Establishment of a hepatitis B virus reporter system harboring a HiBiT-tag in the PreS2 region.

BACKGROUND: Approximately 296 million people suffer from chronic hepatitis B (CHB) caused by hepatitis B virus (HBV). Current standard treatment, nucleos(t)ide analogs, are not efficient enough to eradicate HBV from the hepatocytes. Thus, developing new drugs for CHB is desired to achieve complete cure. METHODS: Here we established a novel HBV reporter system, HBV-HiBiT-PS2, to screen new drugs for CHB. HBV-HiBiT-PS2 was constructed by introducing a HiBiT-tag at the 5'-end of PreS2 and introduced into HepG2-NTCP cells. Culture supernatant containing HBV-HiBiT-PS2 virions was fractionated by a sucrose density gradient ultracentrifugation to characterize their components. Replication kinetics and reporter function of HBV-HiBiT-PS2 were determined by analyzing the parameters for HBV replication in the presence or absence of HBV inhibitors. RESULTS: HBV-HiBiT-PS2 could be used for monitoring most of the replication cycle of HBV. The effects of well-characterized HBV inhibitors could be evaluated by the HiBiT activity. HBV-HiBiT-PS2 could be specialized for screening secretion inhibitors for hepatitis B surface antigen (HBsAg) because most of the HiBiT activity was derived from subviral particles which are the multimers of HBsAg. CONCLUSIONS: We demonstrated that HBV-HiBiT-PS2 would be a robust tool for screening novel drugs, especially HBsAg secretion inhibitors, targeted for CHB.

Successful Retransplantation With Killer Cell Immunoglobulin-like Receptor Ligand-mismatched Cord Blood in Infant Acute Lymphoblastic Leukemia That Relapsed After Transplantation.

The prognosis of children with KMT2A -rearranged ( KMT2A -r) acute lymphoblastic leukemia (ALL) remains dismal. This report describes the successful retransplantation of a patient with infant ALL who relapsed both bone marrow and central nervous system. The patient received HLA-matched cord blood transplantation (CBT) and relapsed 18 months later. After achieving the second remission, the patient received a killer cell immunoglobulin-like receptor ligand-mismatched CBT with a reduced-intensity conditioning regimen and has been in remission for 52 months. Thus, killer cell immunoglobulin-like receptor ligand-mismatched CBT with reduced-intensity conditioning might be a treatment option for patients with KMT2A- r ALL who relapsed after transplantation, even with extramedullary relapse.

Successful blastocyst production by intracytoplasmic injection of sperm after in vitro maturation of follicular oocytes obtained from immature female squirrel monkeys (Saimiri boliviensis).

Advanced reproductive technologies are being applied for the propagation of squirrel monkeys, to ensure their preservation as a genetic resource and the effective use of their gametes in the future. In the present study, oocytes and spermatozoa were collected from live squirrel monkeys, following which piezo intracytoplasmic sperm injection (ICSI) was performed using these gametes. Follicular development was induced by administering equine chorionic gonadotropin (eCG) containing inhibin antiserum to an immature squirrel monkey female. The unilateral ovary was excised after the administration of human chorionic gonadotropin (hCG), to induce ovulation, following which the larger developed follicular oocytes were collected. Follicular oocytes were prepared for ICSI using sperm from the epididymal tail of a unilateral testis extracted from a mature male. The embryos were continuously incubated in CMRL 1066 medium supplemented with 10% (v/v) fetal bovine serum. Embryo culture was performed with cumulus cells. Two experiments of ICSI carried out with three females resulted in 14 mature oocytes from the 49 cumulus-oocyte complexes collected and five embryos, three of which developed into blastocysts. These blastocysts were vitrified, thawed, and transferred to recipient monkeys, but no pregnancies resulted. In conclusion, the present study is the first to successfully produce ICSI-derived blastocysts from MII oocytes obtained by means of hormone administration (a combination of eCG+inhibin antiserum and hCG) and in vitro maturation in immature squirrel monkeys.

Chronic mandibular osteomyelitis caused by Granulicatella adiacens in an immunocompetent child.

We report a pediatric case aged 10 years with Granulicatella adiacens-associated chronic mandibular osteomyelitis. The causative pathogen was uncertain because polymicrobial species were detected from the bacterial culture in bone marrow fluid. In contrast, G. adiacens was predominantly identified in the clone library analysis of the bacterial 16S rRNA gene sequence. Vancomycin to which G. adiacens was reported to be susceptible was not administrated sufficiently to this patient because of its adverse event, whereas linezolid and ciprofloxacin was alternatively effective for the treatment of chronic mandibular osteomyelitis.

ToF-SIMS and Laser-SNMS Imaging of Heterogeneous Topographically Complex Polymer Systems.

Heterogeneous polymer coatings, such as those used in organic electronics and medical devices, are of increasing industrial importance. In order to advance the development of these types of systems, analytical techniques are required which are able to determine the elemental and molecular spatial distributions, on a nanometer scale, with very high detection efficiency and sensitivity. The goal of this study was to investigate the suitability of laser postionization secondary neutral mass spectrometry (Laser-SNMS) with a 157 nm postionization laser beam to image structured polymer mixtures and compare the results with time-of-flight secondary ion mass spectrometry (ToF-SIMS) measurements using Bi3+ primary ions. The results showed that Laser-SNMS is better suited than ToF-SIMS for unambiguous detection and submicrometer imaging of the wide range of polymers investigated. The data also showed that Laser-SNMS has the advantage of being much more sensitive (in general higher by more than an order of magnitude and peaking at up to 3 orders of magnitude) than ToF-SIMS while also showing superior performance on topographically complex structured insulating surfaces, due to significantly reduced field effects and a higher dynamic range as compared to ToF-SIMS. It is concluded that Laser-SNMS is a powerful complementary technique to ToF-SIMS for the analysis of heterogeneous polymers and other complex structured organic mixtures, providing submicrometer resolution and high sensitivity.

Selective DNA Recognition and Cytotoxicity of Water-Soluble Helical Metallosupramolecular Polymers.

Water-soluble helical Fe(II)-based metallosupramolecular polymers ((P)- and (M)-polyFe) were synthesized by 1:1 complexation of Fe(II) ions and bis(terpyridine)s bearing a (R)- and (S)-BINOL spacer, respectively. The binding affinity to calf thymus DNA (ct-DNA) was investigated by titration measurements. (P)-PolyFe with the same helicity as B-DNA showed 40-fold higher binding activity (Kb = 13.08 × 107 M-1) to ct-DNA than (M)-polyFe. The differences in binding affinity were supported by electrochemical impedance spectroscopy analysis. The charge-transfer resistance (Rct) of (P)-polyFe increased from 2.5 to 3.9 kΩ upon DNA binding, while that of (M)-polyFe was nearly unchanged. These results indicate that ionically strong binding of (P)-polyFe to DNA chains decreased the mobility of ions in the conjugate. Unique rod-like images were obtained by atomic force microscopy measurement of the DNA conjugate with (P)-polyFe, likely because of the rigid binding between DNA chains and the polymer. Differences in polymer chirality lead to significantly different cytotoxicity levels in A549 cells. (P)-PolyFe showed higher binding affinity to B-DNA and much higher cytotoxicity than (M)-polyFe. The helicity in metallosupramolecular polymer chains was important not only for chiral recognition of DNA but also for coordination to a biological target in the cellular environment.

Effect of amino groups of mesoporous silica nanoparticles on CpG oligodexynucleotide delivery.

In this study, we proposed to modify mesoporous silica nanoparticles (MSNs) with 3-aminopropyltriethoxysilane (NH2-TES), aminoethylaminopropyltriethoxysilane (2NH2-TES) and 3-[2-(2-aminoethylamino)ethylamino] propyl-trimethoxysilane (3NH2-TES) for binding of cytosine-phosphate-guanosine oligodexynucleotides (CpG ODN), and investigated the effect of different amino groups of MSNs on the CpG ODN delivery. Serum stability, in vitro cytotoxicity, and cytokine interleukin-6 (IL-6) induction by MSN-NH2/CpG, MSN-2NH2/CpG and MSN-3NH2/CpG complexes were investigated in detail. The results showed that three kinds of aminated-MSN-based CpG ODN delivery systems had no cytotoxicity to RAW264.7 cells, and binding of CpG ODN to MSN-NH2, MSN-2NH2 and MSN-3NH2 nanoparticles enhanced the serum stability of CpG ODN due to protection by the nanoparticles. However, three aminated MSN-based CpG ODN delivery systems exhibited different CpG ODN delivery efficiency, and MSN-NH2/CpG complexes had the highest ability to induce IL-6 secretion.

[Recurrent pulmonary edema after umbilical cord blood transplantation in a patient with infant acute lymphoblastic leukemia].

This report describes two infants with recurrent pulmonary edema after umbilical cord blood transplantation (CBT). A 3-month-old boy and a 7-month-old boy with infant acute lymphoblastic leukemia underwent CBT from an unrelated donor in the first complete remission. The conditioning regimen consisted of busulfan, etoposide, and cyclophosphamide. Tacrolimus and short-term methotrexate were administered for the prophylaxis of acute graft-versus-host disease (GVHD). Neutrophil engraftment was achieved on days 17 and 19, respectively. Neither infant developed acute GVHD. They both exhibited tachypnea and weight gain on days 25 and 30, respectively, which were diagnosed as pulmonary edema by chest X rays. The respiratory condition of the patients improved within a few days with the close monitoring of weight changes after the administration of diuretics. However, they suddenly developed dyspnea and pulmonary edema again on days 37 and 59, respectively. Steroid therapy was initiated for both patients. Their respiratory condition again improved quickly after the initiation of steroid therapy. Their symptoms and clinical courses may be classified as a new entity of idiopathic pneumonia syndrome (IPS). Therefore, these cases may represent a new unclassifiable IPS associated with either CBT or infants.

The effects of viewing by scrolling on a small screen on the encoding of objects into visual long-term memory.

The perception of an image obtained by scrolling through a small screen can differ from the typical perception of a wide visual field in a stable environment. However, we do not fully understand image perception by scrolling on a small screen based on psychological knowledge of visual perception and cognition of images. This study investigated how screen size limitations and image shifts caused by scrolling affect image encoding in visual long-term memory. Participants explored the stimulus images under three conditions. Under the scrolling condition, they explored the image through a small screen. Under the moving-window condition, they explored the image by moving the screen over a masked image; this is similar to looking through a moving peephole. Under the no-window condition, participants were able to view the entire image simultaneously. Each stimulus comprised 12 objects. After 1 h, the samples were tested for object recognition. Consequently, the memory retention rate was higher in the scrolling and moving-window conditions than in the no-window condition, and no difference was observed between the scrolling and moving-window conditions. The time required by participants to explore the stimulus was shorter under the no-window condition. Thus, encoding efficiency (i.e., the rate of encoding information into memory in a unit of time) did not differ among the three conditions. An analysis of the scan trace of the scrolling and window movements in relation to the image revealed differences between the scrolling and moving-window conditions in terms of the scan's dynamic features. Moreover, a negative correlation was observed between the memory retention rate and image-scrolling speed. We conclude that perceiving images by scrolling on a small screen enables better memory retention than that obtained through whole-image viewing if the viewing time is not limited. We suggest that viewing through a small screen is not necessarily disadvantageous for memory encoding efficiency depending on the presentation mode, and the results show that participants who scrolled fast tended to have worse memory retention. These findings can impact school education and thus suggest that the use of mobile devices in learning has some merit from the viewpoint of cognitive psychology.

TRIM26 positively affects hepatitis B virus replication by inhibiting proteasome-dependent degradation of viral core protein.

Chronic hepatitis B virus (HBV) infection is a major medical concern worldwide. Current treatments for HBV infection effectively inhibit virus replication; however, these treatments cannot cure HBV and novel treatment-strategies should be necessary. In this study, we identified tripartite motif-containing protein 26 (TRIM26) could be a supportive factor for HBV replication. Small interfering RNA-mediated TRIM26 knockdown (KD) modestly attenuated HBV replication in human hepatocytes. Endogenous TRIM26 physically interacted with HBV core protein (HBc), but not polymerase and HBx, through the TRIM26 SPRY domain. Unexpectedly, TRIM26 inhibited HBc ubiquitination even though TRIM26 is an E3 ligase. HBc was degraded by TRIM26 KD in Huh-7 cells, whereas the reduction was restored by a proteasome inhibitor. RING domain-deleted TRIM26 mutant (TRIM26ΔR), a dominant negative form of TRIM26, sequestered TRIM26 from HBc, resulting in promoting HBc degradation. Taking together, this study demonstrated that HBV utilizes TRIM26 to avoid the proteasome-dependent HBc degradation. The interaction between TRIM26 and HBc might be a novel therapeutic target against HBV infection.

Behavioral analysis of Drosophila transformants expressing human taste receptor genes in the gustatory receptor neurons.

Transgenic Drosophila expressing human T2R4 and T2R38 bitter-taste receptors or PKD2L1 sour-taste receptor in the fly gustatory receptor neurons and other tissues were prepared using conventional Gal4/UAS binary system. Molecular analysis showed that the transgene mRNAs are expressed according to the tissue specificity of the Gal4 drivers. Transformants expressing the transgene taste receptors in the fly taste neurons were then studied by a behavioral assay to analyze whether transgene chemoreceptors are functional and coupled to the cell response. Since wild-type flies show strong aversion against the T2R ligands as in mammals, the authors analyzed the transformants where the transgenes are expressed in the fly sugar receptor neurons so that they promote feeding ligand-dependently if they are functional and activate the neurons. Although the feeding preference varied considerably among different strains and individuals, statistical analysis using large numbers of transformants indicated that transformants expressing T2R4 showed a small but significant increase in the preference for denatonium and quinine, the T2R4 ligands, as compared to the control flies, whereas transformants expressing T2R38 did not. Similarly, transformants expressing T2R38 and PKD2L1 also showed a similar preference increase for T2R38-specific ligand phenylthiocarbamide (PTC) and a sour-taste ligand, citric acid, respectively. Taken together, the transformants expressing mammalian taste receptors showed a small but significant increase in the feeding preference that is taste receptor and also ligand dependent. Although future improvements are required to attain performance comparable to the endogenous robust response, Drosophila taste neurons may serve as a potential in vivo heterologous expression system for analyzing chemoreceptor function.

[Successful treatment with prednisolone for air-leak syndrome following interstitial pneumonia after allogeneic hematopoietic stem cell transplantation].

A 13-year-old boy with T lymphoblastic leukemia underwent allogeneic bone marrow transplantation from an HLA-matched sibling in the second remission phase. After the dose of cyclosporine (CyA) was reduced, dyspnea appeared on Day 117. CT revealed diffuse interstitial shadows on the bilateral lungs. The results of broncho-alveolar lavage suggested Pneumocystis jirovecii pneumonia. The dose of trimethoprim-sulfamethoxazole was increased, and steroid therapy was started. The symptoms transiently subsided, but exacerbated with a reduction in the steroid dose. On Day 139, mediastinal and subcutaneous emphysema appeared. We considered that non-infectious interstitial pneumonia was primarily involved in the pathogenesis for the following reasons: the boy was negative for ß-D-glucan early after onset, and there was a correlation between the steroid-dose reduction and condition. The steroid dose was again increased to 80 mg and the symptoms promptly subsided. When late-onset non-infectious pulmonary complications after hematopoietic stem cell transplantation lead to air-leak syndrome, the mortality rate is very high. However, survival may be achieved by intensifying immunosuppressive therapy in the early stage.

Visualized procollagen Iα1 demonstrates the intracellular processing of propeptides.

The processing of type I procollagen is essential for fibril formation; however, the steps involved remain controversial. We constructed a live cell imaging system by inserting fluorescent proteins into type I pre-procollagen α1. Based on live imaging and immunostaining, the C-propeptide is intracellularly cleaved at the perinuclear region, including the endoplasmic reticulum, and subsequently accumulates at the upside of the cell. The N-propeptide is also intracellularly cleaved, but is transported with the repeating structure domain of collagen into the extracellular region. This system makes it possible to detect relative increases and decreases in collagen secretion in a high-throughput manner by assaying fluorescence in the culture medium, and revealed that the rate-limiting step for collagen secretion occurs after the synthesis of procollagen. In the present study, we identified a defect in procollagen processing in activated hepatic stellate cells, which secrete aberrant collagen fibrils. The results obtained demonstrated the intracellular processing of type I procollagen, and revealed a link between dysfunctional processing and diseases such as hepatic fibrosis.

Two-layered dissolving microneedles for percutaneous delivery of peptide/protein drugs in rats.

PURPOSE: Feasibility study of two-layered dissolving microneedles for percutaneous delivery of peptide/proteins using recombinant human growth hormone (rhGH) and desmopressin (DDAVP). METHODS: Two-layered dissolving microneedles were administered percutaneously to the rat skin. Plasma rhGH and DDAVP concentrations were measured by EIA and LC/MS/MS. In vivo dissolution and diffusion rates of drugs in the skin were studied using tracer dyes, lissamine green B (LG) for rhGH and evans blue (EB) for DDAVP. Diffusion of drugs vertically into the skin was studied using FITC-dextran (MW = 20 kDa)-loaded dissolving microneedles. Stability experiments were performed at -80°C and 4°C. RESULTS: The absorption half-lives, t (1/2a), of rhGH and DDAVP from dissolving microneedles were 23.7 ± 4.3-28.9 ± 5.2 and 14.4 ± 2.9-14.1 ± 1.1 min; the extents of bioavailability were 72.8 ± 4.2-89.9 ± 10.0% and 90.0 ± 15.4-93.1 ± 10.3%, respectively. LG and EB disappeared from the administered site within 2 h and 3 h after administration. Five green fluorescein spots were detected at 15 s and enlarged transversally at 30 s. FITC-dextran was delivered into the microcapillaries at 5 min and 10 min. The rhGH and DDAVP were stable in dissolving microneedles for one month at -80°C and 4°C. CONCLUSIONS: Results suggest that the two-layered dissolving microneedles are useful as an immediate-release transdermal DDS for peptide/protein drugs.

Characterization of the osteoblast-specific transmembrane protein IFITM5 and analysis of IFITM5-deficient mice.

Interferon-inducible transmembrane protein 5 (IFITM5) is an osteoblast-specific membrane protein whose expression peaks around the early mineralization stage during the osteoblast maturation process. To investigate IFITM5 function, we first sought to identify which proteins interact with IFITM5. Liquid chromatography mass spectrometry revealed that FK506-binding protein 11 (FKBP11) co-immunoprecipitated with IFITM5. FKBP11 is the only protein it was found to interact with in osteoblasts, while IFITM5 interacts with several proteins in fibroblasts. FKBPs are involved in protein folding and immunosuppressant binding, but we could not be sure that IFITM5 participated in these activities when bound to FKBP11. Thus, we generated Ifitm5-deficient mice and analyzed their skeletal phenotypes. The skeletons, especially the long bones, of homozygous mutants (Ifitm5(-/-)) were smaller than those of heterozygous mutants (Ifitm5(+/-)), although we did not observe any significant differences in bone morphometric parameters. The effect of Ifitm5 deficiency on bone formation was more significant in newborns than in young and adult mice, suggesting that Ifitm5 deficiency might have a greater effect on prenatal bone development. Overall, the effect of Ifitm5 deficiency on bone formation was less than we expected. We hypothesize that this may have resulted from a compensatory mechanism in Ifitm5-deficient mice.

BMP-2-loaded silica nanotube fibrous meshes for bone generation.

Silica nanotube fibrous meshes were fabricated as multiple functional matrices for both delivering bone morphological protein-2 (BMP-2) and supporting osteoblast attachment and proliferation. The meshes were fabricated via a collagen-templated sol-gel route and consisted of tubular silica with open ends. BMP-2 was loaded to the meshes by soaking in BMP-2 solution. The meshes effectively enabled the attachment and proliferation of osteoblast MC3T3-E1 cells and delivered bioactive BMP-2 to stimulate cell differentiation. These results demonstrate the potential use of the meshes in bone generation applications.

Visualizing Lithium Distribution and Degradation of Composite Electrodes in Sulfide-based All-Solid-State Batteries Using Operando Time-of-Flight Secondary Ion Mass Spectrometry.

Understanding the electrochemical reactions taking place in composite electrodes during cell cycling is essential for improving the performance of all-solid-state batteries. However, comprehensive in situ monitoring of Li distribution, along with measurement of the evolution of degradation, is challenging because of the limitations of the characterization techniques commonly used. This study demonstrates the observation of Li distribution and degradation in composite cathodes consisting of LiNi0.8Co0.15Al0.05O2 (NCA) and 75Li2S·25P2S5 (LPS) during cell operation using operando time-of-flight secondary ion mass spectrometry. The evolution of the nonuniform reaction of NCA particles during charge and discharge cycles was successfully visualized by mapping fragments containing Li. Furthermore, degradation of the NCA/LPS interface was investigated by mapping POx- and SOx- fragments, which are related to the solid electrolyte interphase. We found that during the charge-discharge cycle and application of a high-voltage stress to the composite electrodes, the PO2- and PO3- fragments increased monotonically, whereas the SO3- fragment exhibited a reversible increase-decrease behavior, implying the existence of a redox-active component at the NCA/LPS interface. The demonstrated technique provides insights into both the optimized structures of composite electrodes and the underlying mechanisms of interfacial degradation at active material/solid electrolyte interfaces.

Visualizing Lithiation of Graphite Composite Anodes in All-Solid-State Batteries Using Operando Time-of-Flight Secondary Ion Mass Spectrometry.

A fundamental understanding of lithiation dynamics in composite electrodes is essential for the development of all-solid-state batteries with high capacity and rate capability. However, a comprehensive methodology to monitor the time evolution of lithiation in a composite electrode has not been fully established because of the limitations of the characterization techniques currently employed. In this study, we demonstrate the visualization of the transient distribution of Li in composite graphite anodes during cell operation using operando time-of-flight secondary ion mass spectrometry. Selective detection of Li in the graphite electrode was successfully achieved by mapping the Li- fragment rather than the conventional mapping of positive Li-containing fragments. The results revealed the formation of a Li concentration gradient during charging, indicating that the reaction of the composite electrode is limited by the diffusion of Li ions inside the graphite. The demonstrated technique is expected to facilitate the investigation of the underlying lithiation mechanisms of composite electrodes in operating cells.

[Brain hypothermia therapy for newborns with severe birth asphyxia: an experience from a single neonatal intensive care unit].

The object of this study was to determine the efficacy and safety of brain hypothermia therapy (BHT) for neonates with severe birth asphyxia in our neonatal intensive care unit (NICU). We retrospectively reviewed medical records to analyze the prognosis and the factors affecting the prognosis of 21 patients who underwent BHT at the NICU between 2001 and 2007. The prognosis of those 21 patients at the time of discharge from the NICU was as follows: good-11 patients (52.4%); disability-5 patients (23.8%); and death-5 patients (23.8%). The ten poor prognosis patients (disability: 5, death: 5) had a shorter gestational period, a lower Apgar score, and a significantly higher blood lactate level in comparison with good-prognosis newborns. In particular, a gestational period of less than 34 weeks (3 patients) and a blood lactate level of at least 200 mg/dl (6 out of 7 patients) are considered to be factors for a poor prognosis. In addition, intraventricular hemorrhage was recorded in 7 patients of the 10 poor-prognosis patients and 4 of those patients developed acute renal failure during BHT. Consequently, these disorders are considered to worsen the prognosis. This study supports the efficacy and safety of BHT for neonates with severe birth asphyxia. On the other hand, BHT for the above mentioned types of high-risk patients still requires further consideration for the adoption and methods of BHT.

Visual Search Within a Limited Window Area: Scrolling Versus Moving Window.

Every day we perceive pictures on our mobile phones and scroll through images within a limited space. At present, however, visual perception via image scrolling is not well understood. This study investigated the nature of visual perception within a small window frame. It compared visual search efficiency using three modes: scrolling, moving-window, and free-viewing. The item number and stimulus size varied. Results showed variations in search efficiency depending on search mode. The slowest search occurred under the scrolling condition, followed by the moving-window condition, and the fastest search occurred under the no-window condition. For the scrolling condition, the response time increased the least sharply in proportion to item number but most sharply in proportion to the stimulus size compared to the other two conditions. Analysis of the trace of scan revealed frequent pauses interjected with small and fast stimulus shifts for the scrolling condition, but slow and continuous window movements interjected with a few pauses for the moving-window condition. We concluded that searching via scrolling was less efficient than searching via a moving-window, reflecting differences in dynamic properties of participants' scan.