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The discovery of human pluripotent stem cells (PSCs) at the turn of the century opened the door to a new generation of regenerative medicine research. Among PSCs, the donors available for induced pluripotent stem cells (iPSCs) are greatest, providing a potentially universal cell source for all types of cell therapies including cancer immunotherapies using natural killer (NK cells). Unlike primary NK cells, those prepared from iPSCs can be prepared with a homogeneous quality and are easily modified to exert a desired response to tumor cells. There already exist several protocols to genetically modify and differentiate iPSCs into NK cells, and each has its own advantages with regards to immunotherapies. In this short review, we detail the benefits of using iPSCs in NK cell immunotherapies and discuss the challenges that must be overcome before this approach becomes mainstream in the clinic.

Here, bias stress instability on multilayered MoTe2 field effect transistors (m‐MoTe2 FETs) with encapsulation of hydrophobic polymers (cyclic transparent optical polymer) is systematically investigated under DC and pulse mode operation. During DC mode stress, the threshold voltage shift (ΔVth) under positive bias stress is at least three times larger than that of negative stress due to high hole barrier (∼0.57 eV), compared with electron barrier (∼0.18 eV). However, ΔVth in positive pulse mode stress is significantly reduced with decrease of a duty cycle, as compared with that of negative stress. With isolation from external gas ambient effects such as H2O and O2, shallow electron traps in multilayered MoTe2 field effect transistors are identified as one of strong candidates to cause bias polarity dependency on ΔVth in pulse mode. Moreover, recovery time for traps, involved during positive bias stress, is six times faster than that of negative stress, substantiating that shallow electron traps and their fast detrapping are one of key origins. In practice, optimisation of pulse mode operation via bipolar switching can be potentially utilised for minimisation of device instability during the operation of multilayered MoTe2 field effect transistors devices and their circuits.

In this study, the interfacial reactions and mechanical properties of solder joints after multiple reflows were observed to evaluate the applicability of the developed materials for high-temperature soldering for automotive electronic components. The microstructural changes and mechanical properties of Sn-Cu solders regarding Al(Si) addition and the number of reflows were investigated to determine their reliability under high heat and strong vibrations. Using differential scanning calorimetry, the melting points were measured to be approximately 227, 230, and 231 °C for the SC07 solder, SC-0.01Al(Si), and SC-0.03Al(Si), respectively. The cross-sectional analysis results showed that the total intermetallic compounds (IMCs) of the SC-0.03Al(Si) solder grew the least after the as-reflow, as well as after 10 reflows. Electron probe microanalysis and transmission electron microscopy revealed that the Al-Cu and Cu-Al-Sn IMCs were present inside the solders, and their amounts increased with increasing Al(Si) content. In addition, the Cu6Sn5 IMCs inside the solder became more finely distributed with increasing Al(Si) content. The Sn-0.5Cu-0.03Al(Si) solder exhibited the highest shear strength at the beginning and after 10 reflows, and ductile fracturing was observed in all three solders. This study will facilitate the future application of lead-free solders, such as an Sn-Cu-Al(Si) solder, in automotive electrical components.

The systematic investigations on the value of social wasps as a food resource are deficient, in spite of the long history of the utilization of social wasps as food and pharmaceutical bioresources. Vespa velutina nigrithorax is an invasive alien wasp species that is currently dominating in East Asia and Europe, bringing huge economic damages. As a control over alien species is made when the valuable utilization of the invasive species as a potential resource are discovered, investigations on the potential of V. v. nigrithorax as a useful bioresource are also in demand. Nutritional and heavy metal analyses of the larvae revealed their balanced and rich nutritional value and safety as a food resource. The larval saliva amino acid composition was investigated for further study on amino acid supplementation and exercise enhancement.

Introduction Chemo-responsiveness in pancreatic cancer is known to be dependent on fibrosis and vascularity. The purpose of this study was to assess vascular enhancement in advanced pancreatic adenocarcinoma with or without liver metastasis in computed tomography (CT) and to analyze the correlation between enhancement patterns and chemo-responsiveness. Methods Patients were assigned to either a responder group (partial response or stable disease) or a non-responder group (progressive disease) according to chemo-responsiveness assessed by CT before and after gemcitabine-based chemotherapy. Hounsefield unit (HU) was measured in pancreatic mass and the largest metastatic liver mass using region of interest (ROI). HU differences (ΔHU) between arterial and pre-contrast phase were calculated. Results Of the 101 study subjects, 78(77.2%) were assigned to the pancreas responder group {mean ΔHU (±SD), 36.7(±21.6)} and 23(22.8%) to the pancreas non-responder group {mean ΔHU (±SD), 20.6(±9.9)} (p = 0.001 for ΔHUs). Of the 46 study subjects with liver metastasis, 25(54.3%) were assigned to the liver metastasis responder group {mean ΔHU (±SD), 36.9(±21.0} and 21(45.7%) to the liver metastasis non-responder group {mean ΔHU (±SD), 17.1 (±24.0)}, (p = 0.005 for ΔHUs). Conclusion CT determined mass vascular enhancement patterns may predict chemoresponse in advanced pancreatic cancer.

The potential use of induced pluripotent stem cells (iPSCs) in personalized regenerative medicine applications may be augmented by transgenics, including the expression of constitutive cell labels, differentiation reporters, or modulators of disease phenotypes. Thus, there is precedence for reproducible transgene expression amongst iPSC sub-clones with isogenic or diverse genetic backgrounds. Using virus or transposon vectors, transgene integration sites and copy numbers are difficult to control, and nearly impossible to reproduce across multiple cell lines. Moreover, randomly integrated transgenes are often subject to pleiotropic position effects as a consequence of epigenetic changes inherent in differentiation, undermining applications in iPSCs. To address this, we have adapted popular TALEN and CRISPR/Cas9 nuclease technologies in order to introduce transgenes into pre-defined loci and overcome random position effects. AAVS1 is an exemplary locus within the PPP1R12C gene that permits robust expression of CAG promoter-driven transgenes. Gene targeting controls transgene copy number such that reporter expression patterns are reproducible and scalable by ∼2-fold. Furthermore, gene expression is maintained during long-term human iPSC culture and in vitro differentiation along multiple lineages. Here, we outline our AAVS1 targeting protocol using standardized donor vectors and construction methods, as well as provide practical considerations for iPSC culture, drug selection, and genotyping.

Pseudomembranous colitis (PMC) is a nosocomial and opportunistic infection caused by Clostridium difficile. PMC is related to the use of antibiotics leading to intestinal dysbiosis and an overgrowth of C. difficile. Metronidazole or vancomycin is considered to be the standard therapy for the management of PMC. However, PMC has a 15%–30% recurrence rate and can be refractory to standard treatments, resulting in morbidity and mortality. Here we describe a patient who experienced refractory PMC who was treated with fecal microbiota transplantation. A 69-year-old woman was admitted to the hospital with consistent abdominal pain and diarrhea, which had been present for 5 months. She was diagnosed with PMC by colonoscopy and tested positive for C. difficile toxin. Even though she took metronidazole for 10 days, followed by vancomycin for 4 weeks, her symptoms did not improve. Because of her recurrent and refractory symptoms, we decided to perform fecal microbiota transplantation. Fifty grams of fresh feces from a donor were obtained on the day of the procedure, mixed with 500 mL of normal saline, and then filtered. The filtered solution was administered to the patient's colon using a colonoscope. After the procedure, her symptoms rapidly improved and a follow-up colonoscopy showed that the PMC had resolved without recurrence.

인하대학교병원 1 내과학교실, 2 병리학교실 Interdigitating dendritic cell sarcoma (IDCS) is an extremely rare tumor derived from professional antigen presenting cell and primarily found in lymph nodes, with rarer case report about extranodal presentation of IDCS. A 71-yr-old man was admitted with progressively enlarging and painless mass in the right parotid area for 2 months. Computed tomography of the neck and chest revealed enhancing mass in right parotid gland, multiple lymphadenopathies around neck and mediastinum, and an osteolytic metastasis at thoracic spine. Morphological and immunohistochemical analysis of an excisional biopsy specimen from parotid mass were consistent with a diagnosis of IDCS. Palliative chemotherapy with 6 cycles of CHOP (cyclophosphamide, adriamycin, vincristine, and prednisolone) regimen and 2 cycles of ABVD (adriamycin, bleomycin, vinblastine, and dacarbazine) regimen plus radiotherapy on parotid mass failed in tumor reduction. We describe a rare case of disseminated extranodal IDCS arising from parotid gland.

Summary As the quintessential reprogramming model, OCT3/4, SOX2, KLF4, and c-MYC re-wire somatic cells to achieve induced pluripotency. Yet, subtle differences in methodology confound comparative studies of reprogramming mechanisms. Employing transposons, we systematically assessed cellular and molecular hallmarks of mouse somatic cell reprogramming by various polycistronic cassettes. Reprogramming responses varied in the extent of initiation and stabilization of transgene-independent pluripotency. Notably, the cassettes employed one of two KLF4 variants, differing only by nine N-terminal amino acids, which generated dissimilar protein stoichiometry. Extending the shorter variant by nine N-terminal amino acids or augmenting stoichiometry by KLF4 supplementation rescued both protein levels and phenotypic disparities, implicating a threshold in determining reprogramming outcomes. Strikingly, global gene expression patterns elicited by published polycistronic cassettes diverged according to each KLF4 variant. Our data expose a Klf4 reference cDNA variation that alters polycistronic factor stoichiometry, predicts reprogramming hallmarks, and guides comparison of compatible public data sets., Graphical Abstract, Highlights • Reprogramming vectors inconsistently employ one of two unappreciated Klf4 variants • Polycistronic cassettes encoding Klf4 N-terminal variants drive distinct stoichiometry • Reprogramming initiation and stabilization are sensitive to Klf4 protein levels • Accordingly, gene expression elicited by public vectors forms two distinct clusters, In this article, Woltjen and colleagues reveal the unwitting employment of two different Klf4 cDNAs to construct somatic cell reprogramming vectors. Cloned in a polycistronic cassette, each variant produces different KLF4 protein levels, impacting reprogramming phenotypes. Moreover, comparative gene expression during iPSC derivation bifurcates based on the Klf4 cDNA used, underpinning the importance of factor stoichiometry in defining reprogramming outcomes.

Background: Metformin is considered the first choice oral treatment for type 2 diabetes patients in the absence of contraindications. Rarely, life-threatening complications associated with metformin treatment are seen in some patients with underlying diseases. The aim of this study was to further investigate the clinical profiles and risk factors for metformin-associated lactic acidosis (MALA) and the treatment modalities according to survival. Methods: To identify MALA, we performed a retrospective study in seven diabetic patients who were taking metformin and had been diagnosed with lactic acidosis at Inha University Hospital between 1995 and 2012. For each patient, we recorded the age, sex, daily metformin dosage, laboratory test results, admission diagnosis, and risk factors. Also, concurrent conditions, treatment modalities, and outcomes were evaluated. Results: Six patients had risk factors for lactic acidosis before admission. All patients had renal impairment on admission as a precipitating risk factor. Five patients survived and two patients died despite early renal replacement therapy. Older patients tended to have a poorer prognosis. Conclusion: Renal function must be monitored in elderly type 2 diabetes mellitus patients with underlying diseases and conditions causing renal impairment who begin metformin treatment. Accurate recognition of MALA and initiation of renal replacement are essential for treatment.

When aqueous conjugated-polyelectrolyte colloidal solutions containing an adequate amount of surfactant with an appropriate hydrophile-lipophile balance were sprayed onto latent fingerprints (LFPs), the polymer nanoparticles were readily transferred to the LFPs to reveal highly distinguishable fluorescent images, while the LFPs themselves remained intact.

Background/Aims: This study investigated the safety of endoscopic variceal ligation (EVL) under conscious sedation with midazolam and sequential flumazenil after procedure in these patients. Methods: A total of 279 patients who underwent secondary prophylactic EVL at our institution between April 2012 and June 2014, were enrolled. Conscious sedation was achieved using intravenous midazolam, and flumazenil was routinely used as an antidote immediately after EVL. Patients with sleep (n = 165) and non-sleep (n = 55) endoscopy were matched using propensity score analysis (3:1). Frequencies of overt hepatic encephalopathy (HEP) and patient' satisfactions with EVL were compared between the 2 groups. Results: Of the 279 patients, 155 (55.6%) were of Child-Turcotte-Pugh (CTP) class, B or C, and 224 (80.3%) patients underwent sleep endoscopy. After propensity score analysis, overt HEP was observed in 1 (0.4%) of the 165 patients in the sedated group, but not found in any in the non-sedated group. Patient' satisfaction with EVL was better in the sedated group (p < 0.001). Twenty-nine (65.9%) of the 44 patients with CTP class C underwent sleep endoscopy, and only one (3.4%) experienced overt HEP. Conclusions: Prophylactic EVL under conscious sedation using midazolam and flumazenil is probably safe in cirrhotic patients without experience of HEP, even in those of CTP class C.

Gene-edited induced pluripotent stem cells (iPSCs) provide relevant isogenic human disease models in patient-specific or healthy genetic backgrounds. Towards this end, gene targeting using antibiotic selection along with engineered point mutations remains a reliable method to enrich edited cells. Nevertheless, integrated selection markers obstruct scarless transgene-free gene editing. Here, we present a method for scarless selection marker excision using engineered microhomology-mediated end joining (MMEJ). By overlapping the homology arms of standard donor vectors, short tandem microhomologies are generated flanking the selection marker. Unique CRISPR-Cas9 protospacer sequences nested between the selection marker and engineered microhomologies are cleaved after gene targeting, engaging MMEJ and scarless excision. Moreover, when point mutations are positioned unilaterally within engineered microhomologies, both mutant and normal isogenic clones are derived simultaneously. The utility and fidelity of our method is demonstrated in human iPSCs by editing the X-linked HPRT1 locus and biallelic modification of the autosomal APRT locus, eliciting disease-relevant metabolic phenotypes., ゲノム編集技術を用いたヒトiPS細胞での正確な一塩基置換技術（MhAX法）を開発. 京都大学プレスリリース. 2018-03-06.

Hepatitis-associated aplastic anemia is a rare disease, but can be fatal if it is diagnosed late or untreated. Despite the previous case report of severe aplastic anemia following hepatitis, hepatitis A virus (HAV)-associated aplastic anemia is very rare, and a case with bone marrow transplantation (BMT) has not been reported in the literature. Here, we report the case of a 13-year-old boy, who developed severe aplastic anemia after full recovery of acute HAV infection, and eventually received BMT for aplastic anemia following HAV infection. It is a very rare case and the first case that recovered after BMT. The aim of this case report is to highlight the potential of progression to aplastic anemia after acute HAV infection and the importance of suspicion when pancytopenia is developed.

Acquired cystic kidney disease (ACKD), a common complication in patients with end-stage renal disease, is characterized by more than three kidney cysts and normal or decreased sizes of both kidneys without any familial history of cystic kidney disease. In autosomal dominant polycystic kidney disease (ADPKD), however, both kidneys are usually enlarged. Extrarenal manifestations are common in ADPKD, including hepatic cysts, seminal vesicle cysts, mitral valve prolapse. A 40-year-old man presented to the emergency clinic at Inha University Hospital with severe abdominal pain, nausea, and vomiting for 3 days. He had been undergoing continuous ambulatory peritoneal dialysis (CAPD) for 15 years, but it was recently changed to hemodialysis owing to sclerosing encapsulating peritonitis (SEP). Radiologic imaging studies revealed bilateral enlarged kidneys with multiple eggshell calcified cysts and some hepatic cysts, which suggested ADPKD. He underwent left nephrectomy, and pathological tests revealed ACKD-associated renal cell carcinoma (RCC) confined to the resected kidney. He was treated with steroids for SEP, and the symptoms resolved. We herein report a case of ACKD—resembling ADPKD—that progressed to RCC in a patient with concurrent SEP who had been undergoing CAPD for 15 years.

In this paper, specific molecular design rules are proposed for highly fluorescent, photostable, conjugated polymer dots (CPDs) applicable for the bioimaging of live cells. CPDs are prepared by nanoprecipitation in water using polydiphenylacetylene (PDPA) derivatives and commercial conjugated polymers. Among these, an amorphous, glassy-state PDPA derivative provides highly porous, coarsened nanoparticles. The nanoparticles are dispersed very well in water, and the polymer chains are either hydrodynamically or thermodynamically stable, with a fully relaxed intramolecular stacked structure. This leads to effective radiative emission decays by restraining collisional quenching and vibrational relaxation to achieve an extremely high fluorescence (FL) quantum efficiency. The FL emission quantum yield is as high as 0.76, which is the highest value among those reported for conventional CPDs. The PDPA-based CPD has a very low photobleaching quantum yield (∼10−9), because of its relatively high ionization potential. This aqueous colloidal solution is useful for bioimaging plant and mammalian cells. The excellent FL quantum efficiency, photostability, and cellular uptake suggest that the present CPD is a very promising probe for bioimaging, particularly for long-term imaging and tracking in live cells or experimental animals.

Rosai-Dorfman disease (RDD) is a rare and benign self-limited disorder with pathologic feature of the lymph node sinuses expanded by a proliferation of distinctive histiocytes. The most often involving site is bilateral cervical lymphadenopathy, followed by the skin and soft tissue. Treatment options, including steroid, chemotherapy, radiotherapy and debulking surgery depend on the symptoms or the extent and localization of the lesions. We encountered a very rare case of RDD at the skin lesions, particularly combined with aortic vasculitis, arrhythmia, and valvular heart disease.

Human induced pluripotent stem cells (iPSCs) represent a personalized stem cell source and enable research using the human as a model genetic system. Although iPSCs have been available for nearly a decade, simple application of efficient genome modification—a mainstay of genetics in the long-used mouse model—has only recently come to fruition. Recombinant and programmable nucleases induce targeted DNA damage and exploit native DNA-repair machinery to generate random mutations or designer modifications through a template-mediated process. In this review, we provide an overview of state-of-the-art nuclease technologies such as ZFN, TALEN, and CRISPR/Cas9 and their utility for genome engineering of human iPSCs. We explore how nucleases may be used to edit the genome with base-pair precision, and methods for the detection and avoidance of off-target cleavage. Finally, we highlight sources of genetic and technical variation in iPSCs, and propose resolutions to the question of appropriate isogenic controls.

Abstract 1467 Poster Board I-490 GATA factors function via distinct modes to establish transcriptional networks that control fundamental developmental processes including hematopoiesis. Whereas the master regulator of hematopoiesis GATA-1 is subject to multiple posttranslational modifications, how these modifications influence GATA-1 activity at endogenous loci is poorly understood. GATA-1 is sumoylated at K137, which resides in the N-terminus, but how the N-terminus contributes to GATA-1 function remains unclear. Expression of a GATA-1 mutant lacking amino acids 1-83 of the N-terminus is linked to the development of acute megakaryoblastic leukemia [Wechsler et al. (2002) Nat. Genet. 32, 148], and deletion of the N-terminus preferentially deregulates a subset of target genes [Johnson et al. (2006) PNAS. 103, 15939]. We demonstrate that sumoylation at K137 promotes transcriptional activation only at a subset of its target genes – those requiring the cell type-specific coregulator, Friend of GATA-1 (FOG-1). Interestingly, a GATA-1 mutation that disrupts FOG-1 binding (V205G) and K137 mutations yielded similar phenotypes, although FOG-1 was not required for K137 sumoylation. Both V205 and K137 mutations dysregulated GATA-1 chromatin occupancy at select sites, FOG-1-dependent target gene expression, and were rescued by tethering SUMO-1. While FOG-1- and SUMO-1-dependent genes migrated away from the nuclear periphery upon erythroid maturation, FOG-1- and SUMO-1-independent genes localized at the periphery independent of maturation. These results illustrate how sumoylation of a critical developmental regulator selectively controls its function at specific loci, and members of a target gene ensemble with distinct coregulator and posttranslational modification requirements reside in different subnuclear compartments. Disclosures: No relevant conflicts of interest to declare.

It is known that C3 is required for optimal expansion of T cells during acute viral infections. However, it is not yet determined whether T cell responses to intracellular bacterial infections require C3. Therefore, we have investigated the requirement for C3 to elicit potent T cell responses to Listeria monocytogenes (LM). We show that expansion of Ag-specific CD8 and CD4 T cells during a primary response to LM was markedly reduced in the absence of C3 activity. Further studies indicated that, unlike in an influenza virus infection, the regulation of LM-specific T cell responses by C3 might not involve the downstream effector C5a. Moreover, reduced T cell responses to LM was not linked to defective maturation of dendritic cells or developmental anomalies in the peripheral T cell compartment of C3-deficient mice. Experiments involving adoptive transfer of C3-deficient CD8 T cells into the C3-sufficient environment of wild-type mice showed that these T cells do not have intrinsic proliferative defects, and a paracrine source of C3 will suffice for clonal expansion of CD8 T cells in vivo. However, stimulation of purified C3-deficient CD8 T cells by plastic-immobilized anti-CD3 showed that C3 promotes T cell proliferation directly, independent of its effects on APC. On the basis of these findings, we propose that diminished T cell responses to LM in C3-deficient mice might be at least in part due to lack of direct effects of C3 on T cells. These studies have furthered our understanding of C3-mediated regulation of T cell immunity to intracellular pathogens.

Somatic cell reprogramming to induced pluripotent stem cells (iPSCs) is a revolutionary technology, with repercussions affecting modern functional genomics and regenerative medicine. Still, relatively little is known about the processes underlying this dramatic cellular and molecular metamorphosis. Reprogramming technology based on the implementation of piggyBac (PB) transposons has enabled studies of iPSC reprogramming mechanisms, shedding an increasing light on these processes. Unique characteristics of PB transposons such as efficient genomic integration, unlimited cargo capacity, robust gene expression, and even seamless excision highlight the importance of this transgenic tool in advancing stem cell biology. In this chapter, we provide a detailed overview of versatile primary iPSC generation from mouse somatic cells using PB transposons, and the subsequent establishment of robust secondary reprogramming systems. These protocols are highlighted with examples from recent studies as to how PB has been, and continues to be, conducive to the dissection of reprogramming processes at the cellular and molecular levels.

This paper reports a unique fluorescence (FL) response and diverse applications of conjugated polyelectrolyte (CPE) through nonelectrostatic interaction with appropriate (bio)surfactants in an immiscible two-phase system. A sulfonated microporous conjugated polymer (SMCP) with a conformation-variable intramolecular stacked structure was used as the CPE film. Despite the extremely high hydrophilicity, the SMCP film responded significantly to the hydrophobic circumstances, either physicochemically or electronically, in the presence of water-in-oil (w/o)-type nonionic surfactants with appropriate hydrophile-lipophile balance (HLB) values. The polymer film became fully wet with hydrophobic solvents due to the addition of small amounts of (bio)surfactant to reveal remarkable FL emission enhancement and chromism. Microcontact and inkjet printing using the SMCP film (or SMCP-adsorbed paper) and the surfactant solution as substrate and ink, respectively, provided high-resolution FL images due to the distinctive surfactant-induced FL change (SIFC) characteristic. Moreover, the additional electrostatic interaction of SMCP film with oppositely charged surfactants further enhanced the FL emission. Our findings will help comprehensive understanding of the nonelectrostatic SIFC mechanism of CPEs and development of novel SIFC-active materials.

Transgenics is a mainstay of functional genomics. Conditionally overexpressing genes of interest (GOIs) helps to reveal their roles in the control of complex biological processes. Complemented by findings in classic animal model systems, recent advances in human embryonic stem cell (hESC) and patient-specific induced pluripotent stem cell (hiPSC) differentiation have led to sophisticated in vitro models of human development and disease. Yet, as transgenic elements encoding inducible systems must be introduced de novo into each genetically unique human stem cell line, robust and straightforward solutions to gene delivery are required. Transposons are a family of mobile DNA elements that have been adapted as experimental tools for stable genomic integration of transgenes. The piggyBac (PB) transposon from Trichoplusia ni presents a number of benefits over classic viral or BAC transgenesis: ease of application, simple integration-site mapping, and the unique capacity for traceless excision. Moreover, their large capacity permits the consolidation of multiple transgene components in a single vector system. In this chapter, we outline the features of a panel of "All-in-One" PB transposons designed for drug-inducible gene expression and provide guidelines to establish and validate populations or clones of transgenic hiPSCs.

Study design Animal study. Objective To further investigate the effects of therapeutic hypothermia (TH), the present study compared autophagy and apoptosis after treatment with either therapeutic moderate systemic hypothermia or methylprednisolone sodium succinate (MP) in a rat model of acute spinal cord injury (SCI). Summary of background data The neuroprotective effects of TH have recently become an important topic in the field of SCI research. Methods All rats were subjected to a 25-g/cm spinal cord contusion over the ninth thoracic vertebrae. After the induction of SCI, the control group did not receive any further treatment, TH group immediately received moderate systemic hypothermia for 4 hours, and MP group was administered high-dose MP. The rats were killed either 2 or 7 days after SCI, and the injured spinal cord tissues were obtained. Apoptosis and autophagy were assessed by immunohistochemical analyses and Western blot analyses. In addition, the microarchitecture of the autophagosomes was evaluated using transmission electron microscopy, and the motor activity of the rats was assessed using the Basso-Beattie-Bresnahan (BBB) locomotor rating scale. Results Compared with controls, there was a significant reduction in the expression levels of cleaved caspase-8, -9, and -3 in the TH- and MP-treated groups 2 days after SCI. Moreover, compared with the control group, the expression of LC3II and Beclin-1 exhibited a significant decrease on day 2 after treatment with TH. The numbers of transferase dUTP nicked-end labeling and LC3-positive cells were significantly lower on days 2 and 7. The Basso-Beattie-Bresnahan ratings were significantly higher 6 weeks after SCI in both the TH- and MP-treated groups than in the control group. Conclusion Both TH and MP have neuroprotective effects on injured spinal cord tissues via the inhibition of apoptosis and autophagy. Thus, the application of moderate systemic hypothermia may be a useful treatment modality after acute SCI. Level of evidence N/A.

Transgenics is a mainstay of functional genomics. Conditionally overexpressing genes of interest (GOIs) helps to reveal their roles in the control of complex biological processes. Complemented by findings in classic animal model systems, recent advances in human embryonic stem cell (hESC) and patient-specific induced pluripotent stem cell (hiPSC) differentiation have led to sophisticated in vitro models of human development and disease. Yet, as transgenic elements encoding inducible systems must be introduced de novo into each genetically unique human stem cell line, robust and straightforward solutions to gene delivery are required. Transposons are a family of mobile DNA elements that have been adapted as experimental tools for stable genomic integration of transgenes. The piggyBac (PB) transposon from Trichoplusia ni presents a number of benefits over classic viral or BAC transgenesis: ease of application, simple integration-site mapping, and the unique capacity for traceless excision. Moreover, their large capacity permits the consolidation of multiple transgene components in a single vector system. In this chapter, we outline the features of a panel of "All-in-One" PB transposons designed for drug-inducible gene expression and provide guidelines to establish and validate populations or clones of transgenic hiPSCs.

Summary Since the first generation of induced pluripotent stem cells (iPSCs), several reprogramming systems have been used to study its molecular mechanisms. However, the system of choice largely affects the reprogramming efficiency, influencing our view on the mechanisms. Here, we demonstrate that reprogramming triggered by less efficient polycistronic reprogramming cassettes not only highlights mesenchymal-to-epithelial transition (MET) as a roadblock but also faces more severe difficulties to attain a pluripotent state even post-MET. In contrast, more efficient cassettes can reprogram both wild-type and Nanog−/− fibroblasts with comparable efficiencies, routes, and kinetics, unlike the less efficient reprogramming systems. Moreover, we attribute a previously reported variation in the N terminus of KLF4 as a dominant factor underlying these critical differences. Our data establish that some reprogramming roadblocks are system dependent, highlighting the need to pursue mechanistic studies with close attention to the systems to better understand reprogramming., Graphical Abstract, Highlights • Distinct reprogramming cassettes yield different reprogramming intermediates • MET is not a major rate-limiting step in reprogramming with high KLF4 expression • A lack of endogenous Nanog becomes trivial in efficient reprogramming systems • Roadblocks toward iPSCs depend on the reprogramming systems, In this article, Kaji and colleagues demonstrate that different reprogramming systems with distinct reprogramming efficiencies yield clearly distinguishable intermediate populations and, thus, potentially biased mechanistic views. For example, mesenchymal-to-epithelial transition and lack of endogenous Nanog are obstacles for iPSC generation only in inefficient reprogramming systems. This work highlights the importance of re-assessing molecular mechanisms of reprogramming in the literature heeding the reprogramming systems.

The reciprocal expression of GATA-1 and GATA-2 during hematopoiesis is an important determinant of red blood cell development. Whereas Gata2 is preferentially transcribed early in hematopoiesis, elevated GATA-1 levels result in GATA-1 occupancy at sites upstream of the Gata2 locus and transcriptional repression. GATA-2 occupies these sites in the transcriptionally active locus, suggesting that a "GATA switch" abrogates GATA-2-mediated positive autoregulation. Chromatin immunoprecipitation (ChIP) coupled with genomic microarray analysis and quantitative ChIP analysis with GATA-1-null cells expressing an estrogen receptor ligand binding domain fusion to GATA-1 revealed additional GATA switches 77 kb upstream of Gata2 and within intron 4 at +9.5 kb. Despite indistinguishable GATA-1 occupancy at -77 kb and +9.5 kb versus other GATA switch sites, GATA-1 functioned uniquely at the different regions. GATA-1 induced histone deacetylation at and near Gata2 but not at the -77 kb region. The -77 kb region, which was DNase I hypersensitive in both active and inactive states, conferred equivalent enhancer activities in GATA-1- and GATA-2-expressing cells. By contrast, the +9.5 kb region exhibited considerably stronger enhancer activity in GATA-2- than in GATA-1-expressing cells, and other GATA switch sites were active only in GATA-1- or GATA-2-expressing cells. Chromosome conformation capture analysis demonstrated higher-order interactions between the -77 kb region and Gata2 in the active and repressed states. These results indicate that dispersed GATA factor complexes function via long-range chromatin interactions and qualitatively distinct activities to regulate Gata2 transcription.

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Neural network can be trained to approximate an arbitrary nonlinear function of multivariate data like the mini-mill crown values in Automatic Shape Control. The trained weights of neural network can evaluate or generalize the process data outside the training vectors. Sometimes, the blind modeling of the process data is necessary to compare with the scattered analytical model of mini-mill process in isolated electro-mechanical forms. To come up with a viable model, we propose the blind neural-based range-division domain-clustering piecewise-linear modeling scheme. The basic ideas are: 1) dividing the range of target data, 2) clustering the corresponding input space vectors, 3)training the neural network with clustered prototypes to smooth out the convergence and 4) solving the resulting matrix equations with a pseudo-inverse to alleviate the ill-conditioning problem. The simulation results support the effectiveness of the proposed scheme and it opens a new way to the data analysis technique. By the comparison with the statistical regression, it is evident that the proposed scheme obtains better modeling error uniformity and reduces the magnitudes of errors considerably. Approximatly 10-fold better performance results.

Ginsenoside Rh1 or Rh2 differentiated B16 melanoma or F9 teratocarnoma to phenotypic normal melanocyte-like cells or parietal endoderm-like cells. Ginsenoside Rh3 and Rh4 were recently isolated from Panax ginseng, but their biochemical and pharmacological effects remain unidentified. The present study investigated whether the ginsenoside Rh group (G-Rh1, -Rh2, -Rh3 and -Rh4) having similar structures induce differentiation of HL-60 cells and whether protein kinase C (PKC) is involved in differentiation by ginsenoside. Differentiation was assessed by Wright-Giemsa stain and nitroblue tetrazolium reduction. G-Rh2 and G-Rh3 induced differentiation of HL-60 cells into morphologically and functionally granulocytes but G-Rh1 and G-Rh4 did not. G-Rh2 and G-Rh3 arrested the cell cycle at the G1/S phase, consistent with the ability to induce differentiation in a decreasing order of retinoic acid > G-Rh2 > G-Rh3. During differentiation by G-Rh2, Ca2+/phospholipid-dependent PKC activity was increased in both the cytosol and total cell extract and Ca2+/phospholipid-dependent phosphorylation of 38 and 200 kDa endogenous proteins increased, while phosphorylation of 60, 64, 66 and 97 kDa proteins was Ca2+/phospholipid-independent. When cytosolic PKC isoforms were analyzed by immunoblotting, no significant change was observed in the alpha level, however, the immunoreactive 60 kDa band of a similar mass to the PKC catalytic fragment appeared following treatment with G-Rh2. The beta isoform was gradually increased with prolonged treatment. The gamma isoform was not detected in the cytosol of untreated cells, whereas a small amount was detected 5 days after treatment. It is concluded that G-Rh2 and G-Rh3 can induce differentiation of HL-60 cells into granulocytes and modulation of PKC isoform levels may contribute to differentiation of HL-60 cells by G-Rh2.

Glycosylation of 12beta-acetoxy-dammar-24-en-3beta,20(S)-diol (4), with hepta-O-acetyl-alpha-sophorosyl bromide (5) under catalysis by Ag2CO3 or Ag2O afforded a chromatographically unseparated mixture of the alpha- and beta-linked octaacetates 6 and 7 in an approximately 2.5:1 ratio. After deprotection and chromatographic purification, the free alpha- (8) and beta-glycosides (9) were obtained. Sophoroside 9 was identical in all respects with ginsenoside Rg3, the minor component of Ginseng Radix rubra. All compounds were fully characterized by 1H and 13C NMR spectroscopy.

The involvement of ginsenosides in the signal cascade that stimulates cellular growth was investigated. It was found that ginsenosides Rh1 and Rh2 extracted from the root of Panax ginseng inhibited cellular proliferation in NIH 3T3 fibroblasts. Both ginsenosides Rh1 and Rh2 effectively reduced phospholipase C activity resulting in a decrease in the intracellular level of diacylglycerol, an endogenous activator of protein kinase C. The treatment of cells with Rh1 or Rh2 was thus found to reduce intracellular protein kinase C activity. We also observed that the phosphorylation of myristoylated alanine-rich C kinase substrate, one of the major substrates of protein kinase C in cells, was inhibited by the ginsenosides. Data suggest that the ginsenoside Rh1 or Rh2 exerts antiproliferative effects by inhibiting phospholipase C, which produces second messengers necessary for the activation of protein kinase C.

A possible role of cytochrome P450 (P450) inhibition by red ginseng saponins in carbon tetrachloride (CCl4)-induced lipid peroxidation was investigated in liver microsomes prepared from male Sprague Dawley rats. The total saponin of red ginseng standardized on ginsenosides-Rb1, -Rb2, -Rc, -Rd, -Re, and -Rg1 whose composition was studied in our previous report was used in the present study. The standardized saponin of red ginseng showed inhibitory effects on P450-associated monooxygenase activities in a dose-dependent manner, particularly p-nitrophenol hydroxylase activity which has been known to represent CCl4-activating P450 2E1 enzyme. Meanwhile, silymarin enhanced the activity of P450 2E1 enzyme in liver microsomes. When the lipid peroxidation was induced by incubating rat liver microsomes with CCl4 in the presence of NADPH, the standardized saponin significantly blocked the formation of thiobarbituric acid-reactive substances at the same concentrations showing P450 inhibition in liver microsomes. Silymarin revealed more potent protection against CCl4-induced lipid peroxidation. When the lipid peroxidation was induced by FeCl3, in which metabolic activation may not be required, only silymarin could protect the lipid peroxidation in liver microsomes. Taken together, our present results indicated that the inhibitory effects of red ginseng saponin on P450 enzymes may have a critical role in CCl4-induced lipid peroxidation in rat liver microsomes and that the mechanism of hepatoprotection by red ginseng saponin may be distinct from that of silymarin.

In order to elucidate the cytotoxicity-structure correlation of ginseng-derived components, several prosapogenins and sapogenins were prepared from Korean red ginseng (Panax ginseng) saponins by acid hydrolysis or alkaline cleveage, and their chemical structures were identified by a combination of spectral and physical methods. Some of these degradation products showed the cytotoxic activities against various cancer cell lines, A549, SK-OV-3, SK-Mel-2, P388, L 1210 and K562. The significant difference in cytotoxicity between stereoisomers was not found and the activity was inversely proportional to the number of sugars linked to sapogenins. Diol-type prosapogenins and sapogenins showed higher cytotoxicity than triol-type ones.