Swelling behavior of calcium ion-crosslinked sodium alginate in an in vitro hemostatic tamponade model.

Various types of hemostatic agents are used to manage bleeding in surgery. Many such agents are animal products, which carry the risk of secondary infection. The aim of this study is to develop a novel hemostatic agent from a non-animal source that quickly stops bleeding, is easy to use, and has no risk of infection. In this study, we synthesized calcium ion-crosslinked sodium alginate (Alg-Na/Ca) by partial substitution of Ca ions for Na ions in sodium alginate. We prepared 12 kinds of Alg-Na/Ca powders with different Ca mass ratios, molecular weights, M/G ratios and particle size distributions and measured their swelling ratio and the burst pressure generated. We found that Alg-Na/Ca began to swell immediately after contact with saline, especially Alg-Na/Ca at Ca mass ratios of 74.1-77.0 % showed a high swelling ratio after 2 min and a high burst pressure, over 200 % and 500 mmHg respectively. Also, there is a correlation between the swelling ratio after 2 min and the burst pressure. Our results suggest that, by optimizing the composition conditions, Alg-Na/Ca may be an effective hemostatic agent that could act as a tamponade by absorbing and swelling at a bleeding site to quickly achieve primary hemostasis.

Theoretical Prediction of the Reaction Probabilities of H, O, and OH Radicals on the Polypropylene Surface.

To determine the H-abstraction reaction probabilities of H/O/OH radicals with a polypropylene (PP) surface, a first-principles calculation was performed based on the DLPNO-CCSD(T)/CBS//M06-2X-D3/def-TZVP theory level. The PP chain model used in this study was 2,4,6-trimethylheptane. The rate constants of the H/O/OH radicals with the isolated PP chain model were calculated based on the conventional transition-state theory. By comparing the experimental values and considering the error factors and their compensation, it was concluded that the orders of magnitude of the predicted rate constants were accurate. The resulting rate constants were converted to reaction probabilities between the H/O/OH radicals and the PP surface. The method used in this study is applicable for obtaining theoretical values of surface reaction probabilities based on first-principles calculations. The calculation at the DLPNO-CCSD(T)/CBS theory level has high accuracy but consumes a large amount of computational resources. The study also demonstrated that the double-hybrid functionals, wB97x-2-D3(BJ) and rev-DSD-PBEP86-D3(BJ), with a 3-ζ or 4-ζ basis set, could reproduce the electronic energy values obtained from DLPNO-CCSD(T)/CBS while using only approximately 1/100 of the computational resources required by the latter under our computer configuration.

The clinical efficacy and safety of granulocyte and monocyte adsorptive apheresis in patients with Crohn's disease: A multicenter retrospective pilot study.

INTRODUCTION: A remission induction therapy of granulocyte and monocyte adsorptive apheresis (GMA) was given to patients with Crohn's disease (CD). However, establishing an appropriate treatment strategy for GMA in patients with CD remains unclear. METHODS: This study evaluated the clinical efficacy and subsequent clinical progression after GMA in patients with CD who underwent GMA in seven independent institutions in Japan from 2010 to 2023. RESULTS: Sixteen patients were enrolled. The overall remission and response rates were 25.0% and 68.8%, respectively. All patients responding to GMA received biologics that were continuously used and 36.4% of patients remained on the same biologics 52 weeks after GMA. Notably, all patients who continued the same biologics had previously experienced a loss of response to biologics. CONCLUSION: GMA may exhibit effectiveness even in cases with refractory CD. Moreover, it represents a potential novel therapeutic option for refractory CD with loss of response to biologics.

Development of a concise and reliable method for quantifying the antibody loaded onto lipid nanoparticles modified with Herceptin.

Antibodies are essential components of the immune system with a wide range of molecular targets. They have been recognized as modalities for treating several diseases and more than 130 approved antibody-based therapeutics are available for clinical use. However, limitations remain associated with its efficacy, tissue permeability, and safety, especially in cancer treatment. Nanoparticles, particularly those responsive to external stimuli, have shown promise in improving the efficacy of antibody-based therapeutics and tissue-selective delivery. In this study, we developed a reliable and accurate method for quantifying the amount of antibody loaded onto lipid nanoparticles modified with Herceptin® (Trastuzumab), an antibody-based therapeutic used to treat HER2-positive cancers, using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by silver staining. This method proved to be a suitable alternative to commonly used protein quantification techniques, which are limited by lipid interference present in the samples. Furthermore, the amount of Herceptin modified on the liposomes, measured by this method, was confirmed by Herceptin's antibody-dependent cell-mediated cytotoxicity activity. Our results demonstrate the potential of this method as a critical tool for developing tissue-selective antibody delivery systems, leading to improved efficacy and reduced side effects of antibody-based therapeutics.

Relationships among Postprandial Plasma Active GLP-1 and GIP Excursions, Skeletal Muscle Mass, and Body Fat Mass in Patients with Type 2 Diabetes Treated with Either Miglitol, Sitagliptin, or Their Combination: A Secondary Analysis of the MASTER Study.

BACKGROUND: We previously conducted a pilot randomized controlled trial "the MASTER study" and demonstrated that alpha-glucosidase inhibitor miglitol and a dipeptidyl peptidase-4 inhibitor sitagliptin modified postprandial plasma excursions of active glucagon-like peptide-1 (aGLP-1) and active gastric inhibitory polypeptide (aGIP), and miglitol treatment decreased body fat mass in patients with type 2 diabetes (T2D). However, the details regarding the relationships among postprandial plasma aGLP-1 and aGIP excursions, skeletal muscle mass, and body fat mass are unclear. METHODS: We conducted a secondary analysis of the relationships among skeletal muscle mass index (SMI), total body fat mass index (TBFMI), and the incremental area under the curves (iAUC) of plasma aGLP-1 and aGIP excursions following mixed meal ingestion at baseline and after 24-week add-on treatment with either miglitol alone, sitagliptin alone, or their combination in T2D patients. RESULTS: SMI was not changed after the 24-week treatment with miglitol and/or sitagliptin. TBFMI was reduced and the rates of aGIP-iAUC change were lowered in the two groups treated with miglitol, although their correlations did not reach statistical significance. We observed a positive correlation between the rates of aGIP-iAUC and TBFMI changes and a negative correlation between the rates of TBFMI and SMI changes in T2D patients treated with sitagliptin alone whose rates of aGIP-iAUC change were elevated. CONCLUSIONS: Collectively, although T2D patients treated with miglitol and/or sitagliptin did not show altered SMI after 24-week treatment, the current study suggests that there are possible interrelationships among postprandial plasma aGIP excursion modified by sitagliptin, skeletal muscle mass, and body fat mass.

Rates of Return to Sports and Recurrence in Pediatric Athletes after Conservative Treatment for Lumbar Spondylolysis.

Introduction: Lumbar spondylolysis is common in pediatric athletes, and many athletes can return to sports with conservative treatment. There are two initial treatment strategies: bony union or pain management, but the outcomes of these strategies have not been clarified. The purpose of this study is to investigate the rates of return to sports (RTS) and recurrence in pediatric athletes after conservative treatment for lumbar spondylolysis and to compare both treatment strategies. Methods: A total of 180 patients with lumbar spondylolysis were managed with a trunk brace and cessation of sports activity (bone union [BU] group, n=95) or treated for pain only (pain management [PM] group, n=85). RTS and recurrence rates according to type of conservative treatment were compared. Results: The RTS rate was 98.9% in the BU group and 97.6% in the PM group at 4.7±1.9 and 1.8±1.7 months, respectively. Recurrence occurred in 7.4% of patients in the BU group at 19.0±16.0 months and in 4.8% of the PM group at 17.8±5.2 months. Conclusions: The RTS rate in pediatric athletes with lumbar spondylolysis was high at more than 95%, regardless of type of conservative treatment. The mean time to RTS was longer in the BU group than in the PM group (4.7 vs. 1.8 months) because of the time required for bone healing. There were several cases of recurrence after RTS. Strategies to prevent recurrence of lumbar spondylolysis in pediatric athletes are discussed.

The Role of Genetically Modified Human Feeder Cells in Maintaining the Integrity of Primary Cultured Human Deciduous Dental Pulp Cells.

Tissue-specific stem cells exist in tissues and organs, such as skin and bone marrow. However, their pluripotency is limited compared to embryonic stem cells. Culturing primary cells on plastic tissue culture dishes can result in the loss of multipotency, because of the inability of tissue-specific stem cells to survive in feeder-less dishes. Recent findings suggest that culturing primary cells in medium containing feeder cells, particularly genetically modified feeder cells expressing growth factors, may be beneficial for their survival and proliferation. Therefore, the aim of this study was to elucidate the role of genetically modified human feeder cells expressing growth factors in maintaining the integrity of primary cultured human deciduous dental pulp cells. Feeder cells expressing leukemia inhibitory factor, bone morphogenetic protein 4, and basic fibroblast growth factor were successfully engineered, as evidenced by PCR. Co-culturing with mitomycin-C-treated feeder cells enhanced the proliferation of newly isolated human deciduous dental pulp cells, promoted their differentiation into adipocytes and neurons, and maintained their stemness properties. Our findings suggest that genetically modified human feeder cells may be used to maintain the integrity of primary cultured human deciduous dental pulp cells.

Direct Injection of Recombinant AAV-Containing Solution into the Oviductal Lumen of Pregnant Mice Caused In Situ Infection of Both Preimplantation Embryos and Oviductal Epithelium.

Adeno-associated virus (AAV) vector is an efficient viral-based gene delivery tool used with many types of cells and tissues, including neuronal cells and muscles. AAV serotype 6 (AAV-6), one of numerous AAV serotypes, was recently found to efficiently transduce mouse preimplantation embryos. Furthermore, through coupling with a clustered, regularly interspaced, short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system-a modern genome editing technology-AAV-6 has been shown to effectively create a mutation at a target locus, which relies on isolation of zygotes, in vitro viral infection, and transplantation of the infected embryos to recipient females. Unfortunately, this procedure, termed "ex vivo handling of embryos", requires considerable investment of capital, time, and effort. Direct transduction of preimplantation embryos through the introduction of AAV-6 into the oviductal lumen of pregnant females would be an ideal approach. In this study, we injected various types of recombinant AAV vectors (namely, rAAV-CAG-EGFP-1, -2, -5, and -6, each carrying an enhanced green fluorescent protein [EGFP] cDNA whose expression is under the influence of a cytomegalovirus enhancer + chicken ß-actin promoter) into the ampulla region of oviducts in pregnant female mice at Day 0.7 of pregnancy (corresponding to the late 1-cell stage), and EGFP-derived green fluorescence was assessed in the respective morulae. The highest levels of fluorescence were observed in rAAV-CAG-EGFP-6. The oviductal epithelium was distinctly fluorescent. The fluorescence in embryos peaked at the morula stage. Our results indicate that intra-oviductal injection of AAV-6 vectors is the most effective method for transducing zona pellucida-enclosed preimplantation embryos in situ. AAV-6 vectors could be a useful tool in the genetic manipulation of early embryos, as well as oviductal epithelial cells.

[A case of incipient pancreatic ductal adenocarcinoma concomitant with intraductal papillary mucinous neoplasm, confirmed by multiregion sequencing analysis].

Concomitant pancreatic ductal adenocarcinoma (PDA) is observed in a subset of patients with intraductal papillary mucinous neoplasm (IPMN) of the pancreas, and early detection of those progressing lesions is difficult. We present a case with a de novo carcinoma in situ (CIS) discovered incidentally around the resection margin of IPMNs. A man in his 70s with a history of acute pancreatitis at the age of 50 years and no family history of PDA had a pancreatoduodenectomy for three isolated branch duct IPMNs that caused recurrent pancreatitis. During the 2-year follow-up period, the index lesion in the pancreatic head grew significantly, whereas the other cysts remained small and without mural nodules. The majority of the cysts are histologically composed of low-grade dysplasia and are classified as gastric-type IPMN. CIS with nuclear overexpression of p53 was located in the main pancreatic duct and adjacent brunch duct, which involved the pancreatic resection margin. The precise pathological analysis combined with multiregion sequencing revealed the CIS harbored KRAS G12V and TP53 R248W. Conversely, IPMNs contained GNAS mutant cells as well as components containing additional KRAS mutations. These findings suggested that the CIS formed independently of the multiple IPMNs and appeared to be an early manifestation of concomitant PDA with coexisting IPMNs. Despite widespread agreement on the resection of the radiographically significant IPMN lesion (s), the latent invasive cancer was not eradicated. A detailed pathological and molecular assessment of the resected materials may aid in a better management strategy for concurrent lesions.

Recent Advances in the Production of Genome-Edited Rats.

The rat is an important animal model for understanding gene function and developing human disease models. Knocking out a gene function in rats was difficult until recently, when a series of genome editing (GE) technologies, including zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the type II bacterial clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated Cas9 (CRISPR/Cas9) systems were successfully applied for gene modification (as exemplified by gene-specific knockout and knock-in) in the endogenous target genes of various organisms including rats. Owing to its simple application for gene modification and its ease of use, the CRISPR/Cas9 system is now commonly used worldwide. The most important aspect of this process is the selection of the method used to deliver GE components to rat embryos. In earlier stages, the microinjection (MI) of GE components into the cytoplasm and/or nuclei of a zygote was frequently employed. However, this method is associated with the use of an expensive manipulator system, the skills required to operate it, and the egg transfer (ET) of MI-treated embryos to recipient females for further development. In vitro electroporation (EP) of zygotes is next recognized as a simple and rapid method to introduce GE components to produce GE animals. Furthermore, in vitro transduction of rat embryos with adeno-associated viruses is potentially effective for obtaining GE rats. However, these two approaches also require ET. The use of gene-engineered embryonic stem cells or spermatogonial stem cells appears to be of interest to obtain GE rats; however, the procedure itself is difficult and laborious. Genome-editing via oviductal nucleic acids delivery (GONAD) (or improved GONAD (i-GONAD)) is a novel method allowing for the in situ production of GE zygotes existing within the oviductal lumen. This can be performed by the simple intraoviductal injection of GE components and subsequent in vivo EP toward the injected oviducts and does not require ET. In this review, we describe the development of various approaches for producing GE rats together with an assessment of their technical advantages and limitations, and present new GE-related technologies and current achievements using those rats in relation to human diseases.

Development of an Antibody Delivery Method for Cancer Treatment by Combining Ultrasound with Therapeutic Antibody-Modified Nanobubbles Using Fc-Binding Polypeptide.

A key challenge in treating solid tumors is that the tumor microenvironment often inhibits the penetration of therapeutic antibodies into the tumor, leading to reduced therapeutic efficiency. It has been reported that the combination of ultrasound-responsive micro/nanobubble and therapeutic ultrasound (TUS) enhances the tissue permeability and increases the efficiency of delivery of macromolecular drugs to target tissues. In this study, to facilitate efficient therapeutic antibody delivery to tumors using this combination system, we developed therapeutic antibody-modified nanobubble (NBs) using an Fc-binding polypeptide that can quickly load antibodies to nanocarriers; since the polypeptide was derived from Protein G. TUS exposure to this Herceptin®-modified NBs (Her-NBs) was followed by evaluation of the antibody's own ADCC activity, resulting the retained activity. Moreover, the utility of combining therapeutic antibody-modified NBs and TUS exposure as an antibody delivery system for cancer therapy was assessed in vivo. The Her-NBs + TUS group had a higher inhibitory effect than the Herceptin and Her-NBs groups. Overall, these results suggest that the combination of therapeutic antibody-modified NBs and TUS exposure can enable efficient antibody drug delivery to tumors, while retaining the original antibody activity. Hence, this system has the potential to maximize the therapeutic effects in antibody therapy for solid cancers.

Elemental analysis by Metallobalance provides a complementary support layer over existing blood biochemistry panel-based cancer risk assessment.

Despite the benefit of early cancer screening, Japan has one of the lowest cancer screening rates among developed countries, possibly due to there being a lack of "a good test" that can provide sufficient levels of test sensitivity and accuracy without a large price tag. As a number of essential and trace elements have been intimately connected to the oncogenesis of cancer, Metallobalance, a recent development in elemental analysis utilizing the technique of inductively coupled plasma mass spectrometry has been developed and tested as a robust method for arrayed cancer risk screening. We have conducted case-control epidemiological studies in the prefecture of Chiba, in the Greater Tokyo Area, and sought to determine both Metallobalance screening's effectiveness for predicting pan-cancer outcomes, and whether the method is capable enough to replace the more conventional antigen-based testing methods. Results suggest that MB screening provides some means of classification potential among cancer and non-cancer cases, and may work well as a complementary method to traditional antigen-based tumor marker testing, even in situations where tumor markers alone cannot discernibly identify cancer from non-cancer cases.

Therapeutic potential of d-cysteine against in vitro and in vivo models of spinocerebellar ataxia.

Spinocerebellar ataxia (SCA) is a group of autosomal-dominantly inherited ataxia and is classified into SCA1-48 by the difference of causal genes. Several SCA-causing proteins commonly impair dendritic development in primary cultured Purkinje cells (PCs). We assume that primary cultured PCs expressing SCA-causing proteins are available as in vitro SCA models and that chemicals that improve the impaired dendritic development would be effective for various SCAs. We have recently revealed that D-cysteine enhances the dendritic growth of primary cultured PCs via hydrogen sulfide production. In the present study, we first investigated whether D-cysteine is effective for in vitro SCA models. We expressed SCA1-, SCA3-, and SCA21-causing mutant proteins to primary cultured PCs using adeno-associated viral serotype 9 (AAV9) vectors. D-Cysteine (0.2 mM) significantly ameliorated the impaired dendritic development commonly observed in primary cultured PCs expressing these three SCA-causing proteins. Next, we investigated the therapeutic effect of long-term treatment with D-cysteine on an in vivo SCA model. SCA1 model mice were established by the cerebellar injection of AAV9 vectors, which express SCA1-causing mutant ataxin-1, to ICR mice. Long-term treatment with D-cysteine (100 mg/kg/day) significantly inhibited the progression of motor dysfunction in SCA1 model mice. Immunostaining experiments revealed that D-cysteine prevented the reduction of mGluR1 and glial activation at the early stage after the onset of motor dysfunction in SCA1 model mice. These findings strongly suggest that D-cysteine has therapeutic potential against in vitro and in vivo SCA models and may be a novel therapeutic agent for various SCAs.

Induced Tissue-Specific Stem Cells (iTSCs): Their Generation and Possible Use in Regenerative Medicine.

Induced tissue-specific stem cells (iTSCs) are partially reprogrammed cells which have an intermediate state, such as progenitors or stem cells. They originate from the de-differentiation of differentiated somatic cells into pluripotent stem cells, such as induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs), or from the differentiation of undifferentiated cells. They show a limited capacity to differentiate and a morphology similar to that of somatic cell stem cells present in tissues, but distinct from that of iPSCs and ESCs. iTSCs can be generally obtained 7 to 10 days after reprogramming of somatic cells with Yamanaka's factors, and their fibroblast-like morphology remains unaltered. iTSCs can also be obtained directly from iPSCs cultured under conditions allowing cellular differentiation. In this case, to effectively induce iTSCs, additional treatment is required, as exemplified by the conversion of iPSCs into naïve iPSCs. iTSCs can proliferate continuously in vitro, but when transplanted into immunocompromised mice, they fail to generate solid tumors (teratomas), implying loss of tumorigenic potential. The low tendency of iTSCs to elicit tumors is beneficial, especially considering applications for regenerative medicine in humans. Several iTSC types have been identified, including iTS-L, iTS-P, and iTS-D, obtained by reprogramming hepatocytes, pancreatic cells, and deciduous tooth-derived dental pulp cells, respectively. This review provides a brief overview of iPSCs and discusses recent advances in the establishment of iTSCs and their possible applications in regenerative medicine.

Periodontal surgery using rhFGF-2 with deproteinized bovine bone mineral or rhFGF-2 alone: 2-year follow-up of a randomized controlled trial.

AIM: To compare outcomes of rhFGF-2 + DBBM therapy with rhFGF-2 alone in the treatment of intrabony defects. This study provides 2-year follow-up results from the previous randomized controlled trial. MATERIALS AND METHODS: Defects were randomly allocated to receive rhFGF-2 + DBBM (test) or rhFGF-2 (control). Treated sites were re-evaluated at 2 years postoperatively, using original clinical and patient-centred measures. RESULTS: Thirty-eight sites were available for re-evaluation. At 2 years, both groups showed a significant improvement in clinical attachment level (CAL) from baseline. A gain in CAL of 3.4 ± 1.3 mm in the test group and 3.1 ± 1.5 mm in the control group was found. No significant inter-group difference was noted. Both groups showed a progressive increase in radiographic bone fill (RBF). The test treatment yielded greater RBF (56%) compared with the control group (41%). The control treatment performed better in contained defects in terms of CAL and RBF. There was no significant difference in patient-reported outcomes between groups. CONCLUSIONS: At 2-year follow-up, the test and cotrol treatments were similarly effective in improving CAL, whereas the test treatment achieved a significantly greater RBF. In both treatments, favourable clinical, radiographic, and patient-reported outcomes can be sustained for at least 2 years. TRIAL REGISTRATION: The University Hospital Medical Information Network-Clinical Trials Registry (UMIN-CTR) 000025257.

Ataxic phenotype and neurodegeneration are triggered by the impairment of chaperone-mediated autophagy in cerebellar neurons.

AIMS: Chaperone-mediated autophagy (CMA) is a pathway involved in the autophagy lysosome protein degradation system. CMA has attracted attention as a contributing factor to neurodegenerative diseases since it participates in the degradation of disease-causing proteins. We previously showed that CMA is generally impaired in cells expressing the proteins causing spinocerebellar ataxias (SCAs). Therefore, we investigated the effect of CMA impairment on motor function and the neural survival of cerebellar neurons using the micro RNA (miRNA)-mediated knockdown of lysosome-associated protein 2A (LAMP2A), a CMA-related protein. METHODS: We injected adeno-associated virus serotype 9 vectors, which express green fluorescent protein (GFP) and miRNA (negative control miRNA or LAMP2A miRNA) under neuron-specific synapsin I promoter, into cerebellar parenchyma of 4-week-old ICR mice. Motor function of mice was evaluated by beam walking and footprint tests. Immunofluorescence experiments of cerebellar slices were conducted to evaluate histological changes in cerebella. RESULTS: GFP and miRNA were expressed in interneurons (satellite cells and basket cells) in molecular layers and granule cells in the cerebellar cortices, but not in cerebellar Purkinje cells. LAMP2A knockdown in cerebellar neurons triggered progressive motor impairment, prominent loss of cerebellar Purkinje cells, interneurons, granule cells at the late stage, and astrogliosis and microgliosis from the early stage. CONCLUSIONS: CMA impairment in cerebellar interneurons and granule cells triggers the progressive ataxic phenotype, gliosis and the subsequent degeneration of cerebellar neurons, including Purkinje cells. Our present findings strongly suggest that CMA impairment is related to the pathogenesis of various SCAs.

Volar transfer of the lateral band with transverse retinacular ligament is effective for the correction of swan-neck deformity caused by volar plate injury of the PIP joint.

We introduced a technique with a volar transfer of the lateral band using the transverse retinacular ligament for swan-neck deformity caused by volar plate injury of the PIP joint. A 61-year-old woman injured her 5th finger and was diagnosed with a volar plate injury of the PIP joint. She presented with snapping of the finger together with the appearance of a swan-neck deformity, and surgery was performed. Dorsally located lateral bands were transferred towards the volar aspect of the finger, and their position was maintained using the transverse retinacular ligament. Improvements in the snapping and swan-neck deformities were confirmed by intraoperative active motion of the finger. One year postoperatively, the deformity had not recurred, and there was no contracture of the finger. Surgical transfer of the lateral band using the transverse retinacular ligament is effective for swan-neck deformity caused by volar plate injury of the PIP joint.

Drug-Induced Naïve iPS Cells Exhibit Better Performance than Primed iPS Cells with Respect to the Ability to Differentiate into Pancreatic ß-Cell Lineage.

Pluripotent stem cells are classified as naïve and primed cells, based on their in vitro growth characteristics and potential to differentiate into various types of cells. Human-induced pluripotent stem cells (iPSCs, also known as epiblast stem cells [EpiSCs]) have limited capacity to differentiate and are slightly more differentiated than naïve stem cells (NSCs). Although there are several in vitro protocols that allow iPSCs to differentiate into pancreatic lineage, data concerning generation of ß-cells from these iPSCs are limited. Based on the pluripotentiality of NSCs, it was hypothesized that NSCs can differentiate into pancreatic ß-cells when placed under an appropriate differentiation induction condition. We examined whether NSCs can be efficiently induced to form potentially pancreatic ß cells after being subjected to an in vitro protocol. Several colonies resembling in vitro-produced ß-cell foci, with ß-cell-specific marker expression, were observed when NSC-derived embryoid bodies (EBs) were induced to differentiate into ß-cell lineage. Conversely, EpiSC-derived EBs failed to form such foci in vitro. Intrapancreatic grafting of the in vitro-formed ß-cell foci into nude mice (BALB/c-nu/nu) generated a cell mass containing insulin-producing cells (IPCs), without noticeable tumorigenesis. These NSCs can be used as a promising resource for curing type 1 diabetes.

Low fraction of methane in landfill gas emissions in an industrial waste landfill containing incineration ash and gypsum board waste under anaerobic conditions.

The behaviour of carbon dioxide (CO2) and methane (CH4) emissions at the surface and below the soil cover in an industrial waste landfill under anaerobic operating conditions was evaluated for six years. This landfill contained gypsum board waste and incineration ash - a practice currently allowed because of a change in Japanese regulations. The CO2 and CH4 fluxes decreased throughout the six years of the survey. Almost all of the survey points exhibited fractions of CH4 in landfill gas emissions of <0.5 (mean values: 0.0-0.1 [surface], 0.0-0.3 [subsurface]) under anaerobic conditions. In addition, a relatively high first-order reaction rate constant for the landfill gas emissions (0.3 year-1) was observed. The landfill leachate showed a relatively high sulphate ion (SO4 2-) concentration, although other environmental conditions, such as the pH, oxidation-reduction potential and ammonium concentration, were not at levels that could have inhibited CH4 production. These findings suggest that the low fractions could have been related to the lower amounts of CH4 generation caused by competition between methanogens and sulphate-reducing bacteria (SRB). Therefore, SRB could play a major role in the degradation of organic carbon in the landfill.

Recent Advances and Future Perspectives of In Vivo Targeted Delivery of Genome-Editing Reagents to Germ Cells, Embryos, and Fetuses in Mice.

The recently discovered clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) systems that occur in nature as microbial adaptive immune systems are considered an important tool in assessing the function of genes of interest in various biological systems. Thus, development of efficient and simple methods to produce genome-edited (GE) animals would accelerate research in this field. The CRISPR/Cas9 system was initially employed in early embryos, utilizing classical gene delivery methods such as microinjection or electroporation, which required ex vivo handling of zygotes before transfer to recipients. Recently, novel in vivo methods such as genome editing via oviductal nucleic acid delivery (GONAD), improved GONAD (i-GONAD), or transplacental gene delivery for acquiring genome-edited fetuses (TPGD-GEF), which facilitate easy embryo manipulation, have been established. Studies utilizing these techniques employed pregnant female mice for direct introduction of the genome-editing components into the oviduct or were dependent on delivery via tail-vein injection. In mice, embryogenesis occurs within the oviducts and the uterus, which often hampers the genetic manipulation of embryos, especially those at early postimplantation stages (days 6 to 8), owing to a thick surrounding layer of tissue called decidua. In this review, we have surveyed the recent achievements in the production of GE mice and have outlined the advantages and disadvantages of the process. We have also referred to the past achievements in gene delivery to early postimplantation stage embryos and germ cells such as primordial germ cells and spermatogonial stem cells, which will benefit relevant research.