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The Cas9 nuclease is delivered in the form of either Cas9 protein or mRNA along with CRISPR guide RNA (gRNA: dual-crRNA:tracrRNA or chimeric single-guide RNA) or in a plasmid package encoding both Cas9 and the CRISPR gRNA.We directly compared the efficiency of producing rat blastocysts with homozygous mutations of the Foxn1 locus by pronuclear injection of Cas9 in the form of protein, mRNA, or plasmid DNA. For highly efficient production of rat blastocysts with homozygous Foxn1 mutations, pronuclear injection of Cas9 protein at 60 ng/µl was likely optimal. While blastocyst harvest in the mRNA groups was higher than those in the protein and plasmid DNA groups, genotype analysis showed that 63.6%, 8.7-20.0%, and 25.0% of the analyzed blastocysts were homozygous mutants in the protein, mRNA, and plasmid DNA groups, respectively. The high efficiency of producing homozygous mutant blastocysts in the 60 ng/µl protein group may be associated with primary genome editing being initiated before the first cleavage. In most cases, homozygous mutations at the target Foxn1 locus are triggered by deletion and repair via nonhomologous end joining or microhomology-mediated end joining. Deletion downstream of the Cas9 break site was more likely than deletion in the upstream direction.The Cas9 nuclease in protein form, when coinjected with the CRISPR gRNA (ribonucleoprotein) into a rat zygote pronucleus, can access the target genome site and induce double-strand breaks promptly, resulting in the efficient production of homozygous mutants.

Various liquid biopsy methods have been developed for the non-invasive and early detection of diseases. In particular, the detection of circulating tumor cells (CTCs) and cancer-associated fibroblasts (CAFs) in blood has been receiving a great deal of attention. We have been developing systems and materials to facilitate such liquid biopsies. In this study, we further developed glass filters (with various patterns of holes, pitches, and non-adhesive coating) that can capture CTCs, but not white blood cells. We optimized the glass filters to capture CTCs, and demonstrated that they could be used to detect CTCs from lung cancer patients. We also used the optimized glass filters for detecting CAFs. Additionally, we further developed a system for visualizing the captured cells on the glass filters. Finally, we demonstrated that we could directly culture the captured cells on the glass filters. Based on these results, our high-performance glass filters appear to be useful for capturing and culturing CTCs and CAFs for further examinations.

Background Airway management in children with severe burns is difficult because of airway edema and prolonged duration of ventilatory management. There is insufficient evidence to suggest that tracheostomy is beneficial for children. Case presentation A male child aged 1 year and 4 months was injured when he accidentally fell into a bathtub filled with boiling water. Furthermore, 85% of the burnt area, including the face and neck, consisted of second-degree burns; hence, oral tracheal intubation and resuscitative infusion were required. In this case, the patient was safely switched from oral to nasotracheal intubation using a tracheal tube guide and video laryngoscope, without the use of a bronchoscope, and ventilatory management could be continued for 2 weeks. Conclusion Oral to nasal endotracheal tube exchange using a tracheal tube guide and video laryngoscope may be useful not only for pediatric burn patients but also for adult patients who need to be safely switched from oral to nasotracheal intubation.

Background The fall armyworm, Spodoptera frugiperda (J. E. Smith) (Lepidoptera: Noctuidae), is an invasive alien species in Indonesia. This study aimed to assess the impact of its invasion in Indonesia by measuring the damaged area caused by the pest in maize fields located across East Java and simultaneously determine whether S. frugiperda outcompetes the native Asian armyworm Spodoptera litura (Fabricius). Secondly, the virulence of 14 entomopathogenic fungus (EPF) isolates against S. frugiperda larvae was evaluated in an effort to find effective biocontrol agent candidates. Results The damaged area caused by S. frugiperda was generally higher than that caused by S. litura during the survey period from August 2019 to December 2021. It indicated that S. frugiperda may have dominated the native armyworm and become the primary key pest of maize in Indonesia. Based on a single-concentration assay (106 conidia ml−1), the tested EPF isolates displayed varying degrees of virulence against S. frugiperda larvae, causing larval mortality of 3.5 to 71% at 10-day post-treatment, with the highest mortality rates provided by Beauveria bassiana sensu lato and Trichoderma asperellum sensu lato. At a concentration of 108 conidia ml−1, B. bassiana s.l. and T. asperellum s.l. elicited high larval mortality of 76 and 81%, respectively, at 10-day post-treatment. Nevertheless, the probit analysis based on a concentration–response assay revealed that T. asperellum s.l. had lower LC50 and LC90 values than B. bassiana s.l. Conclusions The attack and invasion of S. frugiperda seem to be a continual threat to the maize agro-ecosystem in Indonesia. As a consequence, Indonesia should mitigate and be well-prepared for future outbreaks of S. frugiperda. Indigenous EPF isolates used in this study may act as promising biocontrol agents of S. frugiperda, especially T. asperellum s.l. This study also serves as the first report documenting the direct lethality of Trichoderma fungus on S. frugiperda larvae.

The Fc portion of immunoglobulin G (IgG) promotes defensive effector functions in the immune system by interacting with Fcγ receptors and complement component C1q. These interactions critically depend on N-glycosylation at Asn297 of each CH2 domain, where biantennary complex-type oligosaccharides contain microheterogeneities resulting primarily from the presence or absence of non-reducing terminal galactose residues. Crystal structures of Fc have shown that a pair of N-glycans is located between the two CH2 domains. Here we applied our metabolic isotope labeling technique using mammalian cells for in-solution structural characterization of mouse IgG2b-Fc glycoforms with a molecular mass of 54 kDa. Based on spectral assignments of the N-glycans as well as polypeptide backbones of Fc, we probed conformational perturbations of Fc induced by N-glycan trimming, especially enzymatic degalactosylation. The results indicated that degalactosylation structurally perturbed the Fc region through rearrangement of glycan-protein interactions. The spectral assignments of IgG2b-Fc glycoprotein will provide the basis for NMR investigation of its dynamic conformations and interactions with effector molecules in solution.

We describe a “left-posterior approach” in which the important steps in laparoscopic distal pancreatectomy (LDP) for left-sided pancreatic cancer are accomplished in the direction caudal and dorsal to the pancreas. The patients who underwent LDP with a left-posterior approach at our hospital from January 2016 to April 2020 were reviewed to evaluate the short-term postoperative outcomes. In LDP, we first dissected retroperitoneal tissues above the left renal vein and superior mesenteric artery, yielding the mobilization of the pancreatic body widely. Then, the splenic artery was divided behind the ventrally lifted pancreas as an artery-first approach. The regional lymphadenectomy was performed in an en bloc manner consecutively in the same operative field. The neck of the pancreas was transected with a linear stapler after mobilization of the spleen. In nine patients (five men and four women) aged 76 years (range: 64–82 years), the operative time was 398 min (276–482 min) with the estimated blood loss of 40 ml (0–80 ml). No patients developed grade B/C pancreatic fistula or delayed gastric emptying. Postoperative complications classified as grade III in the Clavien–Dindo classification occurred in one patient (abdominal abscess). The pathology confirmed R0 resection in all patients who had pancreatic cancer (n = 5), IPMNs (n = 3), and high-grade pancreatic intraepithelial neoplasia (PanIN) (n = 1). The number of retrieved lymph nodes was 35 (11–49). The procedure with a left-posterior approach is a rational surgical technique in LDP for left-sided pancreatic cancer.

Background Borderline resectable pancreatic cancer (BRPC) is a category of pancreatic cancer that is anatomically widely spread, and curative resection is uncommon with upfront surgery. Intensity-modulated radiation therapy (IMRT) is a form of radiation therapy that delivers precise radiation to a tumor while minimizing the dose to surrounding normal tissues. Here, we conducted a phase 2 study to estimate the curability and efficacy of neoadjuvant chemoradiotherapy using IMRT (NACIMRT) for patients with BRPC with arterial abutment (BRPC-A). Methods A total of 49 BRPC-A patients were enrolled in this study and were treated at our hospital according to the study protocol between June 2013 and March 2021. The primary endpoint was microscopically margin-negative resection (R0) rates and we subsequently analyzed safety, histological effect of the treatment as well as survivals among patients with NACIMRT. Results Twenty-nine patients (59.2%) received pancreatectomy after NACIMRT. The R0 rate in resection patients was 93.1% and that in the whole cohort was 55.1%. No mortality was encountered. Local therapeutic effects as assessed by Evans classification showed good therapeutic effect (Grade 1, 3.4%; Grade 2a, 31.0%; Grade 2b, 48.3%; Grade 3, 3.4%; Grade 4, 3.4%). Median disease-free survival was 15.5 months. Median overall survival in the whole cohort was 35.1 months. The only independent prognostic pre-NACIMRT factor identified was serum carbohydrate antigen 19–9 (CA19-9) > 400 U/ml before NACIMRT. Conclusions NACIMRT showed preferable outcome without significant operative morbidity for BRPC-A patients. NACIMRT contributes to good local tumor control, but a high initial serum CA19-9 implies poor prognosis even after neoadjuvant treatment. Trial Registration UMIN-CTR Clinical Trial: https://upload.umin.ac.jp/cgi-open-bin/ctr_e/ctr_view.cgi?recptno=R000011776 Registration number: UMIN000010113. Date of first registration: 01/03/2013

Intravenous administration of magnesium with a short hydration regimen is recommended for patients receiving high-dose cisplatin to protect against cisplatin-induced nephrotoxicity. However, the optimal dose of magnesium supplementation has not been clarified. The aim of this trial was to investigate the safety and efficacy of a short hydration regimen with 20 mEq of magnesium supplementation for lung cancer patients receiving cisplatin-based chemotherapy. The key eligibility criteria included cytologically or histologically diagnosed lung cancer, candidacy for cisplatin-based (≥ 60 mg/m2) chemotherapy or chemoradiotherapy, no prior chemotherapy, aged 20–75 years, and adequate renal function. Cisplatin was administered with pre-hydration with 20 mEq of magnesium sulfate. Mannitol was administered just before cisplatin infusion to enforce diuresis. The primary endpoint was the proportion of patients who underwent cisplatin-based chemotherapy with a short hydration regimen with 20 mEq of magnesium supplementation without a grade 2 or higher elevation in creatinine. Forty patients with a median age of 66 years (range 35–74) were prospectively enrolled. Median baseline creatinine was 0.71 mg/dL. Median dose of cisplatin in the first cycle was 80 mg/m2. In the first cycle, no patients developed grade 2 creatinine toxicity. During the treatment period, one patient developed grade 2 creatinine elevation; thus, the proportion of patients without a grade 2 or higher elevation in creatinine was 97.5% (95% confidence interval 86.8–99.9). A short hydration regimen with 20 mEq of magnesium supplementation is safe and feasible for patients with lung cancer receiving cisplatin-based chemotherapy.

We present the results of theoretical studies of formation and evolution of the current sheet in a colliosionless plasma during magnetic reconnection in relativistic limit. Relativistic magnetic reconnection is driven by parallel laser pulses interacting with underdense plasma target. Annihilation of laser created magnetic field of opposite polarity generates strong non-stationary electric field formed in between the region with opposite polarity magnetic field accelerating charged particles within the current sheet. This laser-plasma target configuration is discussed in regard with the laboratory modeling of charged particle acceleration and gamma flash generation in astrophysics. We present the results of 3-dimensional kinetic simulations and theoretical studies on the formation and evolution of the current sheet in a collisionless plasma during magnetic field annihilation in the ultra-relativistic limit. Annihilation of oppositively directed magnetic fields driven by two laser pulses interacting with underdense plasma target is accompanied by an electromagnetic burst generation. The induced strong non-stationary longitudinal electric field accelerates charged particles within the current sheet. Properties of the laser-plasma target configuration are discussed in the context of the laboratory modeling for charged particle acceleration and gamma flash generation in astrophysics.

When an outer panel of a B-pillar is manufactured with the hot stamping process, reinforcements are spot welded on its inner side. Before reinforcements are added, the microstructure of the outer panel is martensite. However, reheating during spot welding changes the martensite to ferrite, which has a lower hardness in the heat-affected zone than in other areas. If spot welding is conducted before hot stamping for making a spot welded tailored blank, the microstructure in the spot welded tailored blank after hot stamping is martensite. This sequence of processes avoids hardness reduction due to spot welding. In this study, the hardness and microstructure around spot welded parts of the tailored blank were investigated. The results clearly showed that areas close to the spot welded parts are severely stretched during hot stamping. In addition, stretching suppresses the martensitic phase transformation and reduces the hardness. To characterize this phenomenon, a simulation was conducted that considered the effects of pre-strain on the phase transformation. A continuous cooling transformation (CCT) diagram and a deformation continuous cooling transformation (DCCT) diagram were made in order to quantify the effect of the cooling rate and pre-strain on the phase transformation and hardness. The hardness was then calculated using the experimentally measured CCT and DCCT diagrams and the finite element analysis results. The calculated hardness was compared with the experimental hardness. Good agreement was found between the calculated and experimental results.

Fcγ receptor (FcγR) promotes various immune responses through interactions with the Fc portion of immunoglobulin G (IgG). FcγRIIIb is a glycosylphosphatidylinositol-linked protein expressed on neutrophils and triggers degranulation and opsonic phagocytosis. The extracellular region of FcγR is composed of two Ig-fold domains and can be cleaved as a soluble form (sFcγRIIIb), which is also reactive with complement receptor type 3. Although structure and Fc interaction of sFcγRIIIb have been characterized by X-ray crystallography, little has been known about its structure in solution. We herein report the backbone NMR assignments of human sFcγRIIIb to gain basic understanding of functional IgG-FcγRIII interactions of immunological and biopharmaceutical interest regarding the structural investigation.

We present a characteristic function-based method for the estimation of dynamic mixed hitting time model for duration between events and price changes. The model specifies duration between events as the first time a latent component of multivariate Brownian motion crosses a random boundary. Meanwhile, another (correlated) Brownian component generates the prices. The proposed estimation method facilitates computation and overcomes problems related to the discretization error in the moment conditions and the non-tractability in the joint probability density function. An empirical application using transaction level data on stocks of a large capitalization company traded on the Indonesia Stock Exchange is illustrated. Estimation results suggest that durations and return volatility have strong persistence and, further, there is a negative instantaneous relation between volatility and contemporaneous duration. The impact of considering the causality relation between volatilities and durations on instantaneous volatility estimate are also investigated.

Diabetes mellitus is known to adversely affect the bones and be associated with increased fracture risk. We examined whether eldecalcitol (ELD), an active vitamin D3 derivative, could inhibit the diabetic bone loss in streptozotocin-induced type I diabetic rats. ELD (10, 20, or 40 ng/kg), alfacalcidol (ALF; 25, 50, or 100 ng/kg), or vehicle was administered 5 times per week for 12 weeks from 1 week after diabetes induction. Normal control rats received the vehicle. Bone turnover markers, bone mineral density (BMD), and biomechanical strength of the lumbar spine and femur were measured, and bone histomorphometry was performed. Content of advanced glycation end products (AGEs) in the femoral shaft was also determined. In diabetic rats, serum osteocalcin (OC) concentration was lower and urinary excretion of deoxypyridinoline (DPD) tended to be higher than in normal rats. Areal BMD and maximum load of the lumbar vertebrae and femoral shaft were lower in diabetic rats than in normal rats. All doses of ELD and the highest dose of ALF reduced urinary DPD excretion, but had no effect on serum OC. The 20 and 40 ng/kg doses of ELD prevented decreases in BMD and the highest dose of ELD prevented the reduction in maximum load of the lumbar vertebrae, while ALF did not change these parameters. ELD and ALF did not affect areal BMD or biomechanical strength of the femoral shaft. In diabetic rats, bone volume and trabecular thickness in the trabecular bone of the lumbar vertebrae decreased and trabecular separation increased compared to normal rats. ELD and ALF prevented diabetes-induced deterioration of trabecular microstructure. AGE content in the femoral cortical bone increased in the diabetic rats, and ELD and ALF did not change AGE content compared to the diabetic rats. These results indicated that ELD suppressed bone resorption and prevented trabecular bone loss and deterioration of trabecular microstructure, resulting in prevention of reduction in biomechanical strength in type I diabetic rats.

To understand a biological system, it is important to observe structures of biomolecules in the solution where the system is functionalized. Small-Angle X-ray Scattering coupled with Size Exclusion Chromatography (SEC-SAXS) is one of techniques to selectively observe the target molecules in the multi-component system. However, this technique is believed to be available only with a synchrotron-based SAXS instrument due to requirement of high beam intensity and, therefore, the limitation of the beam time was obstacle to satisfy demands from many bio-researchers. We newly developed Laboratory-based Size exclusion chromatography SAXS System (La-SSS) by utilizing a latest laboratory-based SAXS instrument and finely optimization of the balance between flow rate, cell volume, irradiation time and so on. La-SSS succeeded not only decoupling of target protein(s) from non-specific aggregates but also measurement of each concerned component in a multi-component system. In addition, an option: “stopping mode”, which is designed for improving statistics of SAXS profile, realized a high S/N data acquisition for the most interesting protein in a multi-component system. Furthermore, by utilizing a column having small bed volume, the small-scale SEC-SAXS study makes available. Through optimization of instrumental parameters and environments, La-SSS is highly applicable for experimental requirements from various biological samples. It is strongly expected that a La-SSS concept must be a normal option for laboratory-based SAXS in the near future.

Inorganic/organic hybrid membranes for use in fuel cells were synthesized from 3-glycidoxypropyltrimethoxysilane (GPTMS), N-(3-triethoxysilylpropyl)-4,5-dihydroimidazole (NESPHI), and 1,3-propanesultone (PS) through a simple one-pot process. Fourier transform infrared spectroscopy and 13C nuclear magnetic resonance (NMR) measurements confirmed the sultone ring-opening reaction and formation of chemical bonds among the starting materials. The 29Si NMR measurements revealed that a siloxane network had formed within the hybrid membranes via the sol–gel process. The thermogravimetric analysis indicated that the synthesized membranes were thermally stable up to 170 °C. The conductivity of the membranes at low levels of relative humidity (RH) was improved by the addition of NESPHI. The best cell performance at intermediate temperatures and low RHs was achieved by the membrane electrode assembly using the NESPHI/GPTMS/PS = 1:3:4 hybrid membrane with a catalyst ink consisting of Nafion and the hybrid solution at a 1:1 ratio. At 120 °C and 53 % RH, the power density of the NESPHI/GPTMS/PS = 1:3:4 membrane was 6.6 mW/cm2.

In a previous study, we developed a novel in situ analysis and observation system that allows for simultaneous synchrotron X-ray diffraction (XRD) and optical observations of a frictional surface. This in situ system was used to investigate the scuffing phenomena of SUJ2 bearing steel (AISI 52100); characteristic surface deteriorations occurred during the scuffing process, including plastic flow, heat-spot formation, austenite transformation, and a decrease in the width of the XRD peaks (indicating a decrease of dislocations and strain). These surface deteriorations are not observed during normal wear, hence it is possible that they cause catastrophic wear during the scuffing of steel. In this study, to elucidate the scuffing mechanism of steel, we focused on the following two points: (1) whether the above surface deteriorations are unique to SUJ2 steel or whether they occur in general steels as well, and (2) the extent to which these surface deteriorations contribute to the wear amount. To achieve these objectives, we performed scuffing tests on four types of steel using the previously developed in situ system. In particular, we focused on the first stage of the scuffing process. The present test results suggest that these surface deteriorations also occur in general steels, and that plastic flow and heat-spot formation, which originate from the same phenomenon, are the dominant contributors to the wear amount during the scuffing of steel. Furthermore, the wear amount per unit plastic flow appears to be independent of steel composition.