Deficiency of interleukin-19 exacerbates acute lung injury induced by intratracheal treatment of hydrochloric acid.

Interleukin (IL-19) belongs to the IL-10 family of cytokines and plays diverse roles in inflammation, cell development, viral responses, and lipid metabolism. Acute lung injury (ALI) is a severe respiratory condition associated with various diseases, including severe pneumonia, sepsis, and trauma, lacking established treatments. However, the role of IL-19 in acute inflammation of the lungs is unknown. We reported the impact of IL-19 functional deficiency in mice crossed with an ALI model using HCl. Lungs damages, neutrophil infiltration, and pulmonary edema induced by HCl were significantly worse in IL-19 knockout (KO) mice than in wild-type (WT) mice. mRNA expression levels of C-X-C motif chemokine ligand 1 (CXCL1) and IL-6 in the lungs were significantly higher in IL-19 KO mice than in WT mice. Little apoptosis was detected in lung injury in WT mice, whereas apoptosis was observed in exacerbated area of lung injury in IL-19 KO mice. These results are the first to show that IL-19 is involved in acute inflammation of the lungs, suggesting a novel molecular mechanism in acute respiratory failures. If it can be shown that neutrophils have IL-19 receptors and that IL-19 acts directly on them, it would be a novel drug target.

Subcellular dynamics of estrogen-related receptors involved in transrepression through interactions with scaffold attachment factor B1.

Estrogen-related receptor (ERR), a member of the nuclear receptor superfamily, consists of three subtypes (α, ß, γ) and has strong homology with estrogen receptor. No endogenous ligands have been identified for ERRs, but they play key roles in metabolic, hormonal, and developmental processes as transcription factors without ligand binding. Although subnuclear dynamics are essential for nuclear events including nuclear receptor-mediated transcriptional regulation, the dynamics of ERRs are poorly understood. Here, we report that ERRs show subcellular kinetic changes in response to diethylstilbestrol (DES), a synthetic estrogen that represses the transactivity of all three ERR subtypes, using live-cell imaging with fluorescent protein labeling. Upon DES treatment, all ERR subtypes formed discrete clusters in the nucleus, with ERRγ also displaying nuclear export. Fluorescence recovery after photobleaching analyses revealed significant reductions in the intranuclear mobility of DES-bound ERRα and ERRß, and a slight reduction in the intranuclear mobility of DES-bound ERRγ. After DES treatment, colocalization of all ERR subtypes with scaffold attachment factor B1 (SAFB1), a nuclear matrix-associated protein, was observed in dot-like subnuclear clusters, suggesting interactions of the ERRs with the nuclear matrix. Consistently, co-immunoprecipitation analyses confirmed enhanced interactions between ERRs and SAFB1 in the presence of DES. SAFB1 was clarified to repress the transactivity of all ERR subtypes through the ERR-response element. These results demonstrate ligand-dependent cluster formation of ERRs in the nucleus that is closely associated with SAFB1-mediated transrepression. Taken together, the present findings provide a new understanding of the pathophysiology regulated by ERR/SAFB1 signaling pathways and their subcellular dynamics.

Novel metabolic system for lactic acid via LRPGC1/ERRγ signaling pathway.

Lactic acid (LA) is a byproduct of glycolysis resulting from intense exercise or a metabolic defect in aerobic processes. LA metabolism is essential to prevent lactic acidosis, but the mechanism through which LA regulates its own metabolism is largely unknown. Here, we identified a LA-responsive protein, named LRPGC1, which has a distinct role from PGC1α, a key metabolic regulator, and report that LRPGC1 particularly mediates LA response to activate liver LA metabolism. Following LA stimulation, LRPGC1, but not PGC1α, translocates from the cytoplasm to the nucleus through deactivation of nuclear export signals, interacts with the nuclear receptor ERRγ, and upregulates TFAM, which ensures mitochondrial biogenesis. Knockout of PGC1 gene in HepG2 hepatocarcinoma cells decreased the LA consumption and TFAM expression, which were rescued by LRPGC1 expression, but not by PGC1α. These LRPGC1-induced effects were mediated by ERRγ, concomitantly with mitochondrial activation. The response element for LRPGC1/ERRγ signaling pathway was identified in TFAM promoter. Notably, the survival rate of a mouse model of lactic acidosis was reduced by the liver-targeted silencing of Lrpgc1, while it was significantly ameliorated by the pharmacological activation of ERRγ. These findings demonstrate LA-responsive transactivation via LRPGC1 that highlight an intrinsic molecular mechanism for LA homeostasis.

A New Technique to Repair Vesicorectal Fistula: Overlapping Rectal Muscle Plasty by Transanal Endoscopic Surgery.

OBJECTIVE: To evaluate clinical results of a novel surgical technique, we developed to repair vesicorectal fistula (VRF) occurring after prostatectomy, hospital records of the patients, who underwent the new surgical treatment, were assessed. METHODS: The novel surgical technique is called "overlapping rectal muscle plasty," which is performed under transanal endoscopic microsurgery (TEM). During the new procedure, a complete fistulectomy was first performed, and then the proper muscle layer of the rectum was folded, overlapped, and sutured to create a thick wall between the rectum and urinary bladder. This operation was carried out in 15 patients with VRF following radical prostatectomy. RESULTS: The operation was safely performed in all patients with an average time of 127.2 min. Fistula was corrected in 13 patients (86.7%), who were then freed from both urinary and intestinal diversions. CONCLUSIONS: Overlapping rectal muscle plasty by TEM is a safe procedure. The success rate seems to be acceptable in selected patients. This new repair method may be considered as a minimally invasive option in the surgical treatment of VRF after prostatectomy.

Status of growth plates can be monitored by MRI.

BACKGROUND: Growth plate injuries and disorders cause premature closure, resulting in shortened or deformed limbs. Quantitative assessment by MRI might monitor the status of the growth plate and may assist in the prediction of these deformations. PURPOSE: To investigate whether the status of the growth plate can be monitored by quantitative evaluation using MRI of the noninjured region of the growth plate in a physeal injury model. STUDY TYPE: Prospective, longitudinal. ANIMAL MODEL: A 3.0-mm drill was used to create an injury to the central region of the right proximal tibial growth plate in 5-week-old male Japanese white rabbits (N = 18). The left tibia served as the control. FIELD STRENGTH/SEQUENCE: 7.04T, T2 -weighted imaging, diffusion-weighted imaging. ASSESSMENT: Eight of 18 rabbits underwent MRI, proton density-weighted imaging, and T2 -weighted and diffusion-weighted imaging. T2 and apparent diffusion coefficient (ADC) maps were generated for each image. The growth plate height and the T2 and ADC values of the noninjured region were measured. Two rabbits were sacrificed at 2, 4, 6, 8, and 10 weeks postinjury. Proximal tibial bones were evaluated using microcomputed tomography, histological, and immunohistological methods. STATISTICAL TESTS: Data were compared using repeated-measures analysis of variance followed by Tukey post-hoc multiple comparison. RESULTS: Growth plate height decreased at 10 weeks postinjury (P = 0.018) on the injured side. T2 values were greater at 2 weeks postinjury (P = 0.0478) and decreased at 8 and 10 weeks (P = 0.0226, P = 0.0470, respectively) on the injured side. ADC values increased at 6 weeks on the lateral side (P = 0.0304) and decreased at 8 weeks and 10 weeks postinjury (P < 0.01) on the medial and injured sides, respectively. DATA CONCLUSION: Quantitative MRI can help monitor the status of the growth plate and capture its changes early. LEVEL OF EVIDENCE: 1 Technical Efficacy Stage: 3 J. Magn. Reson. Imaging 2020;51:133-143.

CLEAN-NET: a modified laparoendoscopic wedge resection of the stomach to minimize the sacrifice of innocent gastric wall.

BACKGROUND: To avoid excessive sacrifice of the tissue surrounding the submucosal tumor in gastric wedge resection with a stapling device, we perform a "combined laparoscopic and endoscopic approach for neoplasia with a nonexposure technique" (CLEAN-NET). Herein the operative technique of CLEAN-NET is described and its short-term outcomes in 50 patients are evaluated. PATIENTS AND METHODS: Between December 2015 and July 2017 CLEAN-NET was performed in 50 patients with gastric submucosal tumors. During the operation, the seromuscular layer above the tumor is dissected, while the mucosa is kept unbroken. When seromuscular layer is dissected all around the tumor, the full layer is lifted, and the mucosa is stretched. The mucosa is then transected with a stapling device to execute full-thickness resection of the specimen. Finally, the seromuscular defect is repaired by hand-sewn suture. The hospital records of the 50 patients were reviewed to assess the outcomes. The margin width was compared with those measured in another group with 19 patients, who underwent conventional wedge resection with a stapling device. RESULTS: The operation was completed as CLEAN-NET and the tumor was resected en-bloc without rupture in all patients. The average operation time ranged from 50 to 220 min with an average of 105.4 min. The post-operative course was uneventful. Microscopically the surgical margin was tumor-negative (R0 resection) in all cases. The margin width in the CLEAN-NET group was smaller than that in the wedge resection group (5.4 mm ± 2.5 vs. 33.1 mm ± 14.7). CONCLUSIONS: CLEAN-NET can be performed safely with an acceptable operation time. CLEAN-NET can be a useful option in the laparoscopic surgical treatment of gastric submucosal tumors, when excessive sacrifice of the healthy gastric wall surrounding the endophytic tumor should be avoided.

Relationship between the antral follicle count in bovine ovaries from a local abattoir and steroidogenesis of granulosa cells cultured as oocyte-cumulus-granulosa complexes.

The antral follicle count (AFC) is used as an indicator of cow fertility. We herein investigated the relationship between AFC and the steroidogenesis of granulosa cells and confirmed the developmental competence of oocytes derived from early antral follicles (0.5-1.0 mm) using in vitro growth culture. Slaughterhouse-derived ovaries were divided into high (≥ 25) and low (< 25) AFC groups based on AFC (≥ 2.0 mm). Oocyte-cumulus-granulosa complexes (OCGCs) collected from early antral follicles were cultured for 12 days. The total number, viability, and diameter of granulosa cells and estradiol-17ß and progesterone production during the culture were evaluated. Surviving oocytes on day 12 were subjected to in vitro maturation, and their volume and nuclear status were evaluated. Some oocytes were subjected to the evaluation of developmental competence to blastocysts. Although the total number and viability of granulosa cells did not differ between the groups, granulosa cell diameters were smaller in the high AFC group than in the low AFC group. The estradiol-17ß and progesterone ratio on day 8 was higher in the high AFC group than in the low AFC group. Oocyte volumes and nuclear maturation rates were greater in the high AFC group than in the low AFC group. The development rate to blastocysts was 9.1% in the high AFC group, while no oocytes developed to blastocysts in the low AFC group. Therefore, estradiol-17ß production by granulosa cells appears to be greater in high AFC cattle than in low AFC cattle, thereby promoting the acquisition of oocyte competence.

Estrogen-related Receptor ß Reduces the Subnuclear Mobility of Estrogen Receptor α and Suppresses Estrogen-dependent Cellular Function.

Estrogen-related receptor (ERR) is a member of the nuclear receptor superfamily that has strong homology with estrogen receptor (ER) α. ERR has three subtypes (α, ß, and γ) expressed in estrogen-sensitive organs, including ovary, breast, and brain. No endogenous ligands of ERRs have been identified, but these receptors share a common DNA element with ERα and control estrogen-mediated gene transcription. Recent evidence suggests a role of ERRs in estrogen-related pathophysiology, but the detailed mechanisms of ERR functions in estrogen-related tissues are unclear. Using live-cell imaging with fluorescent protein labeling, we found that only ERRß among the ERRs exhibits a punctate intranuclear pattern overlapping with ERα following 17ß-estradiol (E2)-stimulation. Fluorescence recovery after photobleaching showed significant reduction of the mobility of ligand-activated ERα with co-expression of ERRß. Fluorescence resonance energy transfer revealed that ERRß directly interacts with ERα. The N-terminal domain of ERRß was identified as the region that interacts with ERα. We also found a correlation between punctate cluster formation of ERα and interaction between the receptors. Expression of ERRß significantly repressed ERα-mediated transactivity, whereas that of other ERR subtypes had no effect on the transactivity of ERα. Consistent with this finding, E2-stimulated proliferation of MCF-7 breast carcinoma cells and bcl-2 expression was significantly inhibited by expression of ERRß. These results provide strong evidence for a suppressive effect of ERRß on estrogen signaling through reduction of the intranuclear mobility of ERα. The findings further suggest a unique inhibitory role for ERRß in estrogen-dependent cellular function such as cancer cell proliferation.

Long-term outcomes of percutaneous endoscopic intragastric surgery in the treatment of gastrointestinal stromal tumors at the esophagogastric junction.

BACKGROUND: The treatment options for gastrointestinal stromal tumors (GITSs) at the esophagogastric junction (EGJ) are controversial. There have been reports on enucleation for EGJ GISTs in order to avoid gastrectomy. But the number of patients is too small, or the follow-up period is too short to evaluate it. The purpose of this study was to review our experience of 59 patients with EGJ GISTs treated by enucleation by percutaneous endoscopic intragastric surgery (PEIGS) and assess the clinical outcomes. METHODS: PEIGS is performed as described below. Access ports are placed through the abdominal wall and the anterior wall of the stomach. Through the access ports, an endoscope and surgical instruments are inserted into the gastric lumen and tumor enucleation and closure of the defect are carried out. In this study, 59 patients with EGJ GISTs treated by PEIGS between 2005 and 2013 were enrolled. Their hospital records were reviewed, and follow-up data for 8 years were collected to analyze the outcomes. RESULTS: En-bloc enucleation was achieved without tumor rupture in all. Average operation time was 172.3 min. Postoperative complications occurred in 3 (one localized peritonitis, one bleeding, and one surgical site infection). Average tumor size was 35.6 mm. Pathological findings confirmed negative margin in all specimens. The maximum follow-up period was 101 months. Multiple liver metastases were detected in two patients (at 12 and 29 months). The survival rate was 100 %. The disease-free rate was 98.3 % at 12 months and 96.6 % at 29 months, respectively. CONCLUSIONS: As far as the short- and long-term outcomes of our experience are reviewed, PEIGS seems as curative as other aggressive resection methods such as proximal gastrectomy. Tumor enucleation by PEIGS, offering a chance to preserve the stomach, can be a preferable option in carefully selected patients with EGJ GISTs, when performed by a skilled surgeon.

Development of scar-less laparoscopic hernia repair (TAPP-252) facilitated by new 2mm instruments.

INTRODUCTION: To minimize the invasiveness of laparoscopic transabdominal preperitoneal hernia repair (TAPP) for the treatment of adult inguinal hernia, we developed a new operative technique with the use of only one 5 mm port and two 2 mm punctures (TAPP-252). MATERIAL AND METHODS: To facilitate TAPP-252, we developed seven kinds of new 2 mm instruments, including grasping forceps, hook shaped electrode, mesh pusher, needle driver, scissors, laparoscope and port. RESULTS: TAPP-252 was stably performed in 35 patients with minimal abdominal wall destruction and excellent cosmetic result without any recurrence or morbidity. CONCLUSIONS: The newly developed 2 mm devices showed sufficient performance and durability in TAPP-252. Further investigation is necessary to assess durability and long-term outcomes.

Needlescopic intragastric surgery facilitated by newly developed 2mm instruments.

BACKGROUND: Intragastric surgery is a percutaneous endoluminal surgery in the stomach aimed at resection of tumors located at the esophagogastric junction (EGJ). We developed needlescopic intragastric surgery performed via 2 mm, 2 mm, and 5 mm ports (PEIGS-225). MATERIAL AND METHODS: In cooperation with Niti-On Co., Ltd. we developed a series of 2 mm instruments including grasping forceps, a cannula, a laparoscope, an electrocautery, scissors, and a needle holder. OPERATIVE TECHNIQUE: Two 2 mm trocars and a 5 mm one are inserted into the gastric lumen percutaneously. Intragastric procedures are performed by the instruments brought through those three ports. The specimen is extracted via the esophageal-oral route. The defect in the gastroesophageal wall is closed by hand-suture. After the intragastric procedure, the 5 mm stab wound on the gastric wall is closed by hand-suture, while the 2 mm wounds are left untreated. PATIENTS: Between March and August 2015 PEIGS-225 was performed in five patients. RESULTS: There was no operative conversion. The mean operation time was 96 minutes. There were no perioperative complications. Pathological findings indicated that the margin was negative in all cases. CONCLUSION: Needlescopic intragasric surgery performed via the smallest access (2 mm, 2 mm, 5 mm) is enabled by the 2 mm instruments developed by us.

Transanal endoscopic microsurgery for surgical repair of rectovesical fistula following radical prostatectomy.

BACKGROUND: Rectovesical fistula is a rare complication following prostatectomy, associated with significant symptoms such as urinary drainage from anus or faecaluria. While several surgical procedures have been described to treat this condition, none of them has been accepted as the universal standard. Transanal endoscopic microsurgery (TEM) is a well-established endoluminal procedure for local excision of rectal tumors. But its application to the repair of rectovesical fistula has been almost unknown. METHODS: We performed TEM as a surgical repair for refractory rectovesical fistula developing after radical prostatectomy in 10 patients. Under the magnified three-dimensional view, through the stereoscope, the fistula and the surrounding rectal mucosa were precisely resected. The defect and the muscle layer of the rectum were closed by hand-sew technique in four layers. RESULTS: Fistula was completely closed in 7 patients, who eventually underwent enterostomy closure, while in the other 3 patients the fistula recurred. In the three recurrent cases, the fistula was associated with wide, tough scar tissue due to previous irradiation, HIFU, or repeated surgical repair attempts. CONCLUSIONS: Rectovesical fistulas associated with wide, tough scar tissue due to multi-time attempt of surgical repair or any type of energy ablation should not be indicated for repair by TEM. However, for simple fistulas without tough, fibrotic surroundings, TEM can be indicated as a minimally invasive surgical option with very low morbidity, without any incision in healthy tissue for approach.

Insight into the mechanism of reproductive dysfunction caused by neonicotinoid pesticides.

Neonicotinoids, which were developed in the 1990 s as an insecticide having selective toxicity, were later found to cause reproductive abnormalities in experimental animals. In Japan there is an attempt to preserve endangered animals, including the Japanese crested ibis, and there is a question of whether neonicotinoids affect the reproduction of this bird, since they are used in its habitat. Hence, we investigated whether the daily oral administration of the neonicotinoid clothianidin (CTD) has any deleterious effects on the reproductive function of mature male only or both young male and female quails as experimental animals. Vacuolization and the number of germ cells having fragmented DNA in seminiferous tubules, as well as the number and size of vacuoles in hepatocytes, increased dose-dependently. The ovaries showed abnormal histology in the granulosa cells, which produce progesterone. There were significant differences in egg-laying rates and embryo weights between the groups. Glutathione Peroxidase 4 (GPx4) and Manganese Superoxide Dismutase (Mn-SOD), which protect the organism from oxidative damage, showed a dose-dependent decrease. Thus, it is possible neonicotinoids affect the bird's reproductive system through oxidative stress, reflecting an imbalance between the production of reactive oxygen species (ROS) and a biological system's ability to readily detoxify the reactive intermediates or easily repair the resulting damage. Responding to our study, Sado Island has since succeeded in breeding Japanese crested ibis in the wild without the use of neonicotinoids.

Fetal exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin transactivates aryl hydrocarbon receptor-responsive element III in the tyrosine hydroxylase immunoreactive neurons of the mouse midbrain.

Fetal exposure to dioxins and related compounds is known to disrupt normal development of the midbrain dopaminergic system, which regulates behavior, cognition and emotion. The toxicity of these chemicals is mediated mainly by aryl hydrocarbon receptor (AhR) signaling. Previously, we identified a novel binding motif of AhR, the AhR-responsive element III (AHRE-III), in vitro. This motif is located upstream from the gene encoding tyrosine hydroxylase (TH), the rate-limiting enzyme of dopamine biosynthesis. To provide in vivo evidence, we investigated whether 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) could regulate AHRE-III transcriptional activity in midbrain dopaminergic neurons. We produced transgenic mice with inserted constructs of the AHRE-III enhancers, TH gene promoter and the c-myc-tagged luciferase gene. Single oral administrations of TCDD (0-2000 ng kg⁻¹ body weight) to the transgenic dams markedly enhanced TH-immunoreactive (ir) intensity in the A9, A10 and A8 areas of their offspring at 3 days and 8 weeks of age. The offspring of dams treated with 200 ng kg⁻¹ TCDD exhibited significant increases in the numbers of TH- and double (TH and c-myc)-ir neurons in area A9 compared with controls at 8 weeks. These results show that fetal exposure to TCDD upregulates TH expression and increases TH-ir neurons in the midbrain. Moreover, the results suggest that TCDD directly transactivates the TH promoter via the AhR-AHRE-III-mediated pathway in area A9. Fetal exposure to TCDD caused stable upregulation of TH via the AhR-AHRE-III signaling pathway and overgrowth of TH-ir neurons in the midbrain, implying possible involvement in the etiology of neurodevelopmental disorders such as attention-deficit/hyperactivity disorder (ADHD).

A single surgeon's experience with transanal endoscopic microsurgery over 20 years with 153 early cancer cases.

BACKGROUND: The first author performed transanal endoscopic surgery (TEM) in 302 patients in Japan for the last 20 years, 153 of which were early rectal cancer cases. The short- and long-term outcomes of the early rectal cancer cases are herein reported. MATERIAL AND METHODS: The original technique of TEM developed by Gerhard Buess was performed in all cases. The hospital records were reviewed to assess the clinical outcomes. A questionnaire was sent to the patients to analyze the long-term outcomes. RESULTS: One-hundred and fifty-three early cancer cases included 115 T0 and 38 T1 lesions. Full-thickness resection was performed in 36 patients, while 117 underwent submucosal dissection. Conversion to laparoscopic low anterior resection occurred in one case. Mortality was nil. Major operative complication was noted in only one patient, who developed stenosis. Seven patients underwent immediate salvage surgery. Six patients died of recurrence of rectal cancer. Disease-free survival rate at year 5 was 93.7%. CONCLUSIONS: Our study, one of the largest series in the world, confirms that TEM is a preferable option in the surgical treatment of T0 and T1a rectal carcinoma. As long as early cancer cases are treated, submucosal resection seems to be sufficient. When risk of recurrence is found by pathological examination, immediate salvage operation is mandatory to improve the prognosis.

Effects of recombinant osteopontin expressed in Escherichia coli on the recovery of the endometrial epidermal growth factor profile and fertility in repeat breeder dairy cows.

Endometrial epidermal growth factor (EGF) shows a cyclic change with two peaks on days 2-4 and days 13-14 of the estrous cycle. In repeat breeder cows, loss of the peaks has been associated with reduced fertility. By infusing seminal plasma (SP) and osteopontin (OPN) derived from SP and milk into the vagina, their EGF profile and fertility are restored. However, SP is difficult to obtain, and both SP and OPN can transmit infectious diseases. While OPN can be sourced from recombinant protein without this risk, recombinant bovine OPN (rOPN) expressed in Escherichia coli should be examined for its effects on the EGF profile, since it does not undergo posttranslational modification, which is important for its biological activity. In study 1, PBS, SP (0.5 mL), and rOPN (0.3 mg) were infused into the vagina at estrus (day 0) in 74, 37, and 105 repeat breeder Holstein cows, respectively, with an altered EGF profile. The endometrial EGF concentrations were measured on day 3. Some cows (n = 58, 20, and 83, respectively) were inseminated immediately before the infusion and then diagnosed for pregnancy between days 30 and 35. The normalization rate of the EGF profile and conception rate in the rOPN group (58.1 % and 47.0 %, respectively) were not significantly different from those in the SP group (62.2 % and 45.0 %, respectively) but higher than those in PBS group (29.7 % and 28.1 %, respectively) (P < 0.05). In study 2, repeat breeder cows with an altered EGF profile were infused with PBS (n = 18) and rOPN (n = 17), while fertile controls with a normal EGF profile (n = 18) were infused with PBS. Two or three embryos were transferred into cows on day 7 and then recovered on day 14. Embryo recovery rates of the rOPN and fertile groups were comparable (58.7 % vs. 58.3 %) but higher than that of the PBS group (58.7 % vs. 32.0 %) (P < 0.05). The embryo recovery rate of cows with normalized EGF profile was higher than that of cows with unnormalized EGF profile (64.4 % vs. 16.7 %) (P < 0.05). The embryo sizes of cows in the rOPN and fertile groups were comparable but larger than those in the PBS group (P < 0.05). However, the embryo size was not correlated to the corresponding endometrial EGF concentrations. In conclusion, rOPN without posttranslational modifications normalized the EGF profile in repeat breeder cows. Improved fertility by normalization of the EGF profile could be attributed partly to the increased embryo viability up to day 14.

Detection of biomagnetic signals from induced pluripotent stem cell-derived cardiomyocytes using deep learning with simulation data.

The detection of spontaneous magnetic signals can be used for the non-invasive electrophysiological evaluation of induced pluripotent stem cell-derived cardiomyocytes (iPS-CMs). We report that deep learning with a dataset that combines magnetic signals estimated using numerical simulation and actual noise data is effective in the detection of weak biomagnetic signals. To verify the feasibility of this method, we measured artificially generated magnetic signals that mimic cellular magnetic fields using a superconducting quantum interference device and attempted peak detection using a long short-term memory network. We correctly detected 80.0% of the peaks and the method achieved superior detection performance compared with conventional methods. Next, we attempted peak detection for magnetic signals measured from mouse iPS-CMs. The number of detected peaks was consistent with the spontaneous beats counted using microscopic observation and the average peak waveform achieved good similarity with the prediction. We also observed the synchronization of peak positions between simultaneously measured field potentials and magnetic signals. Furthermore, the magnetic measurements of cell samples treated with isoproterenol showed potential for the detection of chronotropic effects. These results suggest that the proposed method is effective and has potential application in the safety assessment of regenerative medicine and drug screening.

Mixed-Culture Propagation of Uterine-Tissue-Resident Macrophages and Their Expression Properties of Steroidogenic Molecules.

Tissue-resident macrophages (Mø) play tissue/organ-specific roles, and the physiological/pathological implications of uterine Mø in fertility and infertility are not yet fully understood. Herein, we report a simple propagation method for tissue-resident Mø by mixed culture with the respective tissue/organ-residing cells as the niche. We successfully propagated mouse uterine Mø by mixed culture with fibroblastic cells that exhibited properties of endometrial stromal cells. Propagated mouse uterine Mø were CD206- and arginase-1-positive; iNOS- and MHC-II-negative, indicating M2 polarization; and highly phagocytic, similar to endometrial Mø. Furthermore, uterine Mø were observed to express steroidogenic molecules including SRD5A1 and exhibited gap junction formation, likely with endometrial stromal cells. Accordingly, uterine Mø propagated by mixed culture may provide a new tool for studying immune-endocrine interactions related to fertility and infertility, particularly androgen's intracrine actions in preparing the uterine tissue environment to support implantation and pregnancy as well as in the etiology of endometriosis.

FoxP2 protein decreases at a specific region in the chick midbrain after hatching.

Forkhead-box subclass P member 2 (FOXP2/FoxP2) protein, a transcription factor, regulates the development of certain brain functions, including human speech and animal vocalization. Although rapid progress has been made in demonstrating a relationship between FoxP2 expression in the brain and vocalization of the zebra finch, a typical vocal learner, the relationship in avian vocal non-learners, including chickens remains elusive. Because the midbrain plays a key role in innate vocalization development, we analyzed the FoxP2 protein in the midbrain of chicks, which do not cheep before hatching but cheep and call after hatching. Western blot analyses showed a significant reduction in FoxP2 protein in the chick midbrain after hatching compared with the findings before hatching. Quantitative immunohistochemistry revealed that FoxP2-immunoreactive (ir) cells significantly decreased at the stratum gris fibrosum (SGFS) of the optic tectum in the chick midbrain after hatching compared with the findings before hatching. These findings support the notion that FoxP2-ir cell numbers decrease at a specific region in the midbrain after hatching may be involved in innate vocalization of avian vocal non-learners.

Oxygen-glucose deprivation-induced glial cell reactivity in the rat primary neuron-glia co-culture.

It has been demonstrated that in vivo brain ischemia induces activation and proliferation of astrocytes and microglia. However, the mechanism underlying the ischemia-induced activation and proliferation of these cells remains to be unclear. Oxygen-glucose deprivation (OGD), an in vitro ischemia mimic, has been extensively used to analyze the hypoxia response of various cell types. This study examined the OGD-induced changes in the expression level of astrocytes and microglia marker proteins and immunoreactivity for Ki-67, a marker protein for cell proliferation, using rat primary hippocampal neuron-glia co-culture (NGC) cells. Furthermore, OGD-induced changes in the expression of M1/M2 microglia phenotype-related genes were also examined. MTT assay indicated that 120 min of OGD decreased cell viability, and immunocytochemistry indicated that 120 min of OGD abolished most microtubule-associated protein 2 (MAP2)-immunopositive neurons. In contrast, glial fibrillary acidic protein (GFAP)-immunopositive astrocytes and ionized calcium-binding adapter protein-1 (Iba-1)-immunopositive microglia, and 2',3'-cyclic nucleotide-3'-phosphodiesterase (CNPase)-immunopositive oligodendrocytes survived OGD. Western blot assays and double-immunofluorescent staining indicated that OGD increased the GFAP expression level and the Ki-67-immunopositive/GFAP-immunopositive cells' ratio. Real-time PCR analysis showed that OGD altered M1 microglia phenotype-related genes. Specifically, OGD decreased the expression level of CD32 and interleukin-1ß (IL-1ß) genes and increased that of the inducible nitric oxide synthase (iNOS) gene. Therefore, applying OGD to NGC cells could serve as a useful in vitro tool to elucidate the molecular mechanisms underlying brain ischemia-induced changes in GFAP expression, astrocyte proliferation, and M1 microglia phenotype-related gene expression.