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Abstract We present a unique case of metastatic metaplastic breast carcinoma responding dramatically to immunochemotherapy. A 46‐year‐old Japanese woman with primary metaplastic carcinoma of the breast, which was immunohistochemically confirmed to be triple‐negative breast cancer, underwent radical surgery, followed by adjuvant chemotherapy with an anthracycline and a taxane. Since multiple lung metastases were detected two months post‐chemotherapy and the primary site was shown to be PD‐L1‐positive, the immune checkpoint inhibitor (ICI) pembrolizumab plus gemcitabine/carboplatin was initiated. While the treatment was discontinued after 15 days due to suspected drug‐induced pneumonitis, the lung metastases significantly shrank with no development of new lesions for three months. The patient remained alive as of approximately 15 months after the recurrence date. This case highlights the potential of immunochemotherapy in treating metaplastic breast carcinomas.

Abstract Primordial germ cells (PGCs) are vital for producing sperm and eggs and are crucial for conserving chicken germplasm and creating genetically modified chickens. However, efforts to use PGCs for preserving native chicken germplasm and genetic modification via CRISPR/Cas9 are limited. Here we show that we established 289 PGC lines from eight Chinese chicken populations with an 81.6% success rate. We regenerated Piao chickens by repropagating cryopreserved PGCs and transplanting them into recipient chickens, achieving a 12.7% efficiency rate. These regenerated chickens carried mitochondrial DNA from female donor PGC and the rumplessness mutation from both male and female donors. Additionally, we created the TYRP1 (tyrosinase-related protein 1) knockout (KO) PGC lines via CRISPR/Cas9. Transplanting KO cells into male recipients and mating them with wild-type hens produced four TYRP1 KO chickens with brown plumage due to reduced eumelanin production. Our work demonstrates efficient PGC culture, cryopreservation, regeneration, and gene editing in chickens.

Abstract Background Distant metastases from lung cancer are commonly found in the brain, bone, and liver. Metastases to the breast from non-mammary malignancies are extremely rare, and their clinical presentations remain unclear. Case presentation We herein report a case of bilateral breast metastases from anaplastic lymphoma kinase-positive advanced lung cancer in a 51-year-old Japanese male patient. During the course of systemic treatment for advanced lung cancer, computed tomography revealed bilateral breast enlargement without contrast enhancement, a finding consistent with gynecomastia. While other metastatic lesions responded to chemotherapy, both breast masses grew vertically like nodules. The breast masses were immunohistochemically diagnosed as metastases from lung cancer and were removed surgically. Simultaneous bilateral breast metastases from malignancies of other organs, like ones in this case, have rarely been described. Conclusions It is important to keep in mind that breast metastases from nonmammary malignancies are a possible explanation for unusual breast findings in a patient with a history of malignancies.

Abstract Background While advanced gastrointestinal stromal tumors (GISTs) are primarily treated with tyrosine kinase inhibitors (TKIs), acquired resistance from specific mutations in KIT or PDGFRA frequently occurs. We aimed to assess the utility of circulating tumor DNA (ctDNA) as a modality of therapeutic decision-making in advanced GIST. Methods We conducted a pooled analysis of SCRUM-Japan studies for advanced GIST patients. We compared patient characteristics analyzed with tissue and blood samples, assessed gene alteration profiles, and evaluated prognostic implications from ctDNA status. Results In 133 patients, tissue and blood samples were analyzed for 89 and 44 patients, respectively. ctDNA was detected in 72.7% of cases; no prior treatment or progressive disease was significantly associated with ctDNA-positivity. ctDNA-positive patients had significantly shorter progression-free survival compared with ctDNA-negative patients (hazard ratio = 3.92; P = 0.007). ctDNA genotyping revealed a complex landscape of gene alterations, characterized by multi-exonic mutations in KIT, compared with tissue-based analysis. Patients who received TKIs matched to the identified KIT mutation in ctDNA demonstrated significantly longer PFS than those with unmatched treatment (median, 8.23 vs. 2.43 months; P

Abstract “NeoRAS WT” refers to the loss of RAS mutations (MTs) following first-line treatment in metastatic colorectal cancer (mCRC). We evaluate the incidence and clinicopathological characteristics of NeoRAS WT mCRC using next-generation sequencing of plasma circulating tumor DNA. Patients with mCRC enrolled in the GOZILA study initially diagnosed with tissue RAS MT mCRC and received subsequent systemic therapy are eligible. NeoRAS WT is defined as the absence of detectable RAS MT in plasma and assessed in all eligible patients (Group A) and in a subgroup with at least one somatic alteration detected in plasma (Group B). Overall, 478 patients are included. NeoRAS WT prevalence is 19.0% (91/478) in Group A and 9.8% (42/429) in Group B. Absence of liver or lymph node metastasis and tissue RAS MTs other than KRAS exon 2 MTs are significantly associated with NeoRAS WT emergence. Overall, 1/6 and 2/6 patients with NeoRAS WT treated with anti-EGFR monoclonal antibodies (mAbs) show partial response and stable disease for ≥6 months, respectively. NeoRAS WT mCRC is observed at a meaningful prevalence, and anti-EGFR mAb-based therapy may be effective.

Abstract Toward drastic enhancement of thermoelectric power factor, quantum confinement effect proposed by Hicks and Dresselhaus has intrigued a lot of researchers. There has been much effort to increase power factor using step-like density-of-states in two-dimensional electron gas (2DEG) system. Here, we pay attention to another effect caused by confining electrons spatially along one-dimensional direction: multiplied 2DEG effect, where multiple discrete subbands contribute to electrical conduction, resulting in high Seebeck coefficient. The power factor of multiple 2DEG in GaAs reaches the ultrahigh value of ~100 μWcm−1 K−2 at 300 K. We evaluate the enhancement rate defined as power factor of 2DEG divided by that of three-dimensional bulk. The experimental enhancement rate relative to the theoretical one of conventional 2DEG reaches anomalously high (~4) in multiple 2DEG compared with those in various conventional 2DEG systems (~1). This proposed methodology for power factor enhancement opens the next era of thermoelectric research.

Abstract This study was designed to perform an association analysis and identify SNP markers associated with production traits of Japanese quail using restriction-site-associated DNA sequencing. Weekly body weight data from 805 quail were collected from hatching to 16 weeks of age. A total number of 3990 eggs obtained from 399 female quail were used to assess egg quality traits. Egg-related traits were measured at the beginning of egg production (first stage) and at 12 weeks of age (second stage). Five eggs were analyzed at each stage. Traits, such as egg weight, egg length and short axes, eggshell strength and weight, egg equator thickness, yolk weight, diameter, and colour, albumen weight, age of first egg, total number of laid eggs, and egg production rate, were assessed. A total of 383 SNPs and 1151 associations as well as 734 SNPs and 1442 associations were identified in relation to quail production traits using general linear model (GLM) and mixed linear model (MLM) approaches, respectively. The GLM-identified SNPs were located on chromosomes 1–13, 15, 17–20, 24, 26–28, and Z, underlying phenotypic traits, except for egg and albumen weight at the first stage and yolk yellowness at the second stage. The MLM-identified SNPs were positioned on defined chromosomes associated with phenotypic traits except for the egg long axis at the second stage of egg production. Finally, 35 speculated genes were identified as candidate genes for the targeted traits based on their nearest positions. Our findings provide a deeper understanding and allow a more precise genetic improvement of production traits of Galliformes, particularly in Japanese quail.

Background Immune checkpoint inhibitor (ICI) combinations represent an emerging treatment strategies in cancer. However, their efficacy in microsatellite stable (MSS) or mismatch repair-proficient (pMMR) colorectal cancer (CRC) is variable. Here, a multiomic characterization was performed to identify predictive biomarkers associated with patient response to ICI combinations in MSS/pMMR CRC for the further development of ICI combinations.Methods Whole-exome sequencing, RNA sequencing, and multiplex fluorescence immunohistochemistry of tumors from patients with MSS/pMMR CRC, who received regorafenib plus nivolumab (REGONIVO) or TAS-116 plus nivolumab (TASNIVO) in clinical trials were conducted. Twenty-two and 23 patients without prior ICI from the REGONIVO and TASNIVO trials were included in this study. A biomarker analysis was performed using samples from each of these studies.Results The epithelial-mesenchymal transition pathway and genes related to cancer-associated fibroblasts were upregulated in the REGONIVO responder group, and the G2M checkpoint pathway was upregulated in the TASNIVO responder group. The MYC pathway was upregulated in the REGONIVO non-responder group. Consensus molecular subtype 4 was significantly associated with response (p=0.035) and longer progression-free survival (p=0.006) in the REGONIVO trial. CD8+ T cells, regulatory T cells, and M2 macrophages density was significantly higher in the REGONIVO trial responders than in non-responders. Mutations in the POLE gene and patient response were significantly associated in the TASNIVO trial; however, the frequencies of other mutations or tumor mutational burden were not significantly different between responders and non-responders in either trial.Conclusions We identified molecular features associated with the response to the REGONIVO and TASNIVO, particularly those related to tumor microenvironmental factors. These findings are likely to contribute to the development of biomarkers to predict treatment efficacy for MSS/pMMR CRC and future immunotherapy combinations for treatment.

ABSTRACT Vanadium dioxide (VO2) material, known for changing physical properties due to metal-insulator transition (MIT) near room temperature, has been reported to undergo a phase change depending on the strain. This fact can be a significant problem for nanoscale devices in VO2, where the strain field covers a large area fraction, spatially non-uniform, and the amount of strain can vary during the MIT process. Direct measurement of the strain field distribution during MIT is expected to establish a methodology for material phase identification. We have demonstrated the effectiveness of geometric phase analysis (GPA), high-resolution transmission electron microscopy techniques, and transmission electron diffraction (TED). The GPA images show that the nanoregions of interest are under tensile strain conditions of less than 0.4% as well as a compressive strain of about 0.7% (Rutile phase VO2[100] direction), indicating that the origin of the newly emerged TED spots in MIT contains a triclinic phase. This study provides a substantial understanding of the strain-temperature phase diagram and strain engineering strategies for effective phase management of nanoscale VO2.

Conservation of chicken germplasm is crucial in supporting commercial breeds for sustainable egg and meat production and preserving the genetic diversity of indigenous breeds for future breeding. Cryopreservation of chicken fertilized eggs or embryos is not feasible, owing to the large yolk-laden structure of the eggs. Primordial germ cells (PGCs), the embryonic precursors of gametes, are the best candidates for the cryobanking of chicken germplasm. Effective cryobanking of chicken PGCs requires an optimal cryopreservation protocol. Cryomedia containing dimethyl sulfoxide (DMSO) or DMSO combined with serum have been widely used for the cryopreservation of chicken PGCs. However, as cryoprotectants are yet to be optimized for chicken PGCs, the efficacy of cryomedia can be further improved. Here, we investigated the cryoprotective effects of propylene glycol (PG), an alternative to DMSO, on chicken PGCs. We found that the addition of non-permeable cryoprotectants, such as trehalose or chicken serum, to DMSO or PG cryomedia improved the recovery and survival rates of post-thawed PGCs. We further investigated the cryoprotective effects of trehalose and chicken serum and found that these additives have different cryoprotective actions. Based on these findings, we designed two different cryomedia: DTS, including 5% DMSO, 0.3 M trehalose, and 1% chicken serum, and PTS, including 7.5% PG, 0.1 M trehalose, and 5% chicken serum. Among the different PGC lines and freshly isolated PGCs, the cryomedia showed similar post-thaw recovery rates. Following transplantation, post-thawed male PGCs can colonize gonads and differentiate into functional sperm. We successfully revived the offspring of Kurokashiwa, a rare chicken breed in Japan, with cryopreserved PGCs. In conclusion, we developed two different cryomedia that achieved > 50% recovery of viable PGCs after thawing while maintaining germline competency.

Abstract Background Previous clinical trials have demonstrated the potential efficacy of poly (ADP-ribose) polymerase (PARP) inhibitors (PARPis) in patients with cancer involving homologous recombination repair (HRR) gene-mutation. Moreover, HRR gene-mutated cancers are effectively treated with immune checkpoint inhibitors (ICIs) with the increase in tumor mutation burden. We have proposed to conduct a multicenter, single-arm phase II trial (IMAGENE trial) for evaluating the efficacy and safety of niraparib (PARPi) plus programmed cell death-1 inhibitor combination therapy in patients with HRR gene-mutated cancers who are refractory to ICIs therapy using a next generation sequencing-based circulating tumor DNA (ctDNA) and tumor tissue analysis. Methods Key eligibility criteria for this trial includes HRR gene-mutated tumor determined by any cancer gene tests; progression after previous ICI treatment; and Eastern Cooperative Oncology Group Performance Status ≤ 1. The primary endpoint is the confirmed objective response rate (ORR) in all patients. The secondary endpoints include the confirmed ORR in patients with HRR gene-mutation of ctDNA using the Caris Assure (CARIS, USA). The target sample size of the IMAGENE trial is 57 patients. Biomarker analyses will be performed in parallel using the Caris Assure, proteome analysis, and T cell repertoire analysis to reveal tumor immunosurveillance in peripheral blood. Expected outcome Our trial aims to confirm the clinical benefit of PARPi plus ICI combination therapy in ICI-resistant patients. Furthermore, through translational research, our trial will shed light on which patients would benefit from the targeted combination therapy for patients with HRR gene-mutated tumor even after the failure of ICIs. Trial registration The IMAGENE trial: jRCT, Clinical trial no.: jRCT2051210120, Registered date: November 9, 2021.

Two-dimensional (2D) material is drawing considerable attention as a promising thermoelectric material. This study establishes the formation method of renewed Ca-intercalated group IV 2D materials, Ca(Ge _1− _x Sn _x ) _2 crystals including germanene-based 2D layers. The solid phase epitaxy allows us to form epitaxial Ca(Ge _1− _x Sn _x ) _2 on Si. Atomic force microscopy reveals that the Ca(Ge _1− _x Sn _x ) _2 has island structures. X-ray diffraction proved the epitaxial growth of the Ca(Ge _1− _x Sn _x ) _2 island structures and the increase of the c -axis lattice constant with Sn content increase. The formation of this renewed intermetallic compound including group IV 2D layer opens an avenue for high performance thermoelectric generator/Si.

Abstract In birds, sex is determined through cell-autonomous mechanisms and various factors, such as the dosage of DMRT1. While the sex-determination mechanism in gonads is well known, the mechanism in germ cells remains unclear. In this study, we explored the gene expression profiles of male and female primordial germ cells (PGCs) during embryogenesis in chickens to predict the mechanism underlying sex determination. Male and female PGCs were isolated from blood and gonads with a purity > 96% using flow cytometry and analyzed using RNA-seq. Prior to settlement in the gonads, female circulating PGCs (cPGCs) obtained from blood displayed sex-biased expression. Gonadal PGCs (gPGCs) also exhibited sex-biased expression, and the number of female-biased genes detected was higher than that of male-biased genes. The female-biased genes in gPGCs were enriched in some metabolic processes. To reveal the mechanisms underlying the transcriptional regulation of female-biased genes in gPGCs, we performed stimulation tests. Retinoic acid stimulation of cultured gPGCs derived from male embryos resulted in the upregulation of several female-biased genes. Overall, our results suggest that sex determination in avian PGCs involves aspects of both cell-autonomous and somatic-cell regulation. Moreover, it appears that sex determination occurs earlier in females than in males.

We used a real-world database (GuardantINFORMTM) to analyze the treatment choices for patients with mCRC who underwent next-generation sequencing of circulating tumor DNA (ctDNA) using a commercially available test (Guardant360®) after first- or second-line therapy. From 18,875 patients with claims for CRC, 1064 had confirmed metastatic disease and sufficient histories for analysis (median age 59 years, 44.8% female, 44.5% left-sided). ctDNA was detectable for 997/1064 (93.7%) patients. Clinically actionable molecular profiles were present for 507/1064 (47.7%) patients, including those who had not received targeted therapy in the previous line (410/926, 44.3%). Second- or third-line targeted therapies were administered to 338/1064 patients (31.8%) and were considered matched for 193/338 (57.1%) patients. Therapies administered after testing were informed by the ctDNA results in 56.7% of patients overall (603/1064). Time to treatment discontinuation was most favorable for patients with a clinically actionable ctDNA profile who received matched therapy. This analysis demonstrates the real-world clinical value of plasma-based comprehensive genomic profiling for selecting appropriate molecular-targeted therapies in mCRC patients with disease progression after first- or second-line therapy.

High temperatures lead to DNA double-strand break accumulation and asynapsis that trigger the meiotic checkpoint and apoptosis of spermatocytes in ex vivo cultures, providing a rationale for the heat sensitivity of sperm production in mammals.

Patients with advanced duodenal carcinoma usually have a poor prognosis due to limited effective chemotherapy options. The study for genotype-directed therapy in patients with duodenal carcinoma is progressing. However, no clinical data assessing the efficacy of molecularly targeted therapy are presently available. We report the case of a 64-year-old woman who was diagnosed with anaplastic lymphocyte kinase (ALK) fusion-positive advanced duodenal carcinoma. Echinoderm microtubule associated protein like-4 (EML4)-ALK rearrangement was detected by comprehensive genomic profiling after resistance to first-line chemotherapy. The patient received alectinib, an ALK inhibitor, with marked shrinkage in primary tumor and liver metastases. She is currently being treated with alectinib for 6 months or more. This is the first report of the efficacy of alectinib in a patient with duodenal carcinoma harboring ALK fusion. Additionally, this case report suggests that the practical use of next-generation sequencing may expand optimal treatment choices in rare solid tumors, including duodenal carcinoma.

Abstract Background Immune checkpoint inhibitors (ICIs) are emerging agents used for the treatment of various malignant tumors. As ICIs are generally used for unresectable malignant tumors, there have been only a few reports of patients who underwent surgery after receiving these drugs. Therefore, it remains unclear how immune-related adverse events (irAEs) affect the postoperative course. Here, we report a patient with advanced gastric cancer who underwent laparoscopic hepatectomy for liver metastases after an objective response with lenvatinib plus pembrolizumab and developed hypothyroidism and hypopituitarism as irAEs in the immediate postoperative period. Case presentation A 73-year-old man had undergone total gastrectomy for pT4aN2M0 gastric cancer followed by adjuvant chemotherapy with S-1 and docetaxel, and developed liver metastases in segments 6 and 7. He was enrolled in phase 2 clinical trial of lenvatinib plus pembrolizumab. He continuously achieved a partial response with the study treatment, and the liver metastases were decreased in size on imaging. The tumors were judged to be resectable and the patient underwent laparoscopic partial hepatectomy for segments 6 and 7. From the 1st postoperative day, the patient continuously presented with fever and general fatigue, and his fasting blood glucose level remained slightly lower than that before the surgery. On the 4th postoperative day, laboratory examination revealed hypothyroidism and hypopituitarism, which were suspected to be irAE caused by lenvatinib plus pembrolizumab after surgery. He received hydrocortisone first, followed by levothyroxine after adrenal insufficiency was recovered. Subsequently, his fever, general fatigue, and any abnormality regarding fasting blood glucose level resolved, and he was discharged on the 12th postoperative day. After discharge, his laboratory data for thyroid and pituitary function remained stable while receiving hydrocortisone and levothyroxine without recurrence of gastric cancer. Conclusion We present a case of laparoscopic hepatectomy after receiving lenvatinib plus pembrolizumab and developed hypothyroidism and hypopituitarism after surgery. Regarding surgery after ICI therapy, it is important to recognize that irAEs might occur in the postoperative period.

Colorectal cancer (CRC) is one of the most common malignant diseases. Generally, stoma construction is performed following surgery for the resection of the primary tumor in patients with CRC. The association of CRC with the gut microbiota has been widely reported, and the gut microbiota is known to play an important role in the carcinogenesis, progression, and treatment of CRC. In this study, we compared the microbiota of patients with CRC between with and without a stoma using fecal metagenomic sequencing data from SCRUM-Japan MONSTAR-SCREEN, a joint industry-academia cancer research project in Japan. We found that the composition of anaerobes was reduced in patients with a stoma. In particular, the abundance of Alistipes, Akkermansia, Intestinimonas, and methane-producing archaea decreased. We also compared gene function (e.g., KEGG Orthology and KEGG pathway) and found that gene function for methane and short-chain fatty acids (SCFAs) production was underrepresented in patients with a stoma. Furthermore, a stoma decreased Shannon diversity based on taxonomic composition but increased that of the KEGG pathway. These results suggest that the feces of patients with a stoma have a reduced abundance of favorable microbes for cancer immunotherapy. In conclusion, we showed that a stoma alters the taxonomic and functional profiles in feces and may be a confounding factor in fecal microbiota analysis.

Managing heat dissipation in nanoscale electronic devices and understanding the underlying mechanisms complicated due to the reduced scale at the interface between the various materials. Here, the authors detect an extremely small interface thermal resistance is in amorphous-(a-) GeS/epitaxial-(e-) PbTe superlattice and perform calculations showing that heat conduction in nanoscale systems with high density interfaces might be controlled by phonon density of states and group velocity similarities.

Abstract Background Previous clinical trials have demonstrated the potential efficacy of rechallenge with anti- epidermal growth factor receptor (EGFR) monoclonal antibodies (mAbs) for patients with RAS/BRAF V600E wild-type metastatic colorectal cancer (mCRC). Moreover, post hoc biomarker analyses of clinical trials has suggested that RAS status in circulating tumor DNA (ctDNA) has a high probability to select patients who could benefit from anti-EGFR mAb rechallenge. Methods This trial is composed of 2 phases: a monitoring phase (REMARRY) and a trial phase (PURSUIT). A monitoring phase, the REMARRY study, aims to evaluate the dynamics of plasma RAS status during the subsequent treatments after refractory to anti-EGFR therapy in patients with mCRC with RAS/BRAF V600E wild-type tumors who have progressed after a response to previous anti-EGFR therapy, using a highly sensitive digital polymerase chain reaction OncoBEAM RAS CRC kit in a central laboratory (Sysmex, Japan). A trial phase, the PURSUIT trial, is a multicenter, single-arm phase II trial to assess the efficacy and safety of rechallenge therapy with panitumumab plus irinotecan in patients without RAS mutations in ctDNA (plasma RAS negative) in the REMARRY study. Key eligibility criteria of the PURSUIT trial include RAS/BRAF V600E wild-type mCRC in tumor tissue refractory or intolerant to fluoropyrimidine, oxaliplatin, and irinotecan; progression after complete or partial response to previous anti-EGFR therapy; plasma RAS negative (defined as plasma mutant allele frequencies [MAF] of all RAS ≤ 0.1%) within 28 days prior to enrollment; 4 months or more between the last administration of previous anti-EGFR mAb and the start of protocol treatment; and Eastern Cooperative Oncology Group (ECOG) Performance Status (PS) ≤ 1. The primary endpoint is the confirmed objective response rate (ORR). The target sample size of the PURSUIT trial is 50 patients. Biomarker analyses will be performed in parallel using the OncoBEAM RAS CRC kit and a next-generation sequencing-based ctDNA analysis (Guardant360). Discussion Our trial aims to confirm the clinical benefit of anti-EGFR mAb rechallenge therapy in patients with plasma RAS negative. Moreover, through biomarker analyses, our trial will shed light on which patients would benefit from rechallenge in addition to being plasma RAS negative. Trial registration The REMARRY study: UMIN, UMIN000036424 . Registered date: April 5, 2019. The PURSUIT trial: jRCT, jRCTs031190096 . Registered date: October 1, 2019.

This study aimed to evaluate the differences between the growth patterns of large- and normal-sized Japanese quail strains and their F1 progeny, by fitting their growth parameter values to five nonlinear regression growth models (Weibull, Logistic, Gompertz, Richards, and Brody). The Richards model presented the best fit for both sexes of the large-sized quail strain, whereas the Gompertz model presented the best fit for both sexes of the normal-sized quail strain, based on goodness-of-fit criteria (higher adjusted R2 and lower Akaike and Bayesian information criteria). Both sexes of F1 birds derived from the cross between normal-sized females and large-sized males were best fitted by the Richards model. In contrast, growth parameters of the F1 birds derived from the cross between large-sized females and normal-sized males were best fitted to the Gompertz model. The data could be fitted nearly as well to the Weibull and Logistic models as to the Richards and Gompertz models. The Brody model presented the poorest fit for the growth parameter values. The results indicated that the Richards and Gompertz models could best describe the growth characteristics of both large- and normal-sized quails. Moreover, the observed growth pattern of the F1 birds was likely inherited from the male parental strain. To the best of our knowledge, this is the first study comparing the growth curves of the reciprocal F1 generations with their parental strains in quails.

Abstract Single-cell level analysis is powerful tool to assess the heterogeneity of cellular components in tumor microenvironments (TME). In this study, we investigated immune-profiles using the single-cell analyses of endoscopically- or surgically-resected tumors, and peripheral blood mononuclear cells from gastric cancer patients. Furthermore, we technically characterized two distinct platforms of the single-cell analysis; RNA-seq-based analysis (scRNA-seq), and mass cytometry-based analysis (CyTOF), both of which are broadly embraced technologies. Our study revealed that the scRNA-seq analysis could cover a broader range of immune cells of TME in the biopsy-resected small samples of tumors, detecting even small subgroups of B cells or Treg cells in the tumors, although CyTOF could distinguish the specific populations in more depth. These findings demonstrate that scRNA-seq analysis is a highly-feasible platform for elucidating the complexity of TME in small biopsy tumors, which would provide a novel strategies to overcome a therapeutic difficulties against cancer heterogeneity in TME.

For realization of new informative systems, the memristor working like synapse has drawn much attention. We developed isolated high-density Fe3O4 nanocrystals on Ge nuclei/Si with uniform and high resistive switching performance using low-temperature growth. The Fe3O4 nanocrystals on Ge nuclei had a well-controlled interface (Fe3O4/GeOx/Ge) composed of high-crystallinity Fe3O4 and high-quality GeOx layers. The nanocrystals showed uniform resistive switching characteristics (high switching probability of ~90%) and relatively high Off/On resistance ratio (~58). The high-quality interface enables electric field application to Fe3O4 and GeOx near the interface, which leads to effective positively charged oxygen vacancy movement, resulting in high-performance resistive switching. Furthermore, we successfully observed memory effect in nanocrystals with well-controlled interface. The experimental confirmation of the memory effect existence even in ultrasmall nanocrystals is significant for realizing non-volatile nanocrystal memory leading to neuromorphic devices.

The Si-nano dot substrates formed using the ultrathin silicon oxide films were applied to fabricate CaSi _2 films. The CaSi _2 formed by this process was identified as the metastable phase 2H as the main component, and the 1H structure existed partially at the grains of the 2H phase. Although no experimental reports exist for the formation of 2H-CaSi _2 crystal, the Si-nano dot substrates are considered as the high-entropy substrate to form the metastable phases. We experimentally determined the lattice parameter of the 2H phase by the annular dark field–scanning transmission electron microscopy observations using the Si as an internal standard sample.

Abstract Background A previous phase I/II C-TASK FORCE study of trifluridine/tipiracil plus bevacizumab for patients with heavily pretreated metastatic colorectal cancer (mCRC) showed promising activity with an acceptable toxicity profile. This retrospective study aimed to investigate the safety and efficacy of trifluridine/tipiracil plus bevacizumab compared with trifluridine/tipiracil monotherapy in patients with heavily pretreated mCRC in clinical settings. Methods Records of patients with mCRC refractory to standard therapies who initiated trifluridine/tipiracil plus bevacizumab from January 2016 to March 2018 or trifluridine/tipiracil monotherapy from June 2014 to December 2015 were retrospectively reviewed at our institution. Results Totally, 60 patients received trifluridine/tipiracil plus bevacizumab and 66 received trifluridine/tipiracil monotherapy. All patients had previously received standard chemotherapy. Median progression-free survival (PFS) was 3.7 months [95% confidence interval (CI), 2.3–5.1] in the trifluridine/tipiracil plus bevacizumab group and 2.2 months (95% CI, 1.8–2.6) in the trifluridine/tipiracil monotherapy group [hazards ratio (HR) 0.69; 95% CI 0.48–0.99]. PFS rate at 16 weeks was 46.6% for the trifluridine/tipiracil plus bevacizumab group and 33.9% for the trifluridine/tipiracil monotherapy group. Although a relatively higher incidence of grade ≥ 3 neutropenia was observed in the trifluridine/tipiracil plus bevacizumab group than that in the other group (50.0% vs. 40.9%, p = 0.371), the incidence of febrile neutropenia was not high (3.3% vs. 7.8%, p = 0.444). Conclusions In real-world settings, trifluridine/tipiracil plus bevacizumab prolonged PFS and helped achieve higher 16-week PFS rate compared with trifluridine/tipiracil monotherapy in patients with heavily pretreated mCRC with manageable toxicities. Trial registration Retrospectively registered.

Abstract Background Clinicopathological and molecular features of responders to nivolumab for advanced gastric cancer (AGC) are not well understood. Methods Patients (pts) with AGC who were treated with nivolumab after two or more chemotherapy regimens in a single institution from September 2017 to May 2018 were enrolled in this study. PD-L1 expression in tumor cells (TC) and mismatch repair (MMR) were analyzed by immunohistochemistry. Epstein-Barr virus (EBV) was detected by in situ hybridization. Cancer genome alterations were evaluated by a next-generation sequencing-based panel. High tumor mutation burden (TMB) was defined as more than 10 mutations/megabase. Results A total of 80 pts were analyzed in this study. Tumor response was evaluated in 72 pts with measurable lesions and 14 pts (19%) had an objective response. Overall response rate (ORR) was significantly higher in pts with ECOGPS 0 in those with PS 1 or 2, MMR-deficient (MMR-D) in those with MMR-proficient (MMR-P), PD-L1+ in TC in those with PD-L1- in TC and PIK3CA mutation in those with PIK3CA wild-type. ORR was 31% in pts with at least one of the following factors; MMR-D, high TMB, EBV+ and PD-L1+ in TC vs. 0% in those without these factors. Progression-free survival was significantly longer in pts with PS 0 than in those with PS 1 or 2, MMR-D than in those with MMR-P, and PD-L1+ in TC than in those with PD-L1- in TC. Conclusions Some features were associated with favorable response to nivolumab for AGC. Combining these features might be useful to predict efficacy.

The design and fabrication of nanostructured materials to control both thermal and electrical properties are demonstrated for high-performance thermoelectric conversion. We have focused on silicon (Si) because it is an environmentally friendly and ubiquitous element. High bulk thermal conductivity of Si limits its potential as a thermoelectric material. The thermal conductivity of Si has been reduced by introducing grains, or wires, yet a further reduction is required while retaining a high electrical conductivity. We have designed two different nanostructures for this purpose. One structure is connected Si nanodots (NDs) with the same crystal orientation. The phonons scattering at the interfaces of these NDs occurred and it depended on the ND size. As a result of phonon scattering, the thermal conductivity of this nanostructured material was below/close to the amorphous limit. The other structure is Si films containing epitaxially grown Ge NDs. The Si layer imparted high electrical conductivity, while the Ge NDs served as phonon scattering bodies reducing thermal conductivity drastically. This work gives a methodology for the independent control of electron and phonon transport using nanostructured materials. This can bring the realization of thermoelectric Si-based materials that are compatible with large scale integrated circuit processing technologies.

Background: The C-TASK-FORCE phase I/II and Danish randomized phase II trials reported the promising efficacy of trifluridine/tipiracil (TAS102) plus bevacizumab (BEV) in patients with chemorefractory metastatic colorectal cancer (mCRC). However, there had been no direct comparative phase III trial to compare the efficacy between TAS102 plus BEV and standard therapy with either TAS102 or regorafenib monotherapy. Methods: We retrospectively reviewed the medical records of patients with mCRC who received TAS102 plus BEV, TAS102 monotherapy, or regorafenib monotherapy after standard chemotherapies during 2013–2019. Results: Patients received TAS102 plus BEV ( n = 139), TAS102 monotherapy ( n = 153), or regorafenib monotherapy ( n = 133). With a median follow-up of 25.3 months, median overall survival (OS) was 11.5 months [95% confidence interval (CI), 9.9–13.9] for TAS102 plus BEV, 8.1 months (95% CI, 6.8–9.2) for TAS102 monotherapy, and 6.8 months (95% CI, 5.7–8.5) for regorafenib monotherapy. The hazard ratios were 0.67 (95% CI, 0.51–0.88) for TAS102 plus BEV versus TAS102 monotherapy and 0.71 (95% CI, 0.54–0.94) for TAS102 plus BEV versus regorafenib monotherapy. Median progression-free survival (PFS) was 4.4 months (95% CI, 3.7–5.4) for TAS102 plus BEV, 2.5 months (95% CI, 1.6–2.3) for TAS102 monotherapy, and 2.1 months (95% CI, 1.6–2.3) for regorafenib monotherapy. The hazard ratios were 0.57 (95% CI, 0.45–0.73) for TAS102 plus BEV versus TAS102 monotherapy and 0.44 (95% CI, 0.34–0.58) for TAS102 plus BEV versus regorafenib monotherapy. On multivariate analysis, TAS102 plus BEV was independently correlated with better OS and PFS. No unexpected adverse events were observed in any group. Conclusion: Our study shows that OS and PFS are longer in patients treated with TAS102 plus BEV than in those treated with TAS102 or regorafenib monotherapy.

Background The efficacy and safety of chemotherapy (CTx) after anti-PD-1 therapy in patients with advanced gastric cancer (AGC) remains unclear.Methods Medical records of consecutive patients with AGC treated with both CTx (taxanes plus ramucirumab, taxanes monotherapy or irinotecan) and anti-PD-1 therapy from June 2015 to April 2019 were retrospectively analysed. Patients were divided into two groups based on prior exposure to anti-PD-1 therapy: anti-PD-1-exposed and anti-PD-1-naïve groups. CTx-related outcomes were compared between two groups in the overall population and each CTx population.Results In total, 233 patients (67 anti-PD-1-exposed, 166 anti-PD-1-naïve) were included. In the overall population, the objective response rate (ORR) to CTX was 44.6% in the anti-PD-1-exposed group and 19.6% in the anti-PD-1-naïve group (p=0.001); the median progression-free survivals (PFS) were 3.7 months and 3.3 months (HR=0.82, p=0.20), respectively. Among patients receiving taxanes plus ramucirumab (n=149), ORR (60.6% vs 20.0%, p

Background: The use of nivolumab or irinotecan as the third-line treatment for patients with advanced gastric cancer (AGC) remains controversial. Methods: This study analyzed patients with AGC treated with nivolumab or irinotecan (nivolumab group or irinotecan group, respectively) from May 2016 to April 2019 following two or more previous lines of chemotherapy. Univariate survival analysis was conducted to identify the clinical and molecular factors associated with progression-free survival (PFS). Results: A total of 156 patients (74 treated with nivolumab and 82 treated with irinotecan) were analyzed. The median PFS was 1.9 months in both treatment groups. The median overall survival (OS) was 7.2 and 6.2 months in the nivolumab and irinotecan groups, respectively. Eastern Cooperative Oncology Group performance status of 1 or more, liver metastasis, a large tumor size at baseline, and HER2-positive status were associated with a worse PFS in the nivolumab group compared with the irinotecan group. The nivolumab group showed a significantly longer PFS (median 3.1 versus 2.0 months) and OS (median 12.9 versus 7.8 months) than the irinotecan group in patients with 0 or 1 of these factors, whereas the irinotecan group showed a significantly longer PFS (median 1.0 versus 1.8 months) and a trend of longer OS (median 3.9 versus 6.1 months) in patients with ⩾2 of these factors. Conclusions: Some clinical and molecular factors were associated with outcomes following nivolumab or irinotecan as the third- or later-line treatment in patients with AGC. These factors must be considered while selecting an optimal treatment option.

The motion analysis of two-finger tip pinching using the thumb and index finger provides crucial data for designing the motion mechanism of electric prosthetic hands. The purpose of this study is to determine the joints that have high mobility during two-finger tip pinching by measuring the flexion angle of each joint. Ten Japanese men with normal hand were selected. CT images were obtained while the hands adopted the following four postures: a basic posture not pinching a cylinder, and three postures pinching wooden cylinders with different diameters (2, 10, and 30 mm). Three-dimensional bone models of the thumb and index finger were created using the CT images and used to measure the flexion angles of the joints. The flexion angles of the proximal interphalangeal and metacarpophalangeal joints of the index finger significantly decreased as the diameter of the cylinder increased. However, even when the diameter of the cylinder changed, the flexion angle of the distal interphalangeal joint of the index finger, and the flexion and rotation angles of all of the thumb joints did not change. When pinching objects of different sizes with a two-finger tip pinch, the posture of the thumb is fixed, and only the posture of the index finger changes. When designing the two-finger tip pinch motion for an electric prosthetic hand, it is sufficient to drive the joints of the index finger only.

The mutant plumage color “extended brown (EB)” of the blue-breasted quail was genetically investigated. Mating experiments revealed that the EB plumage is controlled by an autosomal, incompletely dominant allele, for which we propose the symbol Eb. The EB plumage is characterized by dark brown color, and homozygotes for this mutation generally showed darker pigmentation than the heterozygotes. DNA sequencing and PCR-RFLP analyses of the EB mutants showed a rigid association between the EB plumage and a G-to-A nucleotide substitution at position 274 in the melanocortin 1-receptor gene (MC1R), clearly indicating that MC1R is the candidate gene for the EB plumage color in the blue-breasted quail.

Introduction: Tuberculous meningitis is rare but one of the most severe forms of tuberculosis infection. Case Report: A 78-year-old woman was diagnosed with advanced gastric cancer with multiple lymph node metastases. Four months after the beginning of second-line chemotherapy with weekly paclitaxel, she was admitted to our hospital because of fever and mild drowsiness. She had no other symptoms and no abnormalities in physical examinations. Her blood tests, urinalysis, and blood culture revealed no remarkable abnormal findings. Although her symptoms relieved, her disturbance of consciousness gradually progressed during 2 weeks thereafter. Finally, we diagnosed tuberculous meningitis on the 22nd day of hospitalization by a positive acid-fast bacilli test of the cerebrospinal fluid and tuberculosis-polymerase chain reaction. Although anti-tuberculosis therapy was started, she died on the 37th day of hospitalization because of tumor bleeding. Conclusion: To the best of our knowledge, this is the first report of tuberculous meningitis during chemotherapy for advanced gastric cancer, suggesting that subacute onset of fever followed by disturbance of consciousness may indicate the possibility of tuberculous meningitis even without typical signs of meningitis including headache or meningeal irritation.

Semiconductor nanocrystals are potential nanoelectronic materials but often display nonuniform electric properties due to their anisotropic growths. Here, the authors report cross-sectional cathodoluminescence imaging of a single-crystalline ZnO nanowire to resolve its growth sectors with different carrier concentrations.

In the present study, a simplified, one-way coupled, Discrete Droplet Method is proposed to model multiple rain-droplet motions over the vehicle wall. The Lagrangian method is employed to track the droplet, on which four forces are considered to act, i.e., aerodynamic drag, adhesion, viscous friction, and gravity. Based on the numerical experiment for one droplet, we assumed a “PATH” created by a droplet on the vehicle surface, meaning a wet region, on which the viscous friction was reduced once the droplet had passed. By taking this simple PATH effect into account, better agreements with the corresponding experiment were observed for multiple-droplet cases with different vehicle configurations and flow speeds. This method appears to have a good balance between accuracy and efficiency, and will potentially be applied to other engineering problems such as iced-aircraft.

In this study, the distribution of surface heat transfer induced by dual side-jets injected into a hypersonic flow has been visualized using a temperature sensitive paint. The experiments were performed in both tandem and parallel injector arrangements, and the spacing between the injection holes was taken as a parameter in each arrangement. As a result, the aerodynamic heating in the separated region of the boundary layer and in the horseshoe vortex was clearly visualized. In the tandem arrangements, heat transfer remarkably increased immediately upstream of the front injector. The distributions and the intensity of surface heat transfer were similar to those caused by the single injection. On the other hand, in the parallel arrangements, the extent of the separation nearly doubled, and the maximum heat flux decreased to less than half of that from the single injection. The global distribution of heat transfer varied significantly as the injector spacing was changed. When the injectors were positioned with a large spacing, the interaction between the side-jets was relatively lowered, and thus distribution, as for the single injector, was induced around each injection hole individually. In contrast, with a short spacing, the dual injection behaved as a single obstacle. The most effective reduction of maximum heat flux was achieved with an intermediate injector spacing.

The production method of germline chimeras by transfer of primordial germ cells (PGCs) can be used in gene banking of chickens. However, little is known about the reproductive capability of offspring obtained by mating of male and female germline chimeras. To cryopreserve chicken genetic resources at the cellular level in future, it is necessary to demonstrate whether offspring originating from donor-derived PGCs have normal reproductive capability. In this study, we attempted to obtain the pure Hinai-droi offspring by mating of male and female germline chimeras produced by the transplantation of Hinai-dori PGCs into the Akita White Leghorn line (WL) embryos. Hinai-dori offspring from germline chimeras were screened using the Hinai-dori-specific autosomal microsatellite marker, namely, ADL0315, which allows distinction between the Hinai-dori breed and Akita WL. After sexual maturity of the Hinai-dori offspring originating from donor-derived Hinai-dori PGCs, the reproductive capability (egg production rate, fertilization rate and hatchability) was examined. Percentage hen-day egg production was investigated between 21 and 60 wk of age, and fertilization rate and hatchability were investigated between 43 and 60 wk of age, respectively. Among the 105 progeny obtained by mating of male and female germline chimeras, a single brown-colored female hatchling was obtained. The results from molecular genotyping using the Hinai-dori-specific microsatellite marker ADL0315 were consistent with results from morphological identification, and the brown-colored female hatchling was identified as the pure Hinai-dori breed. The mean percentage hen-day egg production, fertilization rate and hatchability of the Hinai-dori hen were 36.4%, 58.1% and 53.2%, which were similar to those of normal Hinai-dori hens. These results indicated that the pure Hinai-dori offspring originating from donor-derived Hinai-dori PGCs has normal reproductive capability.

Transplantation of primordial germ cells (PGCs), which are the progenitor cells of gametes, is a powerful tool for generation of transgenic chickens. However, the frequencies of transgene integration into the genome of purified PGCs still remain low. An in vitro culture system enabling chicken PGCs to propagate efficiently would be useful for efficient transgenesis of PGCs. In the present study, we optimized the culture conditions for chicken PGCs to enhance the proliferation and evaluated the germline transmission of cultured PGCs that proliferated for long periods of time. PGC-like cells (PGC-LCs), that have remarkably similar morphological characteristics to intact PGCs, could be derived by cultivation of blood containing PGCs obtained from 2.5-day-old chicken embryos according to the protocol of van de Lavoir et al. (2006). We determined which feeder cells and which growth factors were required to improve proliferation of PGC-LCs. Male PGC-LCs survival and proliferation were enhanced during culture in the basic medium containing either basic fibroblast growth factor (bFGF) alone or both bFGF and stem cell factor (SCF) on a feeder of buffalo rat liver (BRL) cells. Male PGC-LCs could be propagated in defined culture condition for extended periods. These cells expressed the germline-specific protein Vasa and undifferentiated cell marker stage-specific embryonic antigen-1 (SSEA-1) and pluripotency genes Nanog and PouV. Furthermore, Male PGC-LCs cultured for 225 d could migrate toward and colonize within recipient gonads and transmit to the next generation following transplantation. We succeeded in produce 3 offspring originating from long-term cultured PGC-LCs from a germline chimeric rooster (6%). The present study represents valuable steps toward defining a culture condition enabling PGC-LCs to propagate efficiently for long periods in vitro with maintenance of their commitment to the germline.

Eminectomy which is one of the popular and most effective treatments for habitual temporomandibular joint luxation was first described by Myrhaug in 1951. There are few reports which described eminectomy being performed under local anesthesia and conscious sedation. We present a case series of habitual luxation of the TMJ treated by eminectomy performed under local anesthesia and conscious sedation and general anesthesia. Five patients were examined and found to have recurrent luxation of the TMJ. The age of patients ranged from 18 to 93 years. Bilateral eminectomy of the TMJ was performed for two patients, and unilateral eminectomy was performed for three patients. Two were examined under intravenous propofol sedation and local anesthesia, while three patients were examined under general anesthesia. One patient died from ileus one month after surgery. The follow-up period except for the case that died from ileus ranged from 12 to 33 months. No recurrent dislocation of the TMJ has been identified. Based on our experience and two other series in the literature, eminectomy with sedation and local anesthesia can be considered and might be a good option in elderly patients.

We numerically simulated the aerodynamic noise generated from the unsteady flow around a generic model of automobile rear-view mirrors in this study. This generic model is made by cutting a hemispherical cylinder into half lengthwise, and placed on a flat plate. The governing equations employed in the present numerical simulation are the compressible Navier-Stokes equations, where no additional models of sound generation and propagation are used. Results show that acoustic pressure waves are generated from the disturbed wake of the mirror model and propagate into the far field. In addition, the spatial distribution of the sound pressure level (SPL) in the far field has a similar tendency to that of the experimental data. These results show that useful data on aerodynamic noise can be obtained by the simple computational method presented in this study, and utilized to make a preliminary design to reduce the aerodynamic noise.

The production of germline chimeric chickens by transfer of primordial germ cells (PGCs) is an effective technique for the preservation and regeneration of genetic resources in chickens. To date, the most widely used method to identify germline chimeric chickens is a testcross using differences in plumage color between the donor and recipient breeds. However, this method is time consuming and laborious. Therefore, simple high-precision molecular techniques for the identification of germline chimeric chickens need to be developed. In this study, we verified the practicability of using a Hinai-dori-specific microsatellite marker that was previously developed to identify germline chimeric chickens. A Hinai-dori-specific microsatellite marker ABR0633 can distinguish the Hinai-dori breed from White Leghorn (WL); thus, these two breeds were used as donor and recipient embryos, respectively, in this study. PGCs obtained from embryonic gonads of Hinai-dori were micro-injected into either the subgerminal cavity or dorsal aorta of WL recipient embryos to produce germline chimeric chickens. A portion of both left and right testes was removed from the manipulated male chicks and was used for genotyping to verify the possibility of identifying germline chimeric chickens. Semen was then obtained from matured manipulated male chickens and was used for genotyping. Simultaneously, these chickens were crossed with Hinai-dori females by artificial insemination to produce offspring. No donor-derived Hinai-dori allele was detected in the testicular tissues from the manipulated male chicks. However, donor-derived Hinai-dori allele was detected in the semen from two manipulated male chickens (2/2) that were produced by microinjection of Hinai-dori PGCs into the dorsal aorta. In the progeny tests, Hinai-dori PGC-derived offspring were obtained from these two chickens. Moreover, only the donor-derived Hinai-dori allele was detected in the offspring that were judged as the Hinai-dori breed by plumage color. Therefore, the offspring were confirmed as the Hinai-dori breed both phenotypically and genotypically. We conclude that a Hinai-dori-specific microsatellite marker is suitable to identify germline chimeric chickens and the use of this method could reduce the time and labor needed for testcrosses.

The purpose of this study is to clarify the effects of freeze-thaw treatment on viability and functionality of primordial germ cells (PGCs) in chickens. PGCs were collected separately from embryonic blood of White Leghorn, Barred Plymouth Rock and Fayoumi breeds. Some PGCs were labeled with the fluorescent lipophilic carbocyanine dye to analyze functionality by transfer assay. 100 PGCs were used for each experiment to ensure accuracy of the test results. In the experimental group, PGCs were slow-frozen, then stored in liquid nitrogen for 1 month. In the unfrozen control group, PGCs were utilized immediately. The recovery rate of PGCs after freeze-thaw was 54.3%. The viability of PGCs in the frozen group was significantly lower than that of the control group (P

In this study, effects of the tip cavity with various depths, widths, and locations on the leakage flow and performance of an axial turbine cascade have been investigated numerically. The blade was a linear model of the tip section of the GE-E3 high-pressure turbine first-stage rotor blade. The Delayed Detached Eddy Simulation (DDES) model was used in the simulations. The computational results showed that the leakage mass flow rate and mass-averaged total pressure loss decreased as the depth and width of the tip cavity increased. And, it was shown that the cavity near the pressure side is more effective than that near the suction side. These effects depend on a vortex generated behind the pressure side in the cavity, which is changed with the depth, width, and location. The vortex entrains the leakage flow through the clearance toward the bottom of the cavity and in the chord wise direction, which reduces the flow leaking out.

The present study aims to control the leakage flow and total pressure loss in high pressure turbines. A new tip shape is proposed. It is based on the triple squealer shape by adopting a new middle squealer, along the camber line, whose first and last thirds were removed. An unstructured, finite volume, multiblock, 3-D, compressible Reynolds-Averaged Navier-Stokes equations solver was used to compute the flow through a high pressure turbine cascade. The turbulent viscosity was calculated by the Delayed Detached Eddy Simulation (DDES) model which is based on the Spalart-Allmaras one equation turbulence model. The performance of the new shape is compared to that of flat tip, double and triple squealers. The results successfully demonstrate that the new shape has the least total pressure loss among them.

Numerous studies in mammalian species have recently been reported that many stem cells have an ability to efficiently efflux the vital DNA-binding dye Hoechst 33342, and it is called side population (SP) cells. However, few study have been reported on the avian SP cells. It could be possible that concentration of hematopoietic stem cells (HSCs) in birds since the characteristic of SP cells should be shared in various tissues and species. In this study, we first attempted the isolation of SP cells from chicken bone marrow and the assessment by gene expression and morphologic analyses. Bone marrow cells (BMCs) were flushed from the femurs and tibias of chicks aged at 10 days with PBS. The BMCs were layered on lymphocyte separation medium and centrifuged for excluding the erythrocytes. The separated cells were adjusted to 106/ml in HBSS. Hoechst 33342 were added (1.25μg/ml) and incubated 60 to 90 minutes at 37°C. Propidium iodide was added (2μg/ml) to exclude dead cells. The SP cells were isolated with flow cytometer. The sorted cells were stained with May-Gruenwald Giemsa (MG) for morphological analysis and RNA was extracted for gene expression analysis. The avian SP cells which was vanished by addition verapamil counld be separated. The percentage of SP cells in chicken bone marrow was about 2.6%. The morphological analysis by MG staining indicated that the SP cells had a larger nuclear and little cytoplasm which were typical characterisation of mouse HSCs. The pattern of gene expressions (CD34, c-Kit, CD4 and CD8) in SP cells also resembled that of the mouse HSCs. These results suggested that the HSCs could be enriched from avian bone marrow cells. Together with these results, it was concluded that SP is one of powerful tools for concentration of avian stem cells.

Individual differentiated somatic cells and undifferentiated stem cells have common genome, although their functions or morphological characters are very different. These differences are derived from difference of gene expression pattern. DNA methylation is generally key factor of suppression of gene and its level is globally change during mammalian early development. But, in birds, whether genome-wide changes in DNA methylation occur during embryonic development is still unknown. Here, we show that genome-wide DNA methylation to assess occurrence during early chick embryonic development. We found that the methylation status at stage 1 was approximately 57%, after which it gradually decreases, reaching a minimum at stage 10 (33%). After stage 10, DNA methylation gradually increased. These results should contribute to clarify the epigenetic mechanisms in birds.

The current study aimed to determine the optimal time of busulfan (1,4-butanediol dimethanesulfonate) administration for the preparation of suitable recipient embryos to generate germline chimeras in chickens. We compared two different administration times, with regards to the degree of endogenous primordial germ cell (PGC) depletion and donor PGC incorporation in recipient embryos. A dose of 75μg busulfan in 50μL sustained-release emulsion was injected into the yolk of chicken embryos incubated for 0h or 24h. Both busulfan treatments resulted in significant reduction in the number of PGCs compared with that of controls (P

The generation of germline chimeras by the transfer of primordial germ cells (PGCs) requires incorporation of the PGCs of the donor into the gonadal tissue of the recipient embryo. We investigated the utility of soft x-irradiation with application of a lead (12×3×0.25mm, ∼0.1g) shield to the embryo proper for the production of chicken-quail germline chimeras. Chicken embryos shielded during irradiation for 120 s (∼7.2Gy) at stages 13 to 17 showed a hatchability of 35% (106/301), whereas the hatchability of unshielded embryos was 26% (27/105). The relative population of gonadal PGCs at stage 30 for embryos irradiated at stage 13 with or without shielding was 13 and 5%, respectively, of the value for nonirradiated controls. Chicken embryos irradiated at stages 13 or 14 with or without shielding and transfused with quail embryonic blood containing PGCs each exhibited ∼130 relative population of donor PGCs in the left gonad at stage 30. Xenotransplanted hatchlings exhibited donor-derived PGCs as detected by Southern hybridization and PCR. Exposure of chicken embryos to ∼7.2Gy of x-radiation at stage 13 with the application of a lead shield to the embryo proper is thus a feasible approach to depletion of endogenous germ cells and the production of chicken-quail germline chimeras.