Sensitivity assessment of diphacinone by pharmacokinetic analysis in invasive black rats in the Bonin (Ogasawara) Archipelago, Japan.

The Bonin Archipelago is a United Nations Educational, Scientific and Cultural Organization's World Natural Heritage Site in Japan with a unique ecosystem; however, the invasive rodents preying on endemic species have been a significant concern. The anticoagulant rodenticide, diphacinone, sprayed by the Ministry of the Environment, has succeeded; however, its repeated use leads to rodenticide resistance. This study evaluated the sensitivity by in vivo pharmacokinetics/pharmacodynamics (PK/PD) analysis and physiologically-based pharmacokinetic modeling to diphacinone in black rats (Rattus rattus) captured on the Bonin Archipelago in February 2022. The Bonin rats exhibited prolonged coagulation time after diphacinone administration. They recovered earlier than susceptible black rats, indicating that Bonin rats were less susceptible, though there were no genetic mutations in Vkorc1, the target enzyme of diphacinone. After the administration of diphacinone, hepatic expression levels of Fsp1, identified as the vitamin K reductase, was decreased, however, the Bonin rats exhibited the most minor suppression. The PK analysis showed that the excretion capacity of the Bonin rats was lower than that of the resistant black rats. In the PBPK modeling, the resistant black rats showed higher clearance than the Bonin and susceptible black rats due to high hepatic metabolic capacity. The Bonin rats demonstrated slow absorption and relatively low clearance. This study highlighted the reduced rodenticide-sensitive tendency of wild black rats in the Bonin Archipelago at an in vivo phenotype level. At the same time, they do not have known rodenticide resistance mechanisms, such as hepatic metabolic enhancement or Vkorc1 mutations. It is crucial to monitor the biological levels to evaluate rodenticide sensitivity accurately.

Primary and immortalized cell lines derived from the Amami rabbit (Pentalagus furnessi) and evolutionally conserved cell cycle control with CDK4 and Cyclin D1.

The Amami rabbit (Pentagulus furnessi) is a dark brown-furred rabbit classified as an endangered species and only found in the Amami Islands of Japan. They are often called living fossils because they retain primitive characteristics of ancient rabbits that lived approximately 1 million years ago, such as short feet and hind legs and small ears. Although the ancient rabbit has disappeared due to the competition with European rabbit (Oryctolagus cuniculus) in the most of the Asian area, Amami rabbit survived since Amami Islands has isolated from Japan and Taiwan. Although Amari rabbit is one of the protected animals, their population decreases each year due to human activities, such as deforestation and roadkill. In this study, we collected roadkill samples of Amami rabbits and established primary and immortalized fibroblast cell lines. Combined expression of human-derived mutant Cyclin-dependent kinase 4, Cyclin D1, and hTERT allowed us to immortalize fibroblasts successfully in three individuals of Amami rabbits. The immortalized fibroblasts dramatically extended the cell culture period, when it was compared with the cell culture period of wild type cells. Furthermore, the immortalized cells maintained their normal chromosomal pattern (2n = 46). Our results suggest that cellular senescence which mainly regulated by p16-RB signaling pathway is conserved in animal evolution at least from 1 million years ago.

Extended proliferation of chicken- and Okinawa rail-derived fibroblasts by expression of cell cycle regulators.

Although immortalized cultured cells are useful for various functional assays or transcriptome analysis, highly efficient and reproducible immortalization methods have not been developed in avian-derived cells. We introduced the simian virus 40 T antigen (SV40T) and human papillomavirus (HPV)-E6E7 to chick and Okinawa rail (endangered species)derived fibroblast. As a result, neither the SV40T nor E6E7 genes could induce avian cell immortality. Accordingly, we attempted to use a recently developed immortalization method, which involved the coexpression of mutant cyclin-dependent kinase 4 (CDK4), Cyclin D, and TERT (K4DT method) in these avian cells. Although the K4DT method could not efficiently induce the efficient immortalization in mass cell population, cellular division until the senescence was significantly extended by K4DT, we succeeded to obtain the immortalized avian cells (chick K4DT: one clone, Okinawa rail K4DT: three clones, Okinawa rail K4DT + telomerase RNA component: one clone) with K4DT expression. We conclude that K4DT expression is used to extend the cell division and immortalization of avian-derived cells.

Chick derived induced pluripotent stem cells by the poly-cistronic transposon with enhanced transcriptional activity.

Induced pluripotent stem (iPS) cell technology lead terminally differentiated cells into the pluripotent stem cells through the expression of defined reprogramming factors. Although, iPS cells have been established in a number of mammalian species, including mouse, human, and monkey, studies on iPS cells in avian species are still very limited. To establish chick iPS cells, six factors were used within the poly-cistronic reprogramming vector (PB-R6F), containing M3O (MyoD derived transactivation domain fused with Oct3/4), Sox2, Klf4, c-Myc, Lin28, and Nanog. The PB-R6F derived iPS cells were alkaline-phosphatase and SSEA-1 positive, which are markers of pluripotency. Elevated levels of endogenous Oct3/4 and Nanog genes were detected in the established iPS cells, suggesting the activation of the FGF signaling pathway is critical for the pluripotent status. Histological analysis of teratoma revealed that the established chick iPS cells have differentiation ability into three-germ-layer derived tissues. This is the first report of establishment of avian derived iPS cells with a single poly-cistronic transposon based expression system. The establishment of avian derived iPS cells could contribute to the genetic conservation and modification of avian species.

Immortalized prairie vole-derived fibroblasts (VMF-K4DTs) can be transformed into pluripotent stem cells and provide a useful tool with which to determine optimal reprogramming conditions.

The cellular conditions required to establish induced pluripotent stem cells (iPSCs), such as the number of reprogramming factors and/or promoter selection, differ among species. The establishment of iPSCs derived from cells of previously unstudied species therefore requires the extensive optimization of programming conditions, including promoter selection and the optimal number of reprogramming factors, through a trial-and-error approach. While the four Yamanaka factors Oct3/4, Sox2, Klf4, and c-Myc are sufficient for iPSC establishment in mice, we reported previously that six reprogramming factors were necessary for the creation of iPSCs from primary prairie vole-derived cells. Further to this study, we now show detailed data describing the optimization protocol we developed in order to obtain iPSCs from immortalized prairie vole-derived fibroblasts. Immortalized cells can be very useful tools in the optimization of cellular reprogramming conditions, as cellular senescence is known to dramatically decrease the efficiency of iPSC establishment. The immortalized prairie vole cells used in this optimization were designated K4DT cells as they contained mutant forms of CDK4, cyclin D, and telomerase reverse transcriptase (TERT). We show that iPSCs derived from these immortalized cells exhibit the transcriptional silencing of exogenous reprogramming factors while maintaining pluripotent cell morphology. There were no observed differences between the iPSCs derived from primary and immortalized prairie vole fibroblasts. Our data suggest that cells that are immortalized with mutant CDK4, cyclin D, and TERT provide a useful tool for the determination of the optimal conditions for iPSC establishment.

Perioperative care with fast-track management in patients undergoing pancreaticoduodenectomy.

BACKGROUND: It has been considered that allowing patients to return to daily life earlier after surgery helps recovery of physiological function and reduces postoperative complications and hospital stay. We investigated the usefulness of fast-track management in perioperative care of patients undergoing pancreaticoduodenectomy (PD). METHODS: Patients (n = 90) who received conventional perioperative management from 2005 to 2009 were included as the 'conventional group' (historical control group), and patients who received perioperative care with fast-track management (n = 100) from 2010 to March 2013 were included as the 'fast-track group'. To evaluate the efficacy of perioperative care with fast-track management, the incidence of postoperative complications and the length of hospital stay were compared between the two groups (comparative study). For statistical analysis, univariate analysis was performed using the χ (2) test or Fisher's exact test. RESULTS: There was no significant difference between the two groups in sex, mean age, presence/absence of diabetes mellitus, preoperative drainage for jaundice, previous disease, operative procedure, mean duration of operation, or blood loss (p < 0.01). The incidence of surgical site infection in the conventional group and fast-track group was 28.9 and 14.0 %, respectively, with a significant difference between the two groups (p = 0.019). In addition, the incidence of pancreatic fistula (grade B, C) significantly differed between the two groups (27.8 % in the conventional group, 9.0 % in the fast-track group; p = 0.001). The mean postoperative hospital stay was 36.3 days in the conventional group and 21.9 days in the fast-track group (p < 0.001). CONCLUSIONS: Perioperative care with fast-track management may reduce postoperative complications and decrease the length of hospital stay in patients undergoing PD.

A splenic volume increase due to preoperative chemotherapy may impair the long-term outcome after hepatectomy in patients with initially non-optimally resectable colorectal cancer liver metastases.

BACKGROUND/AIMS: In patients with colorectal cancer liver metastases (CRCLM), chemotherapy-associated hepatotoxicity (CAH) has been shown to be associated with splenomegaly. The aim of the present study was to investigate whether a higher splenic volume increase (SVI) after preoperative chemotherapy was associated with a worse long-term outcome after hepatectomy in patients with CRCLM. METHODOLOGY: Between 2007 and 2012, there were 36 patients who received preoperative chemotherapy based on a diagnosis of initially non-optimally resectable synchronous CRCLM. The splenic volume was measured by CT-volumetry before preoperative chemotherapy and 12 weeks after the start of the chemotherapy. Long-term outcome was analyzed in these patients. RESULTS: The overall survival was significantly shorter in the SVI ≥30% group than in the SVI <30% group (3-year survival = 52% vs. 28%, p <0.05). The disease-free survival was also significantly shorter in the SVI ≥30% group than in the SVI <30% group (Median = 11 vs. 6 months, p <0.05). The survival after recurrence was significantly shorter in the SVI ≥30% group than in the SVI <30% group (Median = 12.0 vs. 18 months, p<0.05). CONCLUSIONS: In patients with non-optimally resectable CRCLM, the SVI during the first 12 weeks of preoperative chemotherapy may be a significant predictor of the long-term survival after hepatectomy.

Organoids containing neural-like cells derived from chicken iPSCs respond to poly:IC through the RLR family.

There is still much room for development in pluripotent stem cell research on avian species compared to human stem cell studies. Neural cells are useful for the evaluation of risk assessment of infectious diseases since several avian species die of encephalitis derived from infectious diseases. In this study, we attempted to develop induced pluripotent stem cells (iPSCs) technology for avian species by forming organoids containing neural-like cells. In our previous study, we established two types iPSCs from chicken somatic cells, the first is iPSCs with PB-R6F reprogramming vector and the second is iPSCs with PB-TAD-7F reprogramming vector. In this study, we first compared the nature of these two cell types using RNA-seq analysis. The total gene expression of iPSCs with PB-TAD-7F was closer to that of chicken ESCs than that of iPSCs with PB-R6F; therefore, we used iPSCs with PB-TAD-7F to form organoids containing neural-like cells. We successfully established organoids containing neural-like cells from iPSCs using PB-TAD-7F. Furthermore, our organoids responded to poly:IC through the RIG-I-like receptor (RLR) family. In this study, we developed iPSCs technology for avian species via organoid formation. In the future, organoids containing neural-like cells from avian iPSCs can develop as a new evaluation tool for infectious disease risk in avian species, including endangered avian species.

Cultured fibroblasts of the Okinawa rail present delayed innate immune response compared to that of chicken.

The Okinawa rail is endemic to Okinawa Island and is categorized as an endangered animal. In this study, we focused on innate immunity because it is the first line of host defense. In particular, signals recognizing foreign RNA (e.g., viruses) are important for host defense because they activate the host immune system. The retinoic acid-inducible gene I (RIG-I)-like receptor (RLR) families (RIG-I, MDA5, and LGP2) are sensors that activate innate immunity. Therefore, we analyzed these functions in the Okinawa rail using genomic and cellular analyses of fibroblasts. Fibroblasts can be obtained from dead individuals, allowing these cells to be obtained from dead individuals, which is particularly useful for endangered species. The MDA5 gene of Okinawa rail was sequenced using the Sanger method following PCR amplification and extraction of the amplified sequence from agarose gel. Additionally, mRNA expression analysis of cultured fibroblasts exposed to poly I:C was done. The MDA5 gene was found to be a mutated nonfunctional gene in the Okinawa rail. The mRNA expression rates of inflammatory cytokine genes type I IFN, and Mx1 were slower in Okinawa rail than in chicken cultured fibroblasts. Similar to the mRNA expression results, cell number and live cell ratio also slowly decreased in the Okinawa rail compared with chicken cultured fibroblasts, indicating that the innate immune reaction differs between chicken and the Okinawa rail. To the best of our knowledge, this is the first experimental evaluation of the loss of function of the Okinawa rail innate immune genes. In conclusion, our results provide a basis for conservation strategies for the endangered Okinawa rail.

Immortalization of primary cells derived from the endangered Ryukyu long-furred rat.

The Ryukyu long-furred rat is an endangered species confined to the southernmost three small islands of Japan (Amami-Oshima, Tokunoshima, and Okinawa). Its population is rapidly decreasing because of roadkill, deforestation, and feral animals. To date, its genomic and biological information are poorly understood. In this study, we successfully immortalized Ryukyu long-furred rat cells by expressing a combination of cell cycle regulators, mutant cyclin-dependent kinase 4 (CDK4R24C) and cyclin D1, together with telomerase reverse transcriptase or an oncogenic protein, the Simian Virus large T antigen. The cell cycle distribution, telomerase enzymatic activity, and karyotype of these two immortalized cell lines were analyzed. The karyotype of the former cell line immortalized with cell cycle regulators and telomerase reverse transcriptase retained the nature of the primary cells, while that of the latter cell line immortalized with the Simian Virus large T antigen had many aberrant chromosomes. These immortalized cells would be valuable for studying the genomics and biology of Ryukyu long-furred rats.

Localization of estrogen receptor in the central lymphoid organs of chickens during the late stage of embryogenesis.

Immunological function in chicks is greatly affected by estrogen treatment during embryogenesis, but the mechanism of the estrogen effect is not fully understood. To elucidate the effect of estrogen on immune function, we observed estrogen receptor expression in the thymus and bursa of chick embryos by immunohistochemistry. We compared the distribution of estrogen receptor-positive cells with that of keratin-positive epithelial cells. Intense expression of estrogen receptors was detected in thymic and bursal lymphocytes. In peripheral lymphocytes, ER mRNA was detected by RT-PCR analysis. The results of fluorescence-activated cell sorting analysis indicated that the estrogen receptor was expressed in the cytoplasm of the lymphocytes. Furthermore, intense expression of the estrogen receptor was also confirmed in thymic Hassall's corpuscles, bursal follicle-associated epithelial cells, and the bursal interfollicular epithelium. Our results indicate that estrogen affects the differentiation of thymic and bursal lymphocytes, suggesting that the underlying role for estrogen in immune function.

Efficient establishment of pig embryonic fibroblast cell lines with conditional expression of the simian vacuolating virus 40 large T fragment.

The pig is an important animal for both agricultural and medical purposes. However, the number of pig-derived cell lines is relatively limited when compared with mouse- and human-derived lines. We established in this study a retroviral conditional expression system for the Simian vacuolating virus 40 large T fragment (SV40T) which allowed us to efficiently establish pig embryonic fibroblast cell lines. The established cell lines showed high levels of cell proliferation and resistance to cellular senescence. A chromosome analysis showed that 84% of the cells had the normal karyotype. Transient expression of the Cre recombinase allowed us to excise the SV40T fragment from the genome. The development of this research tool will enable us to quickly establish new cell lines derived from various animals.

Lymph node recurrence and re-excision after primary tumor resection of a histiocytic sarcoma of duodenal origin: a case report.

BACKGROUND: Histiocytic sarcoma is a rare malignant tumor that is similar in characteristics to a mature histiocyte/macrophage and is a relatively new disease entity. In approximately one-third of cases, the site of origin is a lymph node; development from the gastrointestinal tract, spleen, soft tissue, and skin has further been reported. The tumor characteristics are not well-understood as reports on its clinical presentation and treatment are limited. We report a case of duodenal primary histiocytic sarcoma. CASE PRESENTATION: An elevated lesion in the second part of the duodenum was detected in a 70-year-old woman during routine examination using upper gastrointestinal tract endoscopy. Blood biochemistry findings were normal for tumor markers. No abnormal findings were observed in the blood count and biochemical examination. Upper gastrointestinal endoscopy revealed a 20-mm elevated lesion with a slight depression in the center, opposite to the papilla of the descending duodenum. The biopsy showed erosions of the mucosal epithelium and inflammatory cell infiltration, but no evidence of malignancy. Ultrasound-guided endoscopy revealed an ischemic tumor of submucosal origin, and bowel biopsy suggested a histiocytic sarcoma. Distant metastasis and lymph node enlargement were absent on abdominal sonography, computed tomography, and magnetic resonance imaging. Duodenal segmental resection was performed. Immunostaining of the excised lesion was positive for CD68, CD163, CD4, CD5, CD15, and CD45 and negative for CD1a, CD21, CD34, MPO, and S-100 protein. Ki-67 positivity was approximately 20%. Based on these findings, the diagnosis of histiocytic sarcoma was confirmed. Ten months after the surgery, a lymph node recurrence in the dorsum of the pancreatic uncus was observed. No evidence of recurrence was found in any other part; hence, we performed pancreaticoduodenectomy. Pathological findings of the excised lymph node confirmed the recurrence of histiocytic sarcoma in the lymph node. CONCLUSIONS: This is the first reported case of a duodenal primary histiocytic sarcoma with recurrence in the lymph node after the primary resection. The patient was treated for recurrence by lymph node excision and pancreaticoduodenectomy.

Induced pluripotent stem cells of endangered avian species.

The number of endangered avian-related species increase in Japan recently. The application of new technologies, such as induced pluripotent stem cells (iPSCs), may contribute to the recovery of the decreasing numbers of endangered animals and conservation of genetic resources. We established novel iPSCs from three endangered avian species (Okinawa rail, Japanese ptarmigan, and Blakiston's fish owl) with seven reprogramming factors (M3O, Sox2, Klf4, c-Myc, Nanog, Lin28, and Klf2). The iPSCs are pluripotency markers and express pluripotency-related genes and differentiated into three germ layers in vivo and in vitro. These three endangered avian iPSCs displayed different cellular characteristics even though the same reprogramming factors use. Japanese ptarmigan-derived iPSCs have different biological characteristics from those observed in other avian-derived iPSCs. Japanese ptarmigan iPSCs contributed to chimeras part in chicken embryos. To the best of our knowledge, our findings provide the first evidence of the potential value of iPSCs as a resource for endangered avian species conservation.

KAv-1 is Better Suited to Chick Fibroblast Culture than DMEM or 199 Media.

Cultured cells are a useful resource for poultry scientists, since these cells allow scientists to evaluate biological responses to conditions such as infectious diseases in vitro while mimicking the whole-body response in birds. However avian cell culture requires an optimized basal medium, and there are currently relatively few options for this basal medium (medium 199 and KAv-1). This means that there is still room for the development of an optimal basal medium for avian cell culture. Here we compare KAv-1 medium, Dulbecco's modified Eagle medium (DMEM) and medium 199 during the culture of chick fibroblasts and determine that KAv-1 remains the optimal medium for these assays. Our results show that DNA damage is reduced in fibroblasts cultured in the KAv-1 medium, when compared to both DMEM and Medium 199 and that these cells also display improved growth dynamics in KAv-1 medium when compared to both DMEM and medium 199. To the best of our knowledge, this is the first study to describe a comparative analysis of culture media for avian cells, which would provide useful information for poultry scientists.

Giant Adrenal Lipoma with Hemorrhage Requiring Extended Surgical Resection.

Adrenal lipoma is a rare, benign tumor, reported to account for 0.7% of primary adrenal tumors. A 69-year-old man presented with left lateral abdominal pain. Computed tomography (CT) was performed, and a huge, irregularly shaped retroperitoneal tumor of uneven internal density was identified, with the border between the tumor and the pancreas and kidney being unclear. Active hemorrhage was also depicted. The tumor consisted mainly of fat, with the exception of the hematoma; it measured 200 mm; and the boundary between it and nearby organs, such as the pancreas, was unclear. Despite angiography being performed twice, the responsible vessel was not identified. Thus, for the purpose of both diagnosis and treatment, we resected the tumor, and considering the possibility of a malignancy, such as liposarcoma, we also resected the pancreatic body and tail and the spleen. The final histopathologic diagnosis was benign adrenal lipoma with hemorrhage, with no invasion to surrounding tissue. Hemorrhage within an adrenal tumor is rare. Most adrenal lipomas are small "incidentalomas" and asymptomatic. With development of a large adrenal lipoma comes the possibility of hemorrhage along with the possibility of features suggestive of malignancy. We encountered a giant adrenal lipoma with hemorrhage and, because of the aforementioned features, performed extended surgical resection, seen in retrospect as oversurgery. The widespread use of CT has led to an increased number of reported cases of adrenal lipoma. We anticipate an accumulation of case reports, which will allow for development of an appropriate treatment algorithm.

Des-gamma-carboxy prothrombin affects the survival of HCC patients with marginal liver function and curative treatment: ACRoS1402.

PURPOSE: Considering the initial treatment of hepatocellular carcinoma (HCC), the best prognostic index for Child-Pugh classes B and C (CP-BC) patients has not been yet established. This study aimed to elucidate the risk factors for disease-free survival (DFS) and overall survival (OS) in multicenter patients with a poor liver functional reserve after curative treatment. METHODS: Between April 2000 and April 2014, 212 CP-BC patients who received treatment in five high-volume centers in Japan were included in this study. CP-B and C patients were 206 and 6, respectively. Cox proportional hazard regression analyses for DFS and OS were performed to estimate the risk factors. RESULTS: The mean observation time was 1132 days. Mean Child-Pugh score and indocyanine green retention rate at 15 min were 7.5 and 31.5%, respectively. Histological chronic hepatitis and liver cirrhosis were observed in 20% and 74% patients, respectively. In the multivariate analysis, the risk factors for DFS were des-gamma-carboxy prothrombin (DCP) [hazard ratio (HR), 1.6; P = 0.012] and treatment without liver transplantation. Moreover, DCP was identified as an independent risk factor for OS (HR, 1.7; P = 0.01). Tumor size, number, tumor thrombus, Milan criteria, liver cirrhosis, and treatment without liver transplantation were not identified as risk factors for OS. The 5-year OS in patients with high serum DCP levels (< 90 mAU/mL) was significantly better than that in those with low serum DCP levels (P = 0.003). CONCLUSIONS: Serum DCP value before treatment predicted both DFS and OS in CP-BC patients with HCC.

Correction: Establishment of immortalized primary cell from the critically endangered Bonin flying fox (Pteropus pselaphon).

[This corrects the article DOI: 10.1371/journal.pone.0221364.].

Rat-derived feeder cells immortalized by expression of mutant CDK4, cyclin D, and telomerase can support stem cell growth.

The maintenance of stem cells often requires the support of feeder cells. Primary mouse embryonic fibroblasts (MEFs) have traditionally been used as feeder cells, and although these MEF-derived feeder cells have exhibited a reasonable performance, they require repeated cell isolation, since MEFs cannot expand indefinitely. To overcome this limitation, immortalized cells, such as STO cells, have been used. However, one major disadvantage is that previously reported immortalized cells can only support stem cell cultures for a relatively short period, typically 4 to 7 days. In this study, we found that our newly established rat-derived fibroblasts immortalized by the expression of mutant cyclin-dependent kinase 4, cyclin D, and telomerase reverse transcriptase, can function as feeder cells for relatively long cell culture periods of approximately 14 days. The rat-derived immortalized cells developed in this study should be a useful source of feeder cells to support stem cell research.

Establishment of immortalized primary cell from the critically endangered Bonin flying fox (Pteropus pselaphon).

The Bonin flying fox (Pteropus pselaphon) is one of the most critically endangered species of animals. The number of this species is estimated to be around 150; being classified at the top rank in the list by International Union of Animal Conservation. Our group previously showed that expression of CDK4, CYCLIN D1, and telomerase reverse transcriptase (TERT) efficiently induce immortalization of human, bovine, swine, monkey, and buffalo-derived cells. In this manuscript, we successfully established the primary cells from Bonin flying fox. We introduced CDK4, CYCLIN D1, and TERT into the primary cells. The established cells showed efficient expression of introduced genes at the protein level. Furthermore, the established cells were free from senescence, indicating it reached to immortalization. Moreover, we showed that interspecies somatic cell nuclear transfer of Bonin flying fox derived cell into bovine embryo allowed the development of the embryo to 8 cell stages. Our established cell has the potential to contribute to species conservation.