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Abstract This study aimed to determine the efficacy of using both the postero-anterior and oblique image intensifier views intra-operatively and late start of post-operative partial weight-bearing (PWB) in reducing the incidence of delayed union in the ischium at one year after curved periacetabular osteotomy. We evaluated computed tomography images to clarify the incidence of delayed union at the osteotomy sites at one year post-operatively for 117 hips in 104 patients. Fifty-eight hips in 54 patients with use of both the postero-anterior and oblique image intensifier views intra-operatively and late start of post-operative PWB were assigned to the oblique view and late PWB group (OL group) and 59 hips in 50 patients with use of only the postero-anterior image intensifier view intra-operatively and early start of post-operative PWB were assigned to the control group (C group). In univariate analyses, the incidence of delayed union in the ischium at one year post-operatively was significantly lower in the OL group (3.5%) than in the C group (22%). Use of both the postero-anterior and oblique image intensifier views during curved periacetabular osteotomy and late start of PWB were effective for reducing delayed union of the ischium at one year post-operatively.

Abstract In vitro reconstruction of highly mature engineered heart tissues (EHTs) is attempted for the selection of cardiotoxic drugs suitable for individual patients before administration. Mechanical contractile force generated in the EHTs is known to be a critical indicator for evaluating the EHT response. However, measuring contractile force requires anchoring the EHT in a tailored force‐sensing cell culture chamber, causing technical difficulties in the stable evaluation of contractile force in long‐term culture. This paper proposes a hydrogel‐sheathed human induced pluripotent stem cell (hiPSC)‐derived heart microtissue (H3M) that can provide an anchor‐free contractile force measurement platform in commonly used multi‐well plates. The contractile force associated with tissue formation and drug response is calculated by motion tracking and finite element analysis on the bending angle of the hydrogel sheath. From the experiment of the drug response, H3M is an excellent drug screening platform with high sensitivity and early testing capability compared to conventionally anchored EHT. This unique platform would be useful and versatile for regenerative therapy and drug discovery research in EHT.

Abstract Fires in petrochemical complexes are inaccessible because of the intense radiant heat from flames. Therefore, water cannon robots require radiant heat countermeasures to perform firefighting safely. Conventional radiant heat countermeasures employ a self-spraying method, wherein the water cannon robot requires a water tank of capacity 1.5 m3 (= 1500 L) to function for 7–8 min in an environment with a 20 kW/m2 radiation heat. However, the water cannon robot has size limitations because it is transported on one transport vehicle (10 t truck) to the site, and only a tank of capacity ~ 0.02 m3 can be installed on the robot. To overcome these drawbacks, this study proposes a method that utilizes a mountable radiant heat-resistant exterior cover that works with a small amount of water. The cover is made of radiant heat-shielding fireproof clothing with an aluminum coating that reflects 90% of the radiant heat on the surface and a mist nozzle that sprays water on the back surface. The remaining 10% is removed by the heat of vaporization of water sprayed on the back of the clothing and natural convection. The amount of water required for cooling was reduced to 1/80th of that compared to self-spraying because of the use of the developed cover. The proposed method of radiation reflection via vaporization and natural convection can be employed to protect firefighting robots.

Abstract Background The relationship between chronological nutritional changes and development of fatty liver after total gastrectomy (TG) in gastric cancer (GC) patients is still unclear. This study aimed to evaluate relationship between development of fatty liver and chronological changes of nutritional parameters during 12 months after TG. Methods We retrospectively analyzed medical records of 59 patients with GC who underwent TG at the Kanazawa Medical University Hospital between January 2009 and December 2017. We defined fatty liver change as a mean liver-to-spleen attenuation ratio (L/S ratio) of less than 1.2 in the computed tomography images at 12 months after TG and divided the patients into fatty liver (FL) and non-FL groups from the L/S ratio. We analyzed serum levels of total protein and albumin, and psoas muscle index (PMI) before TG and at 6 and 12 months after TG in the non-FL and FL groups. Results Six patients showed an L/S ratio of less than 1.2 at 12 months after TG and were included into FL group. There was no significant difference between the groups in serum parameters, L/S ratio, and PMI before TG. In the FL group, the mean levels of total protein and albumin decreased after TG and were significant lower at 6 months, compared with the non-FL group. And then, these levels in the FL group recovered at 12 months. In contrast, the mean levels of total protein and albumin in the non-FL group did not decrease below the preoperative levels throughout the year after surgery. As with laboratory parameters, all patients in the FL group showed decrease of PMI at 6 months after TG. This proportion was significantly higher than that in the non-FL group (100% vs. 40.8%, P = 0.006). Conclusions We evaluated that the patients with fatty liver occurring after TG had significantly lower levels of serum nutritional parameters and skeletal muscle index at 6 months, not but 12 months, after TG.

Summary: We describe a protocol for the efficient culture of human pluripotent stem cells (hPSCs) by supplementing conventional culture medium with L-tryptophan (TRP). TRP is an essential amino acid that is widely available at an affordable cost, thereby allowing cost-effective proliferation of hPSCs compared to using a conventional medium alone. Here, we describe the steps for enhanced proliferation of hPSCs from dermal fibroblasts or peripheral blood cells, but the protocol can be applied to any hPSCs.For complete details on the use and execution of this protocol, please refer to Someya et al. (2021).

Summary: Here we describe a protocol to obtain highly pure cardiomyocytes and neurons from human induced pluripotent stem cells (hiPSCs) via metabolic selection processes. Compared to conventional purification protocols, this approach is easier to perform and scale up and more cost-efficient. The protocol can be applied to hiPSCs and human embryonic stem cells.For complete details on the use and execution of this protocol, please refer to Tohyama et al. (2016) and Tanosaki et al. (2020).

Abstract Introduction In patients with gastric cancer, 6–27% of patients are diagnosed with T4b disease that invades adjacent organs, and curative resection can improve the prognosis of these patients. Case presentation A 70-year-old Japanese man presented with an abdominal tumor and was diagnosed with advanced gastric cancer (L-Circ type 3 T4b N2 M0 H0 stage IVA, based on the 15th edition of the Japanese Classification of Gastric Carcinoma) with extensive abdominal wall invasion. We performed open gastrojejunal bypass for gastric obstruction and initiated a chemotherapeutic regimen comprising S-1 (120 mg/day) and oxaliplatin (100 mg/m2). Upper gastrointestinal endoscopy performed after the administration of six courses of the S-1 and oxaliplatin regimen revealed a persistent primary lower gastric wall lesion; however, the diameter of the abdominal wall invasion and metastatic lymph nodes was significantly reduced, in addition to decreased serum carcinoembryonic antigen and carbohydrate antigen 19-9 levels. Subsequently, the patient underwent distal gastrectomy with D2 lymphadenectomy combined with transverse colon and abdominal wall resection. We performed radical en bloc resection and achieved a tumor-free resection margin. Simple abdominal wall closure was performed without mesh or musculocutaneous flap placement. Histopathological examination of the resected tumor specimen showed direct invasion of the mesocolon and rectus abdominis muscle. The patient was postoperatively diagnosed with L Gre-Ant type5 T4b (SI: rectus abdominis muscle) N2 PM0 DM0 Stage IIIA R0 Grade 2a gastric cancer based on histopathological findings and received S-1 as adjuvant chemotherapy, 2 months postoperatively. No recurrence was detected 6 months postoperatively. Conclusions We report a case of advanced gastric cancer with extensive abdominal wall invasion that was successfully treated with gastrectomy combined with resection of adjacent organs showing tumor invasion after effective systemic chemotherapy. A therapeutic approach comprising curative surgery combined with perioperative chemotherapy is useful in patients with T4b gastric cancer.

Abstract Pluripotent stem cells (PSCs) exhibit promising application in regenerative therapy, drug discovery, and disease modeling. While several protocols for differentiating somatic cells from PSCs exist, their use is limited by contamination of residual undifferentiated PSCs and immaturity of differentiated somatic cells. The metabolism of PSCs differs greatly from that of somatic cells, and a distinct feature is required to sustain the distinct properties of PSCs. To date, several studies have reported on the importance of metabolism in PSCs and their derivative cells. Here, we detail advancements in the field, with a focus on cardiac regenerative therapy.

Background: /Objective: We evaluated the risk of acute cholangitis and/or cholecystitis while waiting for cholecystectomy for gallstones. Methods: We retrospectively enrolled 168 patients who underwent cholecystectomy for gallstones after conservative therapy. We compared clinical data of 20 patients who developed acute cholangitis and/or cholecystitis while waiting for cholecystectomy (group A) with 148 patients who did not develop (group B). We investigated surgical outcomes and risk factors for developing acute cholangitis and/or cholecystitis. Results: Preoperatively, significant numbers of patients with previous history of acute grade II or III cholecystitis (55.0% vs 10.8%; p

Abstract Early gastric mixed neuroendocrine‐non‐neuroendocrine neoplasms (MiNENs) are rare diseases, with no data on their incidence and prognosis. We report the case of intramucosal gastric MiNENs for endoscopic submucosal dissection (ESD) treatment. An 80‐year‐old male underwent esophagogastroduodenoscopy for screening and was suspected of early gastric cancer type 0‐IIa+IIc on the lesser curvature of the antrum, for which ESD treatment was performed. Histopathologically, the diagnosis was MiNENs. Synaptophysin‐positive adenoductal structures were observed in the adenocarcinoma component, suggesting that adenocarcinoma had dedifferentiated into neuroendocrine carcinoma. The tumor was located within the mucosal layer, with lympho‐vascular invasion. The patient was kept under observation; however, 6 months after the ESD, computed tomography scan revealed prominent ascites, enlarged lymph nodes, and liver metastases, and MiNENs were suspected to have poor prognosis. If MiNENs diagnosis is made preoperatively or postoperatively, surgical resection may be considered as treatment regardless of the tumor depth or lympho‐vascular invasion.

A 71-year-old woman was diagnosed with advanced gastroesophageal junction cancer with bulky lymph nodes along the cardiac region and the lower mediastinum (GE-Circ type 3 T3 N3 M0 H0 stage III) and received treatment with S-1 and oxaliplatin (SOX) as first-line chemotherapy. After 3 cycles of SOX, severe anorexia and diarrhea were observed. We converted from this regimen of systemic chemotherapy to ramucirumab (RAM) monotherapy as second-line chemotherapy. This treatment resulted in a reduction in size of the metastatic lymph nodes along the cardiac region and the lower mediastinum. However, progression of lymph node metastasis and the primary tumor was observed following 7 months of RAM monotherapy. Therefore, nivolumab was initiated as third-line chemotherapy 14 months after the initial treatment. After 3 months of nivolumab administration, a 47% reduction in metastatic lymph nodes was achieved and a regression of the primary gastric tumor as seen on an enhanced computed tomography scan. After 7 months of nivolumab monotherapy, the diameter of the target lymph nodes had reduced by 81% from baseline, and there was no evidence of malignancy upon pathological assessment of the primary tumor site biopsy. The patient survived with nivolumab monotherapy for approximately 2 years after her first visit, without any adverse reaction to nivolumab.

Abstract Heart transplantation (HT) is the only radical treatment available for patients with end-stage heart failure that is refractory to optimal medical treatment and device therapies. However, HT as a therapeutic option is limited by marked donor shortage. To overcome this difficulty, regenerative medicine using human-induced pluripotent stem cells (hiPSCs) has drawn increasing attention as an alternative to HT. Several issues including the preparation of clinical-grade hiPSCs, methods for large-scale culture and production of hiPSCs and cardiomyocytes, prevention of tumorigenesis secondary to contamination of undifferentiated stem cells and non-cardiomyocytes, and establishment of an effective transplantation strategy need to be addressed to fulfill this unmet medical need. The ongoing rapid technological advances in hiPSC research have been directed toward the clinical application of this technology, and currently, most issues have been satisfactorily addressed. Cell therapy using hiPSC-derived cardiomyocytes is expected to serve as an integral component of realistic medicine in the near future and is being potentially viewed as a treatment that would revolutionize the management of patients with severe heart failure.

Abstract Background The use of staging laparoscopy (SL) has become widespread in patients with advanced gastric cancer (GC). This study aimed to evaluate the predictive value of the neutrophil/lymphocyte ratio (NLR) for the presence of peritoneal metastasis during staging laparoscopy in patients with advanced GC. Methods This retrospective analysis was performed in 35 patients with advanced GC who underwent SL at Kanazawa Medical University Hospital between January 2009 and December 2017. Clinicopathological characteristics were examined and multivariate analyses were performed to identify preoperative laboratory parameters that were independently associated with the presence of peritoneal metastasis or cytological malignancy (P/CY positive) during SL. Results A P/CY-positive result was confirmed during SL in 16 patients (45.7%). Patients with type 4 or diffuse type 3 tumors showed a significantly higher P/CY-positive rate than those with other tumor types (58.3% vs. 18.2%, P = 0.02). In the univariate analysis for preoperative laboratory parameters, NLR (P 3.5) was associated with a significantly higher P/CY-positive rate than a low NLR (≤ 3.5) (83.3% vs. 33.3%, P = 0.01). Moreover, in patients without type 4 or diffuse type 3 tumors, the P/CY-positive rates were 100% and 0% in patients with NLR > 3.5 and NLR ≤ 3.5, respectively. Conclusions The preoperative NLR was a significant independent predictor of the presence of peritoneal metastasis during SL. Regardless of tumor type, patients with a high NLR could be reasonable candidates for SL. On the other hand, non-diffuse type tumor accompanied by a low NLR may not need to undergo SL.

Key Clinical Message When trastuzumab + capecitabine and cisplatin chemotherapy could not be conducted continuously because of severe adverse reactions to cisplatin, trastuzumab + capecitabine could be an alternative systemic chemotherapy options for metastatic or recurrent gastric cancer patients.

Summary: Human pluripotent stem cells (hPSCs) have a unique metabolic signature for maintenance of pluripotency, self-renewal, and survival. Although hPSCs could be potentially used in regenerative medicine, the prohibitive cost associated with large-scale cell culture presents a major barrier to the clinical application of hPSC. Moreover, without a fully characterized metabolic signature, hPSC culture conditions are not optimized. Here, we performed detailed amino acid profiling and found that tryptophan (TRP) plays a key role in the proliferation with maintenance of pluripotency. In addition, metabolome analyses revealed that intra- and extracellular kynurenine (KYN) is decreased under TRP-supplemented conditions, whereas N-formylkynurenine (NFK), the upstream metabolite of KYN, is increased thereby contributing to proliferation promotion. Taken together, we demonstrate that TRP is indispensable for survival and proliferation of hPSCs. A deeper understanding of TRP metabolism will enable cost-effective large-scale production of hPSCs, leading to advances in regenerative medicine.

Summary: The role of lipid metabolism in human pluripotent stem cells (hPSCs) is poorly understood. We have used large-scale targeted proteomics to demonstrate that undifferentiated hPSCs express different fatty acid (FA) biosynthesis-related enzymes, including ATP citrate lyase and FA synthase (FASN), than those expressed in hPSC-derived cardiomyocytes (hPSC-CMs). Detailed lipid profiling revealed that inhibition of FASN resulted in significant reduction of sphingolipids and phosphatidylcholine (PC); moreover, we found that PC was the key metabolite for cell survival in hPSCs. Inhibition of FASN induced cell death in undifferentiated hPSCs via mitochondria-mediated apoptosis; however, it did not affect cell survival in hPSC-CMs, neurons, or hepatocytes as there was no significant reduction of PC. Furthermore, we did not observe tumor formation following transplantation of FASN inhibitor-treated cells. Our findings demonstrate the importance of de novo FA synthesis in the survival of undifferentiated hPSCs and suggest applications for FASN inhibition in regenerative medicine.

Abstract The objective of this study was to achieve simultaneous localization and mapping (SLAM) of firefighter robots for petrochemical complexes. Consistency of the SLAM map is important because human operators compare the map with aerial images and identify target positions on the map. The global positioning system (GPS) enables increased consistency. Therefore, this paper describes two Rao-Blackwellized particle filters (RBPFs) based on GPS and light detection and ranging (LIDAR) as SLAM solutions. Fast-SLAM 1.0 and Fast-SLAM 2.0 were used in grid maps for RBPFs in this study. We herein propose the use of Fast-SLAM to combine GPS and LIDAR. The difference between the original Fast-SLAM and the proposed method is the use of the log-likelihood function of GPS; the proposed combination method is implemented using a probabilistic mathematics formulation. The proposed methods were evaluated using sensor data measured in a real petrochemical complex in Japan ranging in size from 550–380 m. RTK-GPS data was used for the GPS measurement and had an availability of 56%. Our results showed that Fast-SLAM 2.0 based on GPS and LIDAR in a dense grid map produced the best results. There was significant improvement in alignment to aerial data, and the mean square root error was 0.65 m. To evaluate the mapping consistency, accurate 3D point cloud data measured by Faro Focus 3D (± 3 mm) was used as the ground truth. Building sizes were compared; the minimum mean errors were 0.17 and 0.08 m for the oil refinery and management building area and the area of a sparse building layout with large oil tanks, respectively. Consequently, a consistent map, which was also consistent with an aerial map (from Google Maps), was built by Fast-SLAM 1.0 and 2.0 based on GPS and LIDAR. Our method reproduced map consistency results for ten runs with a variance of ± 0.3 m. Our method reproduced map consistency results with a global accuracy of 0.52 m in a low RTK-Fix-GPS environment, which was a factory with a building layout similar to petrochemical complexes with 20.9% of RTK-Fix-GPS data availability.

A challenging wave energy converter design that utilized the denser energy part of the nearshore breaking wave zone to generate electricity was introduced in 2016 by Shintake. The Okinawa Institute of Science and Technology Graduate University’s project aims to take advantage of breaking wave energy to harness electricity. The 2016 version of the device consisted only of a bare turbine and power generator. Early exploration of the design recorded short periods and high impact wave pressures were experienced by the structure, with the turbine unable to harvest energy effectively. Additional structure to not only reduce incoming impact pressure but also increase the duration of water flow through the turbine was needed. These are the main reasons behind incorporating the duct attachment into the design. This paper show that the duct is capable of halving the impact pressure experienced by the turbine and can increase the energy exposure by up to 1.6 times the bare turbine configuration. Furthermore, it is also said that wave angle (β) = 40° is the critical angle, although the duct still increases wave energy exposure to the power take-off up to β = 60°.

Summary: Cardiac regenerative therapies utilizing human induced pluripotent stem cells (hiPSCs) are hampered by ineffective large-scale culture. hiPSCs were cultured in multilayer culture plates (CPs) with active gas ventilation (AGV), resulting in stable proliferation and pluripotency. Seeding of 1 × 106 hiPSCs per layer yielded 7.2 × 108 hiPSCs in 4-layer CPs and 1.7 × 109 hiPSCs in 10-layer CPs with pluripotency. hiPSCs were sequentially differentiated into cardiomyocytes (CMs) in a two-dimensional (2D) differentiation protocol. The efficiency of cardiac differentiation using 10-layer CPs with AGV was 66%–87%. Approximately 6.2–7.0 × 108 cells (4-layer) and 1.5–2.8 × 109 cells (10-layer) were obtained with AGV. After metabolic purification with glucose- and glutamine-depleted and lactate-supplemented media, a massive amount of purified CMs was prepared. Here, we present a scalable 2D culture system using multilayer CPs with AGV for hiPSC-derived CMs, which will facilitate clinical applications for severe heart failure in the near future. : In this article, Fujita and colleagues show that human induced pluripotent stem cells (hiPSCs) and hiPSC-derived cardiomyocytes (hiPSC-CMs) are stably and efficiently produced using multilayer culture plates with an active gas ventilation system because of stable CO2 and O2 concentration in culture media. Large numbers of obtained hiPSC-CMs are metabolically purified under glucose- and glutamine-depleted and lactate-supplemented conditions. Keywords: iPSCs, regenerative medicine, cardiomyocytes, purification, large scale, 2D culture

Summary: Ronin (THAP11), a DNA-binding protein that evolved from a primordial DNA transposon by molecular domestication, recognizes a hyperconserved promoter sequence to control developmentally and metabolically essential genes in pluripotent stem cells. However, it remains unclear whether Ronin or related THAP proteins perform similar functions in development. Here, we present evidence that Ronin functions within the nascent heart as it arises from the mesoderm and forms a four-chambered organ. We show that Ronin is vital for cardiogenesis during midgestation by controlling a set of critical genes. The activity of Ronin coincided with the recruitment of its cofactor, Hcf-1, and the elevation of H3K4me3 levels at specific target genes, suggesting the involvement of an epigenetic mechanism. On the strength of these findings, we propose that Ronin activity during cardiogenesis offers a template to understand how important gene programs are sustained across different cell types within a developing organ such as the heart. : Fujita et al. find that the transcriptional regulator Ronin (Thap11) appears to control embryonic heart development. Early heart-specific knockout of Ronin leads to growth defects in the developing heart and embryonic lethality, whereas slightly later Ronin loss results in severe dilated cardiomyopathy in the adult. Keywords: transcriptional control, heart development, organ growth, heart disease, dilative cardiomyopathy

Abstract Cold-inducible RNA-binding protein (CIRP) and RNA-binding motif protein 3 (RBM3) are two evolutionarily conserved RNA-binding proteins that are structurally related to hnRNPs and upregulated in response to moderately low temperatures in mammalian cells. Although contributions of splicing efficiency, the gene promoters activated upon mild hypothermia and the transcription factor Sp1 to induction of CIRP have been reported, precise mechanisms by which hypothermia and other stresses induce the expression of mammalian cold-inducible proteins (CIPs) are poorly understood. By screening the serine/arginine-rich splicing factors (SRSFs), we report that the transcript and protein levels of SRSF5 were increased in mammalian cells cultured at 32 °C. Expression of SRSF5 as well as CIRP and RBM3 were also induced by DNA damage, hypoxia, cycloheximide and hypotonicity. Immunohistochemical studies demonstrated that SRSF5 was constitutively expressed in male germ cells and the level was decreased in human testicular germ cell tumors. SRSF5 facilitated production of p19 H-RAS, and increased sensitivity to doxorubicin in human U-2 OS cells. Induction of CIPs was dependent on transient receptor potential vanilloid 4 (TRPV4) channel protein, but seemed independent of its ion channel activity. These findings indicate a previously unappreciated role for the TRP protein in linking environmental stress to splicing.

A new type of wave energy converter which harnesses electricity from onshore breaking waves has been studied at Okinawa Institute of Science and Technology Graduate University (OIST) since 2014. This concept has been demonstrated at a coral beach on the Maldives since 2018. Wave energy conversion is possible when waves approaching the shore steepen due to decreased water depth resulting in wave breaks near the surface. A steepened wave reaches the critical velocity of 4~6 m/sec shoreward before it breaks. A rotating blade takes advantage of this breaking phenomenon to convert the wave energy into electricity. The work presented here includes an experimental and numerical investigation of a prototype model of the wave energy converter. The turbine having five blades of variable chord lengths, twist angles, and constant thickness profile from hub to tip was simulated under similar flow as well as testing conditions, to predict the turbine performance. A commercial computational fluid dynamic tool SolidWorks Flow Simulation 2018 was used for the simulations at various rotation speeds with a uniform inlet velocity. The modified k-ε with a two-scale wall function turbulence closure model was selected. The validation performed for different test cases showed that the present computational results match in good agreement with the experimental results. Additionally, details performance of the turbine running, and generator characteristics have been reported in this paper.

Abstract.: Induced pluripotent stem cell (iPSC) generation is an epoch-making technology. The potential applications for iPSCs are wide-ranging from in vitro disease models to drug discovery. For regenerative medicine in particular, the technology provides great hope for patients with incurable diseases or potentially fatal disorders such as heart failure (HF). However, the true realization of that promise for HF remains uncertain and moving toward the clinical application of iPSCs needs to be stepwise and careful. The establishment of “safe” iPSCs must be a major premise, while genome integration-free and oncogene-free reprogramming is also necessary. Teratoma formation also remains a risk with undifferentiated iPSCs, but it must not happen in patients’ bodies. Thus, regardless of the target organ, the differentiated cells from iPSCs must be purified to exclude any possibility of tumorigenicity. The transplantation strategies used for iPSC-derived cells are very important for the recovery of lost cardiac function. Longer engraftment of transplanted iPSCs-derived cardiomyocytes is essential particularly because their survival could be hampered by ischemia, inflammation, apoptosis, immunological rejection, and other cardiac phenomena. Providing these multistep solutions will open the new frontier of regenerative therapies with iPSCs for patients with severe HF. Keywords:: induced pluripotent stem cell, cardiomyocyte, regenerative medicine, cell transplantation, cell survival

Background. Members of the Runx gene family encode transcription factors that bind to DNA in a sequence-specific manner. Among the three Runx proteins, Runx2 comprises 607 amino acid (aa) residues, is expressed in bone, and plays crucial roles in osteoblast differentiation and bone development. We examined whether the Runx2 gene is also expressed in testes. Methods. Murine testes from 1-, 2-, 3-, 4-, and 10-week-old male mice of the C57BL/6J strain and W∕Wv strain were used throughout the study. Northern Blot Analyses were performed using extracts form the murine testes. Sequencing of cDNA clones and 5′-rapid amplification of cDNA ends were performed to determine the full length of the transcripts, which revealed that the testicular Runx2 comprises 106 aa residues coding novel protein. Generating an antiserum using the amino-terminal 15 aa of Runx2 (Met1 to Gly15) as an antigen, immunoblot analyses were performed to detect the predicted polypeptide of 106 aa residues with the initiating Met1. With the affinity-purified anti-Runx2 antibody, immunohistochemical analyses were performed to elucidate the localization of the protein. Furthermore, bioinformatic analyses were performed to predict the function of the protein. Results. A Runx2 transcript was detected in testes and was specifically expressed in germ cells. Determination of the transcript structure indicated that the testicular Runx2 is a splice isoform. The predicted testicular Runx2 polypeptide is composed of only 106 aa residues, lacks a Runt domain, and appears to be a basic protein with a predominantly alpha-helical conformation. Immunoblot analyses with an anti-Runx2 antibody revealed that Met1 in the deduced open reading frame of Runx2 is used as the initiation codon to express an 11 kDa protein. Furthermore, immunohistochemical analyses revealed that the Runx2 polypeptide was located in the nuclei, and was detected in spermatocytes at the stages of late pachytene, diplotene and second meiotic cells as well as in round spermatids. Bioinformatic analyses suggested that the testicular Runx2 is a histone-like protein. Discussion. A variant of Runx2 that differs from the bone isoform in its splicing is expressed in pachytene spermatocytes and round spermatids in testes, and encodes a histone-like, nuclear protein of 106 aa residues. Considering its nuclear localization and differentiation stage-dependent expression, Runx2 may function as a chromatin-remodeling factor during spermatogenesis. We thus conclude that a single Runx2 gene can encode two different types of nuclear proteins, a previously defined transcription factor in bone and cartilage and a short testicular variant that lacks a Runt domain.

Following publication of the original article [1], the authors reported that one of the authors’ name is spelled incorrectly. In this Correction the incorrect and correct author name are shown. The original publication of this article has been corrected.

Cell transplantation therapy will mean a breakthrough in resolving the donor shortage in cardiac transplantation. Cardiomyocyte (CM) transplantation, however, has been relatively inefficient in restoring cardiac function after myocardial infarction (MI) due to low engraftment of transplanted CM. In order to ameliorate engraftment of CM, the novel transplantation strategy must be invented. Gelatin hydrogel (GH) is a biodegradable water-soluble polymer gel. Gelatin is made of collagen. Although we observed that collagen strongly induced the aggregation of platelets to potentially cause coronary microembolization, GH did not enhance thrombogenicity. Therefore, GH is a suitable biomaterial in the cell therapy after heart failure. To assess the effect of GH on the improvement of cardiac function, fetal rat CM (5×10(6) or 1x10(6) cells) were transplanted with GH (10 mg/ml) to infarcted hearts. We compared this group with sham operated rats, CM in phosphate buffered saline (PBS), only PBS, and only GH-transplanted groups. Three weeks after transplantation, cardiac function was evaluated by echocardiography. The echocardiography confirmed that transplantation of 5×10(6) CM with GH significantly improved cardiac systolic function, compared with the CM+PBS group (fractional area change: 75.1±3.4% vs. 60.7±5.9%, p

Myocardial perfusion scintigraphy (MPS) plays an important role in the evaluation and quantification of myocardial ischemia, and those with significant ischemia (SI) benefit most from revascularization procedures. This study aimed to identify the clinical factors and anatomical features associated with SI in patients with stable ischemic heart disease (SIHD).Data were analyzed from 4197 SIHD patients undergoing percutaneous coronary intervention (PCI). Ischemia was based on MPS findings prior to PCI, with SI defined as an ischemic region of more than 10% of the total left ventricular area. Logistic regression analysis was performed to identify any clinical factors associated with SI. MPS was used to evaluate 1070 (25.5%) patients pre-procedurally. Patients with a history of heart failure, stroke, or anginal symptoms with Canadian Cardiovascular Society class 2 or more were more likely to have SI (odds ratio [OR] 1.63, p = 0.025, OR: 1.85, p = 0.009, and OR: 1.49, p = 0.003, respectively). When angiographic variables were considered, a proximal left anterior descending artery (pLAD) lesion was the sole factor associated with SI (OR: 1.45, p = 0.012). Importantly, those with SI had more in-hospital complications (p = 0.006), most notably post-PCI infarcts (p = 0.008).Patients' background data, such as stronger anginal symptoms or a pLAD lesion, were associated with SI. Patients with SI must be treated with PCI to improve their long-term prognosis; however, procedure-related complications happen more frequently in SI patients than in non-SI patients. Physicians must give their full attention when performing the PCI procedure in SI patients to minimize their complication rate.

Recently, induced pluripotent stem cells (iPSCs) were established as promising cell sources for revolutionary regenerative therapies. The initial culture system used for iPSC generation needed fetal calf serum in the culture medium and mouse embryonic fibroblast as a feeder layer, both of which could possibly transfer unknown exogenous antigens and pathogens into the iPSC population. Therefore, the development of culture systems designed to minimize such potential risks has become increasingly vital for future applications of iPSCs for clinical use. On another front, although donor cell types for generating iPSCs are wide-ranging, T cells have attracted attention as unique cell sources for iPSCs generation because T cell-derived iPSCs (TiPSCs) have a unique monoclonal T cell receptor genomic rearrangement that enables their differentiation into antigen-specific T cells, which can be applied to novel immunotherapies. In the present study, we generated transgene-free human TiPSCs using a combination of activated human T cells and Sendai virus under defined culture conditions. These TiPSCs expressed pluripotent markers by quantitative PCR and immunostaining, had a normal karyotype, and were capable of differentiating into cells from all three germ layers. This method of TiPSCs generation is more suitable for the therapeutic application of iPSC technology because it lowers the risks associated with the presence of undefined, animal-derived feeder cells and serum. Therefore this work will lead to establishment of safer iPSCs and extended clinical application.

Induced pluripotent stem cells (iPSCs) have been proposed as novel cell sources for genetic disease models and revolutionary clinical therapies. Accordingly, human iPSC-derived cardiomyocytes are potential cell sources for cardiomyocyte transplantation therapy. We previously developed a novel generation method for human peripheral T cell-derived iPSCs (TiPSCs) that uses a minimally invasive approach to obtain patient cells. However, it remained unknown whether TiPSCs with genomic rearrangements in the T cell receptor (TCR) gene could differentiate into functional cardiomyocyte in vitro. To address this issue, we investigated the morphology, gene expression pattern, and electrophysiological properties of TiPSC-derived cardiomyocytes differentiated by floating culture. RT-PCR analysis and immunohistochemistry showed that the TiPSC-derived cardiomyocytes properly express cardiomyocyte markers and ion channels, and show the typical cardiomyocyte morphology. Multiple electrode arrays with application of ion channel inhibitors also revealed normal electrophysiological responses in the TiPSC-derived cardiomyocytes in terms of beating rate and the field potential waveform. In this report, we showed that TiPSCs successfully differentiated into cardiomyocytes with morphology, gene expression patterns, and electrophysiological features typical of native cardiomyocytes. TiPSCs-derived cardiomyocytes obtained from patients by a minimally invasive technique could therefore become disease models for understanding the mechanisms of cardiac disease and cell sources for revolutionary cardiomyocyte therapies.

BACKGROUND: Although inflammation is an important feature of pulmonary arterial hypertension (PAH), the usefulness of local inflammatory markers as biomarkers for PAH is unknown. In this study, we tested whether plasma concentrations of human pentraxin 3 (PTX3), a local inflammatory marker, would be a useful biomarker for detecting PAH. METHODS: Plasma PTX3 concentrations were evaluated in 50 PAH patients (27 with idiopathic PAH, 17 with PAH associated with connective tissue disease (CTD-PAH), and six with congenital heart disease), 100 age and sex-matched healthy controls, and 34 disease-matched CTD patients without PAH. Plasma concentrations of B-type natriuretic peptide (BNP) and C-reactive protein (CRP) were also determined. RESULTS: Mean PTX3 levels were significantly higher in all PAH patients than in the healthy controls (4.40±0.37 vs. 1.94±0.09 ng/mL, respectively; P

Indocyanine green (ICG) fluorescence imaging represents a promising method for sentinel node (SN) biopsy in laparoscopic gastric surgery due to its signal stability. In the present study, the suitability and optimal settings of ICG fluorescence imaging for SN biopsy in early gastric cancer were determined. Patients with single primary superficial-type adenocarcinoma of the stomach, lesions <5 cm in diameter, and no evident nodal metastasis and out of indication for endoscopic submucosal dissection were enrolled. The day prior to surgery, ICG solution was endoscopically injected into four quadrants of the submucosal layer of the tumor. The Photodynamic Eye was used to detect ICG fluorescence. Bright nodes were defined as clearly fluorescent nodes. A total of 72 patients were enrolled; 11 cases presented with metastasis, and of these, 10 could be diagnosed by bright node biopsy. The adequate concentration and injection volume of ICG was determined to be 50 µg/ml (x100) and 0.5 mlx4 points, respectively. There was 1 false-negative case, and this was attributed to the failure of the frozen section diagnosis. These results suggested that ICG fluorescence imaging for SN biopsy in laparoscopic surgery for early gastric cancer is feasible. However, a weakness of ICG fluorescence imaging is the subjectivity of bright node evaluation. [ABSTRACT FROM AUTHOR]

Aims We investigated whether neural crest-derived cardiac resident cells contribute to the restoration of intrinsic adrenergic function following transplantation in mice. Transplanted heart shows partial restoration of cardiac adrenergic activity with time. Both the intrinsic cardiac adrenergic system and extrinsic sympathetic re-innervation contribute to neuronal remodelling in the transplanted heart. Little is known about the origin and function of the intrinsic system in the trans- planted heart. Methods and results Heart from the protein 0-Cre/Floxed-Enhanced Green Fluorescent Protein double-transgenic mouse was transplanted onto the abdominal aorta of the non-obese diabetic/severe combined immunodeficient mouse to trace the fate of cardiac resident neural crest-derived cells. Sympathetic nerve fibres, which are predominantly localized to the epicardial surface of the heart, disappeared in the transplanted heart. Intramyocardial neural crest cells increased immediately, while neural crest-derived nucleated tyrosine hydroxylase (TH)-immunoreactive cells increased over 2 weeks following transplantation. The mRNA expression levels of TH, dopamine-β-hydroxylase and phenylethanolamine N-methyl-transferase, and the tissue content of catecholamines in the transplanted hearts increased with time in association with an increase in the number of neural crest-derived nucleated TH-immunoreactive cells and tissue nerve growth factor levels. Iodine-123-metaiodobenzylguanidine scintigraphy showed that the uptake ability of transplanted heart for catecholamines also recovered with time. Finally, the chronotropic response to tyramine both in vivo and ex vivo reappeared 2 weeks after transplantation. Conclusion Neural crest-derived adrenergic cells increased following heart transplantation. The restoration of cardiac sympathetic activities in transplanted heart is tightly coupled with an increase in the number of neural crest-derived adrenergic cells. [ABSTRACT FROM AUTHOR]

Heart failure (HF) is the leading cause of death in developed countries. Regenerative medicine has the potential to drastically improve treatment for advanced HF. Stem cell-based medicine has received attention as a promising candidate therapy over the past decade; however, it has not yet realized this potential in terms of reliability. The cell sheet is an innovative technology for constructing aligned graft cells, and several cell sources have been investigated for making a feasible cell sheet. The most representative thus far is skeletal myoblast, although such cells raise the issue of arrhythmogenicity. Regenerative cardiomyocytes (CMs) derived from pluripotent stem cells (PSCs), such as embryonic stem cells or induced PSCs, are the most promising, because a myocardial cell sheet (MCS) constructed with regenerative CMs can potentially enable contraction recovery and electromechanical coupling with host CMs. The functional outcomes of experimental MCS are reduction of ventricular wall stress and paracrine effects rather than contraction recovery. Several technical obstacles still hamper the clinical application of MCSs, with graft survival the most pivotal issue. Ischemia, apoptosis, inflammation, and immune response can all cause graft cell death, and a stable blood supply to the MCS is critical for successful engraftment. Ventricular tachycardia must also be considered in any myocardial cell therapy, and multiple layering of MCS (>3 layers) is necessary to reconstruct human myocardium. Innervation is also a potential issue. The future application of myocardial cell therapy with MCS for advanced HF depends on resolving these difficulties. [ABSTRACT FROM AUTHOR]

The administration of granulocyte colony-stimulating factor (G-CSF) after myocardial infarction (MI) improves cardiac function and survival rates in mice. It was also reported recently that bone marrow (BM)-derived c-kit+ cells or macrophages in the infarcted heart are associated with improvement of cardiac remodeling and function. These observations prompted us to examine whether BM-derived hematopoietic cells mobilized by G-CSF administration after MI play a beneficial role in the infarct region. A single hematopoietic stem cell from green fluorescent protein (GFP)-transgenic mice was used to reconstitute hematopoiesis in each experimental mouse. MI was then induced, and the mice received G-CSF for 10 days. In the acute phase, a number of GFP+ cells showing the elongated morphology were found in the infarcted area. Most of these cells were positive for vimentin and α-smooth muscle actin but negative for CD45, indicating that they were myofibroblasts. The number of these cells was markedly enhanced by G-CSF administration, and the enhanced myofibroblast-rich repair was considered to lead to improvements of cardiac remodeling, function, and survival rate. Next, G-CSF-mobilized monocytes were harvested from the peripheral blood of GFP-transgenic mice and injected intravenously into the infarcted mice. Following this procedure, GFP+ myofibroblasts were observed in the infarcted myocardium. These results indicate that cardiac myofibroblasts are hematopoietic in origin and could arise from monocytes/macrophages. MI leads to the recruitment of monocytes, which differentiate into myofibroblasts in the infarct region. Administration of G-CSF promotes this recruitment and enhances cardiac protection. [ABSTRACT FROM AUTHOR]

Dividing cardiomyocytes are observed in autopsied human hearts following recent myocardial infarction, however there is a lack of information in the literature on the division of these cells. In this study we used a rat model to investigate how and when adult mammalian cardiomyocytes proliferate by cell division after myocardial infarction. Myocardial infarction was induced in Wistar rats by ligation of the left coronary artery. The rats were sacrificed periodically up to 28 days following induced myocardial infarction, and the hearts subjected to microscopic investigation. Cardiomyocytes entering the cell cycle were assayed by observation of nuclear morphology and measuring expression of Ki-67, a proliferating cell marker. Ki-67 positive cardiomyocytes and dividing nuclei were observed initially after 1 day. After 2 days dividing cells gradually increased in number at the ischemic border zone, reaching a peak increase of 1.12% after 3 days, then gradually decreasing in number. Dividing nuclei increased at the ischemic border zone after 3 days, peaked by 0.14% at day 5, and then decreased. In contrast, Ki-67 positive cells and dividing nuclei were limited in number in the non-ischemic area throughout all experiments. In conclusion, mitogenic cardiomyocytes are present in the adult rat heart following myocardial infarction, but were spatially and temporally restricted. [ABSTRACT FROM AUTHOR]

Colitis-associated cancer (CAC) is caused by chronic intestinal inflammation and is reported to be associated with refractory inflammatory bowel disease (IBD). Defective apoptosis of inflammatory cell populations seems to be a relevant pathogenetic mechanism in refractory IBD. We assessed the involvement of stress response protein cold-inducible RNA-binding protein (Cirp) in the development of intestinal inflammation and CAC. In the colonic mucosa of patients with ulcerative colitis, expression of Cirp correlated significantly with the expression of TNFα, IL23/IL17, antiapoptotic proteins Bcl-2 and Bcl-xL, and stem cell markers such as Sox2, Bmi1, and Lgr5. The expression of Cirp and Sox2 was enhanced in the colonic mucosae of refractory ulcerative colitis, suggesting that Cirp expression might be related to increased cancer risk. In human CAC specimens, inflammatory cells expressed Cirp protein. Cirp-/- mice given dextran sodium sulfate exhibited decreased susceptibility to colonic inflammation through decreased expression of TNFα, IL23, Bcl-2, and Bcl-xL in colonic lamina propria cells compared with similarly treated wild-type (WT) mice. In the murine CAC model, Cirp deficiency decreased the expression of TNFα, IL23/IL17, Bcl-2, Bcl-xL, and Sox2 and the number of Dclk1+ cells, leading to attenuated tumorigenic potential. Transplantation of Cirp-/- bone marrow into WT mice reduced tumorigenesis, indicating the importance of Cirp in hematopoietic cells. Cirp promotes the development of intestinal inflammation and colorectal tumors through regulating apoptosis and production of TNFα and IL23 in inflammatory cells. [ABSTRACT FROM AUTHOR]