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Objectives: The objective is to evaluate the feasibility and safety of ArtiSential for performing minimally invasive surgeries for gynecological cancers. Materials and Methods: We conducted a prospective observational study at 10 Tertiary Institutional Hospitals in Korea between November 2021 and April 2022. Eligible patients were 18 years or older and planned to undergo minimally invasive surgery for gynecologic cancer. We collected baseline characteristics, surgical information, and postoperative outcomes. The primary endpoint was to compare the operation time required for gynecologic cancer surgery using ArtiSential with the reported operation time for surgery using conventional laparoscopic instruments or robots. The secondary endpoints were to evaluate the surgical outcomes of gynecologic cancer surgery using ArtiSential compared to conventional laparoscopic instruments or robots and collect operator feedback on equipment improvements during surgery. Results: Forty patients were enrolled in this study, including 19 with endometrial cancer, 15 with cervical cancer, and 6 with ovarian cancer. The average operation time was 187.0 ± 49.2 min, with no complications encountered during surgery. Pelvic lymph nodes were assessed in 34 patients, with the ArtiSential device utilized in 22 (64.7%) of these patients, at an average assessment time of 40.3 ± 19.4 min. Most surgeons using the ArtiSential device reported that it performed slightly better than conventional laparoscopic instruments. Conclusion: The use of the ArtiSential device in minimally invasive surgery has been demonstrated to be both feasible and safe for the treatment of early-stage gynecologic cancer.

The Conical 9 well dish (C9 well dish) is characterized by a decreasing cross-sectional area towards the base. This design was hypothesized to enhance embryonic development by emulating the in vivo physical environment through density modulation. Comparative analyses revealed no significant difference in nuclear maturation rates between the C9 well dish and the 5-well dish. Reactive oxygen species (ROS) generation was lower in the C9 well dish compared to the 5-well dish; however, this difference was not statistically significant. On the second day of in vitro culture, the cleavage rate in the C9 well dish was 4.66% higher, although not statistically significant, and the rates of blastocyst development were similar across both dishes. No significant differences were observed in the intracellular levels of glutathione (GSH) and ROS, as well as in the total cell number within the blastocysts between the dish types. The expression of mitogen-related factors, TGFα and IGF-1, in the blastocysts was consistent between the dishes. However, PDGFβ expression was significantly lower in the C9 well dish compared to the 35 mm petri dish. Similarly, the expression of the apoptosis factor Bax/Bcl2l2 showed no significant differences between the two dishes. Despite the marked difference in PDGFβ expression, its impact on blastocyst formation appeared negligible. The study also confirmed the feasibility of culturing a small number of oocytes per donor, collected via Ovum Pick-Up (OPU), with reduced volumes of culture medium and mineral oil, thus offering economic advantages. In conclusion, the present study indicates that the C9 well dish is effective for in vitro development of a small quantity of oocytes and embryos, presenting it as a viable alternative to traditional cell culture dishes.

Background: KRAS , TP53 , CDKN2A , and SMAD4 have been the main driver mutations in pancreatic ductal adenocarcinoma (PDAC). Studies on the clinical significance and treatment response to 5-fluorouracil, leucovorin, irinotecan, and oxaliplatin (FOLFIRINOX) regimen in terms of the presence of these mutations remain inconclusive. Objectives: This study aimed to compare the survival outcome and response to FOLFIRINOX chemotherapy based on the presence of four driver mutation genes. Design: A multi-center retrospective study conducted at two tertiary medical centers. Methods: This study analyzed PDAC patients who were treated with FOLFIRINOX chemotherapy as the initial treatment. Tumor specimens were analyzed by a targeted next-generation sequencing platform at two tertiary referral hospitals from January 2016 to March 2022. Patients’ demographics, survival outcomes, and chemotherapeutic response were investigated and compared according to the presence of driver mutations. Results: The analysis included 100 patients. KRAS mutation was identified in 92 (92.0%) patients, followed by TP53 , CDKN2A , and SMAD4 in 63 (63.0%), 18 (18.0%), and 17 (17.0%) patients, respectively. The TP53 wild-type group demonstrated longer overall survival (OS) than the TP53 mutated group (median OS: 29 vs 19 months, p = 0.03), and TP53 served as a prognostic factor for survival (hazard ratio = 1.74, 95% confidence interval: 1.00–3.00, p = 0.048). The difference in OS according to TP53 mutation was intensified in localized pancreatic adenocarcinoma (37 vs 19 months, p = 0.01). The TP53 wild-type group demonstrated a higher objective response rate to FOLFIRINOX chemotherapy than the TP53 mutation group in localized pancreatic adenocarcinoma (50.0% vs 17.6%, p = 0.024). Conclusion: PDAC patients with wild-type TP53 demonstrated longer OS than those with TP53 mutation, and this trend was intensified in patients with localized disease. This result may be due to an impaired response to FOLFIRINOX chemotherapy in patients with TP53 mutation.

Abstract SH-SY5Y, a neuroblastoma cell line, can be converted into mature neuronal phenotypes, characterized by the expression of mature neuronal and neurotransmitter markers. However, the mature phenotypes described across multiple studies appear inconsistent. As this cell line expresses common neuronal markers after a simple induction, there is a high chance of misinterpreting its maturity. Therefore, sole reliance on common neuronal markers is presumably inadequate. The Alzheimer's disease (AD) central gene, amyloid precursor protein (APP), has shown contrasting transcript variant dynamics in various cell types. We differentiated SH-SY5Y cells into mature neuron-like cells using a concise protocol and observed the upregulation of total APP throughout differentiation. However, APP transcript variant-1 was upregulated only during the early to middle stages of differentiation and declined in later stages. We identified the maturity state where this post-transcriptional shift occurs, terming it "true maturity." At this stage, we observed a predominant expression of mature neuronal and cholinergic markers, along with a distinct APP variant pattern. Our findings emphasize the necessity of using a differentiation state-sensitive marker system to precisely characterize SH-SY5Y differentiation. Moreover, this study offers an APP-guided, alternative neuronal marker system to enhance the accuracy of the conventional markers.

Abstract Androgenetic alopecia (AGA), also known as male pattern baldness, is a common hair loss condition influenced by genetic and hormonal factors. Variations in gene expression and androgen responsiveness have been observed between the frontal and occipital regions of AGA patients. However, obtaining and cultivating frontal hair follicles is challenging. Therefore, no matched frontal and occipital dermal papilla (DP) cell lines have been reported yet. This study aimed to establish matched immortalized human frontal and occipital scalp DP cell lines from AGA patients. Simian virus 40 large T antigen (SV40T-Ag) and human telomerase reverse transcriptase (hTERT) were introduced into primary human DP cells. The obtained cell lines were characterized by assessing their gene expression patterns, androgen receptor (AR) levels, and the presence of 5-alpha reductase (5αR). Additionally, we examined their response to dihydrotestosterone (DHT) and evaluated cell viability. The conditioned medium from the frontal DP cell line inhibited human hair follicle growth, leading to reduced keratinocyte proliferation and increased apoptosis. Furthermore, when the cells were cultured in a 3D environment mimicking in vivo conditions, the 3D cultured frontal DP cell line exhibited weaker sphere aggregation than the occipital DP cell line due to the increased expression of matrix metalloproteinase 1 (MMP1), MMP3, and MMP9. Additionally, the expression of DP signature genes was inhibited in the 3D cultured frontal DP cell line. These matched frontal and occipital DP cell lines hold significant potential as valuable resources for research on hair loss. Their establishment allows us to investigate the differences between frontal and occipital DP cells, contributing to a better understanding of the molecular mechanisms underlying AGA. Furthermore, these cell lines may be valuable for developing targeted therapeutic approaches for hair loss conditions.

Abstract Differentiating inflammatory bowel disease (IBD) from other inflammatory diseases is often challenging. Programmed cell death protein-1 (PD-1) is expressed in T cells and is an indicator of their exhaustion. The role of PD-1 expression in diagnosing IBD and predicting the response of biologic agents remains inconclusive. In this study, endoscopic biopsy samples of 19 patients diagnosed with IBD, intestinal tuberculosis, and intestinal Behcet’s disease were analyzed using multiplexed immunohistochemistry. Additionally, a separate "vedolizumab (VDZ) cohort" established in ulcerative colitis patients who underwent endoscopic biopsy before VDZ administration was analyzed to predict response to VDZ. In the immunohistochemistry analysis, the cell density of T cell subsets, including PD-1 + cells, was investigated and compared between IBD and other inflammatory diseases (OID). Cell densities of PD-1 + cells (p = 0.028), PD-1 + helper T cells (p = 0.008), and PD-1 + regulatory T cells (p = 0.024) were higher in IBD compared with OID. In the VDZ cohort, patients with a 14-week steroid-free clinical response had higher levels of PD-1 + cells (p = 0.026), PD-1 + helper T cells (p = 0.026), and PD-1 + regulatory T cells (p = 0.041) than the no response group. PD-1 + immune cells may contribute to the diagnosis of IBD and could be used to predict response to VDZ in ulcerative colitis patients.

Immune checkpoint inhibitors have dramatically revolutionized the therapeutic landscape for patients with advanced malignancies. Recently, convincing evidence has shown meaningful influence of gut microbiome on human immune system. With the complex link between gut microbiome, host immunity and cancer, the variations in the gut microbiota may influence the efficacy of immune checkpoint inhibitors. Indeed, some bacterial species have been reported to be predictive for cancer outcome in patients treated with immune checkpoint inhibitors. Although immune checkpoint inhibitors are currently proven to be an effective anti-tumor treatment, they can induce a distinct form of toxicity, termed immune-related adverse events. Immune-related colitis is one of the common toxicities from immune checkpoint inhibitors, and it might preclude the cancer therapy in severe or refractory cases. The manipulation of gut microbiome by fecal microbiota transplantation or probiotics administration has been suggested as one of the methods to enhance anti-tumor effects and decrease the risk of immune-related colitis. Here we review the role of gut microbiome on immune checkpoint inhibitor therapy and consequent immune-related colitis to provide a new insight for better anti-cancer therapy.

ABSTRACTObesity and overweight among companion animals are significant concerns, paralleling the issues observed in human populations. Recent research has highlighted the potential benefits of various probiotics in addressing weight-related changes, obesity, and associated pathologies. In this study, we delved into the beneficial probiotic mechanisms in high-fat-induced obese canines, revealing that Enterococcus faecium IDCC 2102 (IDCC 2102) and Bifidobacterium lactis IDCC 4301 (IDCC 4301) have the capacity to mitigate the increase in body weight and lipid accumulation in obese canines subjected to a high-fat diet and hyperlipidemic Caenorhabditis elegans (C. elegans) strain VS29. Both IDCC 2102 and IDCC 4301 demonstrated the ability to reduce systemic inflammation and hormonal disruptions induced by obesity. Notably, these probiotics induced modifications in the microbiota by promoting lactic acid bacteria, including Lactobacillaceae, Ruminococcaceae, and S24-7, with concomitant activation of pyruvate metabolism. IDCC 4301, through the generation of bacterial short-chain fatty acids and carboxylic acids, facilitated glycolysis and contributed to ATP synthesis. Meanwhile, IDCC 2102 produced bacterial metabolites such as acetic acid and butyric acid, exhibiting a particular ability to stimulate dopamine synthesis in a canine model. This stimulation led to the restoration of eating behavior and improvements in glucose and insulin tolerance. In summary, we propose novel probiotics for the treatment of obese animals based on the modifications induced by IDCC 2102 and IDCC 4301. These probiotics enhanced systemic energy utilization in response to high caloric intake, thereby preventing lipid accumulation and restoring stability to the fecal microbiota. Consequently, this intervention resulted in a reduction in systemic inflammation caused by the high-fat diet.IMPORTANCEProbiotic supplementation affected commensal bacterial proliferation, and administering probiotics increased glycolysis and activated pyruvate metabolism in the body, which is related to propanate metabolism as a result of pyruvate metabolism activation boosting bacterial fatty acid production via dopamine and carboxylic acid specialized pathways, hence contributing to increased ATP synthesis and energy metabolism activity.

Amniotic fluid mesenchymal stem cells (AF-MSCs), which can be obtained from fetal tissue, reportedly have self-renewal capacity and multi-lineage differentiation potential. The aim of this study was to identify the biological characteristics of AF-MSCs and evaluate their stability and safety in long-term culture. To confirm the biological characteristics of AF-MSCs, morphology, proliferation capacity, karyotype, differentiation capacity, gene expression level, and immunophenotype were analyzed after isolating AF-MSCs from equine amniotic fluid. AF-MSCs were differentiated into adipocytes, chondrocytes, and osteocytes. Immunophenotype analyses revealed expression levels of ≥95% and ≤ 2% of cells for a positive and negative marker, respectively. Analysis of the MSCs relative gene expression levels of AF-MSCs was approximately at least twice that of the control. The endotoxin level was measured to verify the safety of AF-MSCs and was found to be less than the standard value of 0.5 EU/ml. AF-MSCs were cultured for a long time without any evidence of abnormalities in morphology, proliferation ability, and karyotype. These results suggest that amniotic fluid is a competent source for acquiring equine MSCs and that it is valuable as a cell therapy due to its high stability.

The purpose of this study is to design a Swarm Control algorithm for the effective mission performance of multiple unmanned surface vehicles (USVs) used for marine research purposes at sea. For this purpose, external force information was utilized for the control of multiple USV swarms using a lead–follow-formation technique. At this time, to efficiently control multiple USVs, the LSTM algorithm was used to learn ocean currents. Then, the predicted ocean currents were used to control USVs, and a study was conducted on behavioral-based control to manage USV formation. In this study, a control system model for several USVs, each equipped with two rear thrusters and a front lateral thruster, was designed. The LSTM algorithm was trained using historical ocean current data to predict the velocity of subsequent ocean currents. These predictions were subsequently utilized as system disturbances to adjust the controller’s thrust. To measure ocean currents at sea as each USV moves, velocity, azimuth, and position data (latitude, longitude) from the GPS units mounted on the USVs were utilized to determine the speed and direction of the hull’s movement. Furthermore, the flow rate was measured using a flow rate sensor on a small USV. The movement and position of the USV were regulated using an Artificial Neural Network-PID (ANN-PID) controller. Subsequently, this study involved a comparative analysis between the results obtained from the designed USV model and those simulated, encompassing the behavioral control rules of the USV swarm and the path traced by the actual USV swarm at sea. The effectiveness of the USV mathematical model and behavior control rules were verified. Through a comparison of the movement paths of the swarm USV with and without the disturbance learning algorithm and the ANN-PID control algorithm applied to the designed simulator, we analyzed the position error and maintenance performance of the swarm formation. Subsequently, we compared the application results.

Spinal Cord Injury (SCI) refers to complete or incomplete damage to the spinal cord, which comprises the central nervous system. SCI in dogs, like humans, is mostly caused by external trauma, and the degree of impact is dependent on the location of the injury in the spine. Stem cell therapy is a promising avenue for SCI research. In this report, we investigate the therapeutic potential of amniotic fluid-derived mesenchymal stem cells (AF-MSCs) in dogs with spinal cord injuries. A 2-year-old male beagle dog presented with sensory and motor incomplete symptoms resulting in an inability to control the legs, hips, and genitourinary system due to an injury in the lumbar region of the spinal cord. In addition to the administration of surgical decompression, AF-MSCs were directly injected into the damaged spinal tissue. Approximately 15–16 weeks after stem cell transplantation, the dog's hind limb movement improved, and spinal cord regeneration was confirmed through magnetic resonance imaging (MRI). Eventually, the dog was able to walk independently, although not perfectly. In conclusion, AF-MSC-based stem cell transplantation may be beneficial for SCIs.

Abstract Canine hip dysplasia (HD) is a multifactorial disease caused by interactions between genetic and environmental factors. HD, which mainly occurs in medium- to large-sized dogs, is a disease that causes severe pain and requires surgical intervention. However, the procedure is not straight-forward, and the only way to ameliorate the situation is to exclude individual dogs with HD from breeding programs. Recently, prime editing (PE), a novel genome editing tool based on the CRISPR-Cas9 system, has been developed and validated in plants and mice. In this study, we successfully corrected a mutation related to HD in Labrador retriever dogs for the first time. We collected cells from a dog diagnosed with HD, corrected the mutation using PE, and generated mutation-corrected dogs by somatic cell nuclear transfer. The results indicate that PE technology can potentially be used as a platform to correct genetic defects in dogs.

Abstract In this work, we proposed a new damage model for estimating radiation-induced direct damage to biomolecular systems and validated its the effectiveness for pBR322 plasmids. The proposed model estimates radiation-induced damage to biomolecular systems by: (1) simulation geometry modeling using the coarse-grained (CG) technique to replace the minimum repeating units of a molecule with a single bead, (2) approximation of the threshold energy for radiation damage through CG potential calculation, (3) calculation of cumulative absorption energy for each radiation event in microscopic regions of CG models using the Monte Carlo track structure (MCTS) code, and (4) estimation of direct radiation damage to biomolecular systems by comparing CG potentials and absorption energy. The proposed model replicated measured data with an average error of approximately 14.2% in the estimation of radiation damage to pBR322 plasmids using the common MCTS code Geant4-DNA. This is similar to the results of previous simulation studies. However, in existing damage models, parameters are adjusted based on experimental data to increase the reliability of simulation results, whereas in the proposed model, they can be determined without using empirical data. Because the proposed model proposed is applicable to DNA and various biomolecular systems with minimal experimental data, it provides a new method that is convenient and effective for predicting damage in living organisms caused by radiation exposure.

The recent seismic events that occurred in South Korea have increased the interest in the re-evaluation of the seismic capacity of nuclear power plant (NPP) equipment, which is often conservatively estimated. To date, various approaches—including the Bayesian method proposed by the United States (US) Electric Power Research Institute —have been developed to quantify the seismic capacity of NPP equipment. Among these, the Bayesian approach has advantages in accounting for both prior knowledge and new information to update the probabilistic distribution of seismic capacity. However, data availability and region-specific issues exist in applying this Bayesian approach to Korean NPP equipment. Therefore, this paper proposes to construct an earthquake experience database by combining available earthquake records at Korean NPP sites and the general location of equipment within NPPs. Also, for the better representation of the seismic demand of Korean earthquake datasets, which have distinct seismic characteristics from those of the US at a high-frequency range, a broadband frequency range optimization is suggested. The proposed data construction and seismic demand optimization method for seismic capacity re-evaluation are demonstrated and tested on a 480 V motor control center of a South Korea NPP.

The sociomedical environment is changing. In the traditional physician-patient relationship, the physician was authoritative and the patient was obedient. The contractual relationship featured patient consent to the physician’s decision. Today, the physician must explain fully the planned medical treatment, and any alternative, to the patient, who has the right to choose her treatment after considering the benefits and side-effects. The Korean Society of Gynecologic Oncology thus decided to standardize the surgical consent forms to meet the legal requirements of modern medicine, improve patient understanding of the surgical details, and protect medical staff from legal disputes. To determine the format and content, subcommittees for each cancer type collected and reviewed all relevant articles and the current consent forms of domestic medical institutions. After several meetings, 16 basic items to be included for each type of gynecologic cancer were selected. Also, to help patients understand the surgical details, figures were included. The revised forms were legally reviewed in terms of the appropriateness of the format and content. We also developed English versions to provide adequate information for foreign patients. We hope that these efforts will promote trust between patients and physicians, and contribute to effective treatment by laying a foundation of mutual respect.

It is important to make a differential diagnosis between inflammatory diseases of the bowel with similar clinical and endoscopic features. The profiling of immune cells could be helpful for accurately diagnosing inflammatory bowel diseases. We compared immune marker expression between Crohn’s disease (CD), intestinal Behcet’s disease (BD), and intestinal tuberculosis (TB) and evaluated the usefulness of immune profiling in differentiating between these diseases. Biopsy specimens were acquired around ulcerations on the terminal ileum or cecum from five patients with each disease. Panel 1 included multiplex immunohistochemistry staining for CD8, CD4, Foxp3, CD20, programmed death-1, and granzyme B. CD56, CD68, CD163, CD11c, and HLA-DR were analyzed in panel 2. The differences in cytotoxic T cells (CD8+CD4−Fopx3−CD20−), helper T cells (CD8−CD4+Fopx3−CD20−), and regulatory T cells (CD8−CD4+Fopx3+CD20−) were also not significant. However, M1 macrophage (CD68+CD163−HLA−DR−) cell densities were significantly higher in intestinal BD than in other diseases. The expression level of dendritic cells (CD56−CD68−CD163−CD11c+HLA-DR+) was highest in intestinal TB and lowest in intestinal BD. The expression of immune cells, including M1 macrophages and dendritic cells, was different between CD, intestinal BD, and intestinal TB. Immune profiling can be helpful for establishing differential diagnoses of inflammatory bowel diseases.

BackgroundThe complete resection rate of pancreatic cancer has increased because of the advent of efficacious first-line treatments for unresectable pancreatic cancer. Still, strategies regarding adjuvant therapy after neoadjuvant FOLFIRINOX treatment remain to be established.MethodsData on 144 patients with borderline resectable and locally advanced pancreatic cancer who underwent resection after neoadjuvant FOLFIRINOX between January 2013 and April 2021 were retrospectively reviewed.ResultsAmong the study patients, 113 patients (78.5%) were diagnosed with borderline resectable pancreatic cancer and 31 patients (21.5%) were diagnosed with locally advanced pancreatic cancer. Seventy-five patients (52.1%) received radiotherapy before surgery. After radical resection, 84 patients (58.3%) received 5-fluorouracil-based adjuvant therapy and 60 patients (41.7%) received non-5-fluorouracil-based adjuvant therapy. Adjuvant therapy with 5-fluorouracil-based regimen [hazard ratio (HR), 0.43 (95% CI, 0.21–0.87); p = 0.019], preoperative assessment as locally advanced pancreatic cancer [HR, 2.87 (95% CI, 1.08–7.64); p = 0.035], positive resection margin [HR, 3.91 (95% CI, 1.71–8.94); p = 0.001], and presence of pathologic lymph node involvement [HR, 2.31 (95% CI, 1.00–5.33), p = 0.050] were associated with decreased recurrence-free survival. Adjuvant therapy with 5-fluorouracil-based regimen [HR, 0.35 (95% CI, 0.15–0.84); p = 0.018], positive resection margin [HR, 4.14 (95% CI, 1.75–9.78); p = 0.001], presence of pathologic lymph node involvement [HR, 3.36 (95% CI, 1.23–9.15); p = 0.018], poor differentiation [HR, 5.69 (95% CI, 1.76–18.36); p = 0.004], and dose reduction during adjuvant therapy [HR, 1.78 (95% CI, 1.24–24.37); p = 0.025] were associated with decreased overall survival.ConclusionsThe 5-fluorouracil-based adjuvant therapy seems to be the proper adjuvant therapy for patients who received neoadjuvant FOLFIRINOX for borderline resectable and locally advanced pancreatic cancer.

Abstract Although solution-processed Cu(In,Ga)(S,Se)2 (CIGS) absorber layers can potentially enable the low-cost and large-area production of highly stable electronic devices, they have rarely been applied in photodetector applications. In this work, we present a near-infrared photodetector functioning at 980 nm based on solution-processed CIGS with a potassium-induced bandgap grading structure and chalcopyrite grain growth. The incorporation of potassium in the CIGS film promotes Se uptake in the bulk of the film during the chalcogenization process, resulting in a bandgap grading structure with a wide space charge region that allows improved light absorption in the near-infrared region and charge carrier separation. Also, increasing the Se penetration in the potassium-incorporated CIGS film leads to the enhancement of chalcopyrite crystalline grain growth, increasing charge carrier mobility. Under the reverse bias condition, associated with hole tunneling from the ZnO interlayer, the increasing carrier mobility of potassium-incorporated CIGS photodetector improved photosensitivity and particularly external quantum efficiency more than 100% at low light intensity. The responsivity and detectivity of the potassium-incorporated CIGS photodetector reach 1.87 A W−1 and 6.45 $$\times$$ × 1010 Jones, respectively, and the − 3 dB bandwidth of the device extends to 10.5 kHz under 980 nm near-infrared light.

Although somatic cell nuclear transfer (SCNT) is frequently employed to produce cloned animals in laboratories, this technique is expensive and inefficient. Therefore, the handmade cloning (HMC) technique has been suggested to simplify and advance the cloning process, however, HMC wastes many oocytes and leads to mitochondrial heteroplasmy. To solve these problems, we propose a modified handmade cloning (mHMC) technique that uses simple laboratory equipment, i.e., a Pasteur pipette and an alcohol lamp, applying it to porcine embryo cloning. To validate the application of mHMC to pig cloning, embryos produced through SCNT and mHMC are compared using multiple methods, such as enucleation efficiency, oxidative stress, embryo developmental competence, and gene expression. The results show no significant differences between techniques except in the enucleation efficiency. The 8-cell and 16-cell embryo developmental competence and Oct4 expression levels exhibit significant differences. However, the blastocyst rate is not significantly different between mHMC and SCNT. This study verifies that cloned embryos derived from the two techniques exhibit similar generation and developmental competence. Thus, we suggest that mHMC could replace SCNT for simpler and cheaper porcine cloning.

Cumulus-oocyte complexes (COCs), which contain immature oocytes, are matured in vitro for in vitro embryo production. Oocyte and cumulus cells are then separated using hyaluronidase. To date, there have only been a few reported cases of the toxic effects of hyaluronidase on porcine oocytes. The aim of this study was to compare the effects of bovine testis-derived hyaluronidase and recombinant human hyaluronidase on oocyte denudation and quality. Porcine COCs were matured for 44 h and denuded using different hyaluronidase concentrations and exposure times. Then, oocytes were activated by electrical parthenogenesis. In experiment 1, COCs were denuded using bovine-derived, ovine-derived (Hirax), and human recombinant (ALT-BC4) hyaluronidases for 10 and 20 min. In experiment 2, bovine-derived and human recombinant (ALT-BC4 and ICSI Cumulase®) hyaluronidases were used to denude the COCs for 2 and 20 min. In both experiments the oocytes were all completely denuded, and there was no degeneration. Rate of embryo development was significantly increased in group treated ALT-BC4 for 2 min and not significantly different in other treatment groups. In general it slightly decreased with longer exposure times. These results have confirmed that different sources of hyaluronidase do not have detrimental effects on the quality of porcine oocytes and suggest that the human recombinant hyaluronidase ALT-BC4 is suitable for oocyte denudation with an increased blastocyst rate.

Many damages due to flow-accelerated corrosion and cracking have been observed during recent in-service inspections of nuclear power plants. To determine the operability or repair for damaged pipes, an integrity evaluation related to the damaged piping system should be performed by using already proven code and standards. One of them, the ASME Code Case is most popularly used to integrity assessment in nuclear power plants. However, the recent version of CC N-513 still recommends the simplified method which means a damaged elbow is assumed as an equivalent straight pipe. In addition, to enhance the accuracy integrity assessment in elbow, several previous studies recommend that the SIF and elastic COD values for an elbow with relatively large crack could be predicted by an interpolation technique. However, those estimates for elbow with relatively large crack might be derived to inaccurate results for crack growth analysis, such as for the allowable crack size and life estimation. Therefore, in this paper, the effect of crack length (0.3≤θ1/π ≤ 0.5) on SIF and elastic COD for elbow is systematically investigated. Then, for large crack in elbow, accurate estimates for SIF and elastic COD, which are widely used to assess the integrity of elbows, are proposed. Those proposed solutions are expected to be the technical basis for revisions of CC N-513-4 through the validation.

Abstract Background The differentiation of human mesenchymal stem cells (hMSCs) into osteoblasts (OBs) is a prerequisite for bone formation. However, little is known about the definitive surface markers for OBs during osteogenesis. Methods To study the surface markers on OBs, we generated and used monoclonal antibodies (MAbs) against surface molecules on transforming growth factor-β1 (TGF-β1)-treated cancer cells. The generated MAbs were further selected toward expression changes on hMSCs cultured with TGF-β1/bone morphogenetic protein-2 (BMP-2) or osteogenic differentiation medium (ODM) by flow cytometry. Immunoprecipitation and mass spectrometry were performed to identify target antigens of selected MAbs. Expression changes of the target antigens were evaluated in hMSCs, human periodontal ligament cells (hPDLCs), and human dental pulp cells (hDPCs) during osteogenic and adipogenic differentiation by quantitative polymerase chain reaction (qPCR) and flow cytometry. hMSCs were also sorted by the MAbs using magnetic-activated cell sorting system, and osteogenic potential of sorted cells was evaluated via Alizarin Red S (ARS) staining and qPCR. Results The binding reactivity of MR14-E5, one of the MAbs, was downregulated in hMSCs with ODM while the binding reactivity of ER7-A7, ER7-A8, and MR1-B1 MAbs was upregulated. Mass spectrometry and overexpression identified that MR14-E5, ER7-A7/ER7-A8, and MR1-B1 recognized integrin α2, α3, and αV, respectively. Upon osteogenic differentiation of hMSCs, the expression of integrin α2 was drastically downregulated, but the expression of integrin α3 and αV was upregulated in accordance with upregulation of osteogenic markers. Expression of integrin α3 and αV was also upregulated in hPDLCs and hDPCs during osteogenic differentiation. Cell sorting showed that integrin αV-high hMSCs have a greater osteogenic potential than integrin αV-low hMSCs upon the osteogenic differentiation of hMSCs. Cell sorting further revealed that the surface expression of integrin αV is more dramatically induced even in integrin αV-low hMSCs. Conclusion These findings suggest that integrin α3 and αV induction is a good indicator of OB differentiation. These findings also shed insight into the expression dynamics of integrins upon osteogenic differentiation of hMSCs and provide the reason why different integrin ligands are required for OB differentiation of hMSCs.

Efforts have constantly been made to incorporate AI into teaching and learning; however, the successful implementation of new instructional technologies is closely related to the attitudes of the teachers who lead the lesson. Teachers’ perceptions of AI utilization have only been investigated by only few scholars due an overall lack of experience of teachers regarding how AI can be utilized in the classroom as well as no specific idea of what AI-adopted tools would be like. This study investigated how teachers perceived an AI-enhanced scaffolding system developed to support students’ scientific writing for STEM education. Results revealed that most STEM teachers positively experienced AI as a source for superior scaffolding. On the other hand, they also raised the possibility of several issues caused by using AI such as the change in the role played by the teachers in the classroom and the transparency of the decisions made by the AI system. These results can be used as a foundation for which to create guidelines for the future integration of AI with STEM education in schools, since it reports teachers’ experiences utilizing the system and various considerations regarding its implementation.

Human pluripotent stem cells (hPSCs) exist in at least two distinct states in mammals: naïve pluripotency that represents several molecular characteristics in pre-implantation epiblast and primed pluripotency that corresponds to cells poised for differentiation in post-implantation epiblast. To identify and characterize the surface molecules that are necessary for the maintenance of naïve hPSCs, we generated a panel of murine monoclonal antibodies (MAbs) specific to the naïve state of hPSCs. Flow cytometry showed that N1-A4, one of the MAbs, bound to naïve hPSCs but not to primed hPSCs. Cell surface biotinylation and immunoprecipitation analysis identified that N1-A4 recognized heat shock protein 60 (HSP60) expressed on the surface of naïve hPSCs. Quantitative polymerase chain reaction (qPCR) analysis showed that HSP60 expression was rapidly downregulated during the embryoid body (EB) differentiation of primed hPSCs. HSP60 knockdown led to a decrease in the expression of pluripotency genes in primed hPSCs. HSP60 depletion also led to a decrease in the expression of pluripotency genes and representative naïve-state-specific genes in naïve hPSCs. Taken together, the results suggest that HSP60 is downregulated during differentiation of hPSCs and is required for the maintenance of pluripotency genes in both primed and naïve hPSCs, suggesting that HSP60 is a regulator of hPSC pluripotency and differentiation.

We aimed to establish screening and efficacy test techniques for use in the development of hair-promoting agents. To this end, we used the dermal papilla cell (DPc)-derived immortalized cell line (SV40T-hTERT DPc) and neonatal foreskin-derived keratinocyte cell line (Ker-CT) to form an immortalized cell-based hair follicle-like structure. The SV40T-hTERT DPc spheroids exhibited a higher cell ratio in the spheroids than primary DPc spheroids, and SV40T-hTERT DPc aggregated with spheroids larger in diameter than primary DPc when the same cell number was seeded into the low-adhesion plate. Microscopic imaging and fluorescence staining results indicated that both primary and immortalized cell combinations form a hair follicle-like structure with a long-stretched keratinocyte layer under the condition that the spheroids have the same diameter as that of in vivo dermal papillary tissue in the hair follicle. The hair follicle-like structure elongation was increased upon treatment with three known hair follicle growth-promoting compounds (minoxidil, tofacitinib, and ascorbic acid) compared with that in the control group. Therefore, using immortalized cells to generate a coherent follicle-like structure, we have developed models for screening and evaluating hair-care materials commonly used in the industry.

Abstract Evaporative drying (ED) is an alternative technique for long-term preservation of mammalian sperm, which does not require liquid nitrogen or freeze-drying equipment, but offers advantages for storage and shipping at ambient temperature and low cost. However, the development of zygotes generated from these sperms was poor. Here, we demonstrated that the supplementation of tauroursodeoxycholic acid (TUDCA), an endogenous bile acid, during embryo culture improved the developmental competency of embryos derived from in vitro matured pig oocytes injected intracytoplasmically with boar ED spermatozoa by reducing the production of reactive oxygen species, the DNA degradation and fragmentation, and the expression of apoptosis-related gene Bax and Bak, and by increasing the transcription of anti-apoptosis gene Bcl-XL and Bcl-2. Furthermore, TUDCA treatment promoted the blastocyst quality manifested by the total cell numbers and the ratio of inner cell mass. Taken together, our data suggest that evaporative drying would be a potentially useful method for the routine preservation of boar sperm in combination with further optimization of subsequently embryo culture conditions.

Purpose This study aimed to clarify the association between human epidermal growth factor receptor 2 (HER-2) status and the clinicopathologic factors of patients who underwent curative intent gastrectomy. Methods From June 2011 to May 2015, curative intent gastrectomy was performed in 441 patients at Konyang University Hospital. Among them, we evaluated the HER-2 status in 113 patients. Data on clinicopathologic parameters such as age, sex, histological subtype, endoscopic Lauren classification, tumor location, size, presence of lymphovascular invasion, invasion depth, pathologic stage, HER-2 overexpression, recurrence and survival were obtained. In this study, pathological HER-2 intensity scores of 0, 1+, and 2+ were assumed to be negative, 3+ only was to be positive for overexpression. Results In a total of 113 cases who underwent curative intent gastrectomy with HER-2 testing, 16 (14.2%) cases had positivity of HER-2 overexpression. HER-2 overexpression had significant associations with tumor stage (19.0% in I-IIIb vs. 2.9% in IIIc-IV, P=0.036). Survival analysis of HER2 overexpression has no significant difference. Conclusion In this study, HER-2 overexpression rate was 14.2% and patient tumor stage had significant association with HER-2 overexpression.

Continued CRISPR/Cas9-mediated editing activity that allows differential and asynchronous modification of alleles in successive cell generations expands allelic complexity. To understand the earliest events during CRISPR/Cas9 editing and the allelic selection among the progeny of subsequent cell divisions, we inspected in detail the genotypes of 4- and 8-cell embryos and embryonic stem cells (ESCs) after microinjection of a CRISPR toolkit into the zygotes. We found a higher editing frequency in 8-cell embryos than in 4-cell embryos, indicating that the CRISPR/Cas9 activity persisted through the 8-cell stage. Analysis of a CRISPR/Cas9 transgenic founder mouse revealed that four different alleles were present in its organs in different combinations and that its germline included three different mutant alleles, as shown by the genotypes of the pups. The indel depth, which measured the extent of indels at the sequence level within single embryos, decreased significantly as the embryos advanced to form ESCs, suggesting that exclusion of fatal indels occurred in the subsequent cell generations. Interestingly, we discovered that the CRISPR sites frequently contained introduced retroelement sequences and that this occurred preferentially with certain classes of retroelements. Therefore, in addition to CRISPR/Cas9's innate mechanism of separate, differential enzymatic modifications of alleles, the frequent retroelement insertions that occur in early mouse embryos during CRISPR/Cas9 editing further expand the allelic diversity and mosaicism in the resulting transgenic founders.

In vitro culture (IVC) for porcine embryo development is inferior compared to in vivo development because oxidative stress can be induced by the production of excessive reactive oxygen species (ROS) under high oxygen tension in the in vitro environment. To overcome this problem, we investigated the effect of lycopene, an antioxidant carotenoid, on developmental competence and the mechanisms involved in mitochondria-dependent apoptosis pathways in porcine embryos. In vitro fertilized (IVF) embryos were cultured in IVC medium supplemented with 0, 0.02, 0.05, 0.1, or 0.2 μM lycopene. The results indicate that 0.1 μM lycopene significantly increased the rate of blastocyst formation and the total cell numbers, including trophectoderm cell numbers, on Day In terms of mitochondria-dependent apoptosis, IVF embryos treated with 0.1 μM lycopene exhibited significantly decreased levels of ROS, increased mitochondrial membrane potential, and decreased expression of cytochrome c on Days 2 and Furthermore, 0.1 μM lycopene significantly decreased the number and percentage of caspase 3-positive and apoptotic cells in Day-6 blastocysts. In addition, Day-2 embryos and Day-6 blastocysts treated with 0.1 μM lycopene showed significantly reduced mRNA expression related to antioxidant enzymes (SOD1, SOD2, CATALASE) and apoptosis (BAX/BCL2L1 ratio). These results indicate that lycopene supplementation during the entire period of IVC enhanced embryonic development in pigs by regulating oxidative stress and mitochondria-dependent apoptosis.

[This corrects the article DOI: 10.1371/journal.pone.0169091.].

Probabilistic safety assessment (PSA) of nuclear facilities on external multi-hazards has become a major issue after the Fukushima accident in 2011. However, the existing external hazard PSA methodology is for single hazard events and cannot cover the impact of multi-hazards. Therefore, this study proposes a methodology for quantifying multi-hazard risks for nuclear energy plants. Specifically, we developed an efficient multi-hazard PSA methodology based on the probability distribution-based Boolean algebraic approach and sampling-based method, which are currently single-hazard PSA methodologies. The limitations of the probability distribution-based Boolean algebraic approach not being able to handle partial dependencies between the components are solved through this sampling-based method. In addition, we devised an algorithm that was more efficient than the existing algorithm for improving the limits of the current sampling-based method, as it required a significant computational time. The proposed methodology was applied from simple examples to single- and multi-hazard PSA examples of actual nuclear power plants. The results showed that the proposed methodology was verified in terms of accuracy and efficiency perspectives. Regarding the sampling-based method, it was confirmed that the proposed algorithm yielded fragility and risk results that have similar degrees of accuracy, even though it extracted a smaller number of samples than the existing algorithm.

Somatic cell nuclear transfer (SCNT) is a well-known laboratory technique. The principle of the SCNT involves the reprogramming a somatic nucleus by injecting a somatic cell into a recipient oocyte whose nucleus has been removed. Therefore, the nucleus donor cells are considered as a crucial factor in SCNT. Cell cycle synchronization of nucleus donor cells at G0/G1 stage can be induced by contact inhibition or serum starvation. In this study, acteoside, a phenylpropanoid glycoside compound, was investigated to determine whether it is applicable for inducing cell cycle synchronization, cytoprotection, and improving SCNT efficiency in canine fetal fibroblasts. Primary canine fetal fibroblasts were treated with acteoside (10, 30, 50 μM) for various time periods (24, 48 and 72 hours). Cell cycle synchronization at G0/G1 stage did not differ significantly with the method of induction: acteoside treatment, contact inhibition or serum starvation. However, of these three treatments, serum starvation resulted in significantly increased level of reactive oxygen species (ROS) (99.5 ± 0.3%) and apoptosis. The results also revealed that acteoside reduced ROS and apoptosis processes including necrosis in canine fetal fibroblasts, and improved the cell survival. Canine fetal fibroblasts treated with acteoside were successfully arrested at the G0/G1 stage. Moreover, the reconstructed embryos using nucleus donor cells treated with acteoside produced a healthy cloned dog, but not the embryos produced using nucleus donor cells subjected to contact inhibition. In conclusion, acteoside induced cell cycle synchronization of nucleus donor cells would be an alternative method to improve the efficiency of canine SCNT because of its cytoprotective effects.

Many studies have shown that the mycoplasmal membrane protein p37 enhances cancer cell migration, invasion, and metastasis. Previously, we generated 6 monoclonal antibodies (MAbs) against the mycoplasmal protein p37 and showed the presence of mycoplasma-infected circulating tumor cells in the blood of hepatocellular carcinoma patients by using CA27, one of the six MAbs. When mycoplasmas were incubated with cancer cells in the presence of CA27, mycoplasma infection was completely inhibited, suggesting that CA27 is a neutralizing antibody inhibiting mycoplasma infection. To examine the neutralizing epitope of CA27, we generated a series of glutathione S-transferase (GST)-fused p37 deletion mutant proteins in which p37 was partly deleted. To express p37-coding sequences in E.coli, mycoplasmal TGA codons were substituted with TGG in the p37 deletion mutant genes. GST-fused p37 deletion mutant proteins were then screened to identify the epitope targeted by CA27. Western blots showed that CA27 bound to the residues 216-246 on the middle part of the p37 protein while it did not bind to the residues 183-219 and 216-240. Fine mapping showed that CA27 was able to bind to the residues 226-246, but its binding activity was relatively weakened as compared to that to the residues 216-246, suggesting that the residues 226-246 is essential for optimal binding activity of CA27. Interestingly, the treatment of the purified GST-tagged epitopes with urea showed that CA27 binding to the epitope was sodium dodecyl sulfate-resistant but urea-sensitive. The same 226-246 residues were also recognized by two other anti-p37 MAbs, suggesting that the epitope is immunodominant. The identification of the novel neutralizing epitope may provide new insight into the interaction between the p37 protein and host receptors.

Previously, we showed that B-cell receptor associated protein 31 (BAP31), an endoplasmic reticulum (ER) membrane chaperone, is also expressed on the cell surface by two monoclonal antibodies (MAbs) 297-D4 and 144-A8. Both MAbs recognize the same linear epitope on the C-terminal domain of BAP31, although they were independently established. Here, flow cytometric analysis showed that 144-A8 had additional binding properties to some cells, as compared to 297-D4. Quantitative antigen binding assays also showed that 144-A8 had higher antigen binding capacity than 297-D4. Affinity measurement revealed that 144-A8 had 1.54-fold higher binding affinity than 297-D4. Analysis of the heavy- and light-chain variable region sequences of two MAbs revealed that both MAbs belonged to the same heavy chain (Igh-V3660 VH3) and light chain subgroup (IGKV21) with just two amino acid differences in each framework region, indicating that both MAbs arise from the same germline origin. Seven amino acid differences were found between the complementarity determining regions (CDRs) of the two MAbs. Molecular modeling of the epitope-paratope complexes revealed that the epitope appeared to reside in closer proximity to the CDRs of 144-A8 than to those of 297-D4 with the stronger hydrogen bond interactions with the former than the latter. More interestingly, an additional hydrophobic interaction appeared to be established between the leucine residue of epitope and the paratope of 144-A8, due to the substitution of H-Tyr101 for H-Phe101 in 144-A8. Thus, the different binding specificity and affinity of 144-A8 appeared to be due to the different hydrogen bonds and hydrophobic interaction induced by the alterations of amino acids in CDRs of 144-A8. The results provide molecular insights into how the binding specificities and affinities of antibodies evolve with the same epitope in different microenvironments.

This study investigated the development of rat oocytes in vitro and in vivo following intracytoplasmic injection of heads from spermatozoa heat-dried at 50°C for 8 h and stored at 4°C in different gas phases. Sperm membrane and chromosome are damaged by the process of heat-drying. Oocyte activation and cleavage of oocytes were worse in oocytes injected with spermatozoa heat-dried and stored for 1 week than unheated, fresh spermatozoa, but in heat-dried spermatozoa, there were no differences in these abilities of oocytes between the samples stored in nitrogen gas and in air. The oocytes injected with heat-dried spermatozoa stored for 1 week could develop to the morula and blastocyst stages without difference between the samples stored in nitrogen gas and in air after artificial stimulation. Cleavage of oocytes and development of cleaved embryos were higher when heat-dried spermatozoa were stored for 3 and 6 months in nitrogen gas than in air. However, the ability of injected oocytes to develop to the morula and blastocyst stages was not inhibited even when heat-dried spermatozoa stored in both atmosphere conditions for as long as 6 months were used. When 2-cell embryos derived from oocytes injected with heads from spermatozoa heat-dried and stored for 1 week and 1 month were transferred, each 1 of 4 recipients was conceived, and the conceived recipients delivered 1 live young each. These results demonstrate that rat oocytes can be fertilized with heat-dried spermatozoa and that the fertilized oocytes can develop to term.

Alzheimer’s disease is considered one of the major neurodegenerative diseases and is characterized by the production of β-amyloid (Aβ) proteins and progressive loss of neurons. Biochanin A, a phytoestrogen compound found mainly in Trifolium pratense, was used in the present study as a potential alternative to estrogen replacement therapy via the investigation of its neuroprotective effects against Aβ25–35-induced toxicity, as well as of its potential mechanisms of action in PC12 cells. Exposure of these cells to the Aβ25–35 protein significantly increased cell viability loss and apoptosis. However, the effects induced by Aβ25–35 were markedly reversed in the present of biochanin A. Pretreatment with biochanin A attenuated the cytotoxic effect of the Aβ25–35 protein by decreasing viability loss, LDH release, and caspase activity in cells. Moreover, we found that expression of cytochrome c and Puma were reduced, alongside with the restoration of Bcl-2/Bax and Bcl-xL/Bax ratio in the presence of biochanin A, which led to a decrease in the apoptotic rate. These data demonstrate that mitochondria are involved in the protective effect of biochanin A against Aβ25–35 and that this drug attenuated Aβ25–35-induced PC12 cell injury and apoptosis by preventing mitochondrial dysfunction. Thus, biochanin A might raise a possibility as a potential therapeutic agent for Alzheimer’s disease and other related neurodegenerative diseases.

We propose a new electrical-phase-change magnetic material, namely Fe-doped In-Sb-Te (FIST), for possible non-volatile multi-bit memory applications. FIST was formed by typical co-sputter method with Fe 10 at.% doping in In3Sb1Te2. FIST offers the electrical-phase-change and magnetic properties by way of the change of In 4d chemical bonding density and embedded Fe nanoclusters with the size of 4∼5 nm, respectively. It maintained the amorphous phase on the electrical-phase-change. Chemical state of In was only changed to increase the density of In-In chemical bonding during the electrical-phase-change without Fe nanoclusters contribution. Also, the magnetic property by Fe nanoclusters was not changed by the electrical-phase-change. On this basis, we propose the FIST material with the individual electrical-phase-change and magnetic properties for the multi-bit nonvolatile memory materials.

Background: Bulky or multiple lymph node (LN) metastases are associated with poor prognosis in cervical cancer, and the size or number of LN metastases is not yet reflected in the staging system and therapeutic strategy. Although the therapeutic effects of surgical resection of bulky LNs before standard treatment have been reported in several retrospective studies, well-planned randomized clinical studies are lacking. Therefore, the aim of the Korean Gynecologic Oncology Group (KGOG) 1047/DEBULK trial is to investigate whether the debulking surgery of bulky or multiple LNs prior to concurrent chemoradiation therapy (CCRT) improves the survival rate of patients with cervical cancer IIICr diagnosed by imaging tests. Methods: The KGOG 1047/DEBULK trial is a phase III, multicenter, randomized clinical trial involving patients with bulky or multiple LN metastases in cervical cancer IIICr. This study will include patients with a short-axis diameter of a pelvic or para-aortic LN ≥2 cm or ≥3 LNs with a short-axis diameter ≥1 cm and for whom CCRT is planned. The treatment arms will be randomly allocated in a 1:1 ratio to either receive CCRT (control arm) or undergo surgical debulking of bulky or multiple LNs before CCRT (experimental arm). CCRT consists of extended-field external beam radiotherapy/pelvic radiotherapy, brachytherapy and LN boost, and weekly chemotherapy with cisplatin (40 mg/m2), 4--6 times administered intravenously. The primary endpoint will be 3-year progression-free survival rate. The secondary endpoints will be 3-year overall survival rate, treatment-related complications, and accuracy of radiological diagnosis of bulky or multiple LNs. [ABSTRACT FROM AUTHOR]