissues, expose the extracellular matrix (ECM) due to a defect of mesothelial cells on their surface, which may explain why peritoneal implantation of cancer cells preferentially takes place at MS. We recently reported that adenovirus vector-mediated intraperitoneal production of NK4 strongly suppressed MS-selective implantation of cancer cells and subsequent peritoneal dissemination, without histological evidence of angiogenesis inhibition. The present study was conducted to clarify the mechanisms underlying the suppressive effects of NK4 on peritoneal implantation. In mice intraperitoneally injected with CT26 cells that were genetically modified to produce NK4 (CT26-NK4), peritoneal dissemination was significantly suppressed with survival prolongation. A decreased cell implantation to omental MS was also detected and evaluated by green fluorescence protein (GFP) imaging. In an in vitro adhesion assay, hepatocyte growth factor-stimulated adhesion to ECM components, such as fibronectin and collagen, was inhibited in CT26-NK4 compared to control cells. These results strongly suggest an inhibition of cancer cell adhesion to the ECM in the suppression of peritoneal implantation by NK4.

Peritoneal dissemination is the most common mode of metastasis in gastric cancer. We previously reported the importance of milky spots (MS), peritoneal lymphoid tissues, as selective sites of cancer implantation in peritoneal dissemination. In the present study, we first demonstrated that intraperitoneal injection of adenovirus vector encoding the GFP gene into tumor-free nude mice resulted in GFP expression at omental and mesenteric MS; MS macrophages were target cells for adenovirus infection. We confirmed that intraperitoneal injection of adenovirus vector encoding the NK4 gene (AdNK4) resulted in NK4 production localized to the peritoneal cavity, especially the omentum. Adenovirus vector-mediated MS-selective transgene expression was markedly impaired in tumor-bearing mice whose MS had already been replaced by infiltrating cancer cells. However, prior injection of AdNK4 successfully inhibited MS-selective cancer cell implantation, resulting in suppression of peritoneal dissemination and prolongation of survival. Adenovirus vector-mediated MS-selective delivery of a therapeutic gene may prevent peritoneal dissemination of gastric cancer.

All of the cells and tissues from GFP transgenic mice, with the exception of erythrocytes and hair, express green fluorescent protein. Additionally, lymphokine activated killer cells induced from splenocytes of the mice (GFP-LAK) express green fluorescence under the observation by fluorescent microscopy. In the present study, we studied the biodistribution of LAK in the two adoptive immunotherapy models by injecting GFP-LAK into non-GFP expressing syngeneic mice. In peritoneal dissemination model of B16 melanoma cells, intraperitoneally injected GFP-LAK accumulated densely on the tumor. On the other hand, in the lung metastases model, intravenously injected GFP-LAK stayed scattered around the tumor, although they were observed abundantly in the spleen. Our adoptive transfer model using GFP transgenic mice is useful for understanding antitumor mechanisms induced by adoptively transferred immune cells, without any troublesome marking procedures.

NK4 suppresses invasion and metastasis of tumor cells by means of dual actions as HGF antagonist and angiogenesis inhibitor. Our previous studies showed that NK4 suppresses the implantation of tumor cells to the peritoneal milky spots (MS) by intraperitoneal injection (i.p.) of adenovirus vector expressing NK4 (Ad-NK4) or NK4 gene-transfected tumor cells. In the present study, we investigated the antitumor mechanisms of NK4 in the suppression of peritoneal implantation. When evaluated by a fluorescent microscopy, a prior injection of Ad-NK4 suppressed peritoneal implantation immediately after the injection of GFP-expressing tumor cells. DNA microarray analyses also demonstrated a reduced expression of some adhesion molecules in NK4 gene-transfected tumor cells as compared to neomycin gene-trasfected cells (control). In the in vitro adhesion assay, the adhesion to some types of the extra cellular matrixs (ECM) was significantly decreased in NK4 gene-transfected cells as compared to the control. These results suggest that NK4 may suppress peritoneal implantation by inhibiting adhesion of tumor cells to ECM around MS.

Dendritic cells (DC) are potent antigen-presenting cells capable of stimulating T cell mediated immunity. Gene transfer of tumor specific antigens or cytokines into DC would be a useful strategy for immunotherapeutical purposes. In the present study, in vitro transfection of human DCs (hDC) with the complex of biodegradable cationized gelatin and an EGFP gene was performed. Flow cytometric analyses revealed that approximately 14% of DC was positively expressed for EGFP, and the mRNA expression of EGFP gene in transfected hDC was detected by RT-PCR. Additionally, when evaluated by allogeneic MLR, the antigen-presenting capacity of transfected DC was equal to that of control DC. Cationized gelatin is a promising nonviral vector for gene transfer into DC.

Tumor-stromal interactions, which are regulated by stromal-derived HGF and tumor-derived HGF inducers, are essential for tumor cell acquisition of such malignant properties as invasion and metastasis. NK4, a proteolytic cleavage product of HGF, has antitumor activities as both an HGF antagonist and an angiogenesis inhibitor. In this study, we examined the in vitro and in vivo behaviors of mouse colon adenocarcinoma C T26 cells modified by gene transfer to secrete NK4, and investigated the influence of NK4 on expression of HGF and HGF inducers associated with tumor-stromal interactions. In vitro cell proliferation rates of NK4 transfectant (C T26-NK4) and mock transfectant (C T26-NEO) were essentially the same, and scattering and invasion were stimulated by HGF in C T26-NEO, but not in C T26-NK4. In syngeneic BALB/c female mice, subcutaneous tumor growth of C T26-NK4 was potently suppressed, and the survival was prolonged significantly. Immunohistochemistry showed significantly decreased microvessels and increased apoptotic cells in C T26-NK4 tumor compared with control. Interestingly, HGF, strongly expressed in C T26-NEO tumor stroma, was reduced in C T26-NK4. In vitro, conditioned medium of C T26-NK4 inhibited fibroblast-derived HGF production, which was increased by that of C T26-NEO. Moreover, although similar constitutive expression levels of PDGF and TGF-alpha (both HGF inducers) were detected in C T26-NK4 and C T26-NEO in semiquantitative RT-PCR analyses, the expression was up-regulated by HGF in C T26-NEO, but not C T26-NK4. These results suggest that NK4 may exert antitumor activities not only by antagonizing HGF, but also by inhibiting HGF amplification via tumor-stromal interactions. Continuous, abundant NK4 production induced at a tumor site by gene transfer should show multiple antitumor activities with potential therapeutic benefit.

It is important to develop an efficient adjuvant therapy for the prevention of the postoperative peritoneal recurrence of gastrointestinal cancers such as gastric cancer or pancreatic cancer. Milky sports (MS) are peritoneal lymphoid tissues broadly distributed on the peritoneal tissues such as the omentum, mesentery, or Douglas pouch, and are the selective implantation sites of disseminated cancer cells. In this study, we introduced GFP gene into various cancer cell lines and host-derived cells such as peritoneal macrophages and dendritic cells by adenovirus vetor and injected them i.p. into mice. We then investigated the sites of GFP expression on the peritoneum by fluorescence microscopy. The results showed that green fluorescence was detected specifically for the MS sites that stained black with activated carbon particles (CH40), despite the differences in the cell type injected. These findings indicate that 1) the physioanatomical characteristics of MS may play an essential role in the formation of the initial disseminated lesions at MS, and 2) the host-derived cells accumulating near MS may be available as carriers of a therapeutic gene in intraperitoneal gene therapy against peritoneal dissemination.

An HGF antagonist, NK4, inhibits not only invasion and metastasis of tumor cells driven by HGF-Met receptor binding, but also tumor angiogenesis. To address the antitumor activities of NK4, we investigated the biological behaviors of CT26 transfected with the NK4 gene (CT26-NK4) in vitro and in vivo. In the in vitro assay, the invasion in MOCK transfected cells (control) was stimulated by HGF; however, in CT26-NK4 cells, these effects were completely inhibited. In the in vivo assay, the tumor growth of CT26-NK4 was strongly suppressed and the survival of CT26-NK4 tumor-bearing mice was significantly prolonged. Immunohistochemical analysis revealed that while proliferating cells (PCNA immunostaining) of CT26-NK4 tumors were weakly suppressed, the micro-vessel number (CD31/PECAM-1 immunostaining) in those tumors was significantly suppressed as compared with the control tumors. In conclusion, NK4 exerts potent antitumor effects via anti-angiogenesis rather than inhibition of biological events of tumor cells stimulated by HGF.