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.

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.

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.

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.

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.

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.

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.

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.

Visual Feature Integration of Three Attributes in Stimulus-Response Mapping Is Distinct From That of Two.

In the human visual system, different attributes of an object are processed separately and are thought to be then temporarily bound by attention into an integrated representation to produce a specific response. However, if such representations existed in the brain for arbitrary multi-attribute objects, a combinatorial explosion problem would be unavoidable. Here, we show that attention may bind features of different attributes only in pairs and that bound feature pairs, rather than integrated object representations, are associated with responses for unfamiliar objects. We found that in a mapping task from three-attribute stimuli to responses, presenting three attributes in pairs (two attributes in each window) did not significantly complicate feature integration and response selection when the stimuli were not very familiar. We also found that repeated presentation of the same triple conjunctions significantly improved performance on the stimulus-response task when the correct responses were determined by the combination of three attributes, but this familiarity effect was not observed when the response could be determined by two attributes. These findings indicate that integration of three or more attributes is a distinct process from that of two, requiring long-term learning or some serial process. This suggests that integrated object representations are not formed or are formed only for a limited number of very familiar objects, which resolves the computational difficulty of the binding problem.

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.

3D-Ridge Stocked Layers of Nitrogen-Doped Mesoporous Carbon Nanosheets for Ultrasensitive Monitoring of Dopamine Released from PC12 Cells under K+ Stimulation.

3D-ridge nanosheets of N-doped mesoporous carbon (NMCS)-based electrodes are fabricated as ultrasensitive biosensors for in vitro monitoring of dopamine (DA) released from living cells. The large-scale ranges of dense-layered sheets are arranged linearly with a thickness of <10 nm, soft tangled edges, stocked layer arrangements, and tunable mesoporous frameworks with 3D orientations. The intrinsic features of the active interfacial surface of the electrode based on NMCS along with polarized surfaces, dense surface-charged matrices, fast electron transfer, and easy molecular diffusion, are present in the highly active electrode for biosensing applications. The designed electrode based on the NMCS shows high sensitivity and selectivity for DA sensing even in the presence of physiological interference molecules, such as ascorbic acid and/or uric acid, at a low applied potential of 0.25 V versus Ag/AgCl. The large-scale NMCS-based electrode shows low detection limits as low as 10 nmol L-1 , wide linear range up to 0.5 mmol L-1 , long-term stability for more than 15 d (relative standard deviation (RSD)= 5.8%), and a low cytotoxicity with high biocompatibility. The findings demonstrated that the NMCS-based electrode is a reliable modified electrode for ultratrace sensitivity of DA, which is secreted normally from dopaminergic cells (PC12) or under a stimulating agent (K+ ).

One-step selective screening of bioactive molecules in living cells using sulfur-doped microporous carbon.

A metal-free electrode using heteroatom-doped microporous carbon was fabricated for the ultrasensitive monitoring of mono-bioactive molecules and the selective signaling of dopamine (DA) secreted by living cells. The constructed electrode based on sulfur-doped microporous carbon (S-MC) shows a high surface area, a spherical construction, numerous carbon chain defects, and microporous structures, which are the key factors of the interactive signaling transducer, fast response, and active interfacial surfaces. The intrinsic features of S-MC with different %S-doping (S-MC-1, and S-MC-2) through the sp2-carbon chain create abundant catalytic active sites, facilitate molecular diffusion through the microporous structure, promote strong binding with the targeted molecules, and induce interactions at electrolyte-electrode interfaces. The S-MC-1 provides selective signaling in a tertiary mixture of DA, ascorbic acid (AA), and uric acid (UA) with a high sensitivity and a wide linear range of 0.01-5, 10-4000, and 1-2000 µM, respectively. The detection limits were set at 3 nM, 1.26 µM, and 0.23 µM for DA, AA, and UA respectively. The S-MC-1 demonstrated a selective screening of DA released from PC12 cells under a K+ ion- stimulator with high sensitivity and promoted high biocompatibility, low cytotoxicity, high stability, and reliable reproducibility (%RSD ranged from 1 to 2.7). Our findings indicated that the S-MC-1 can be utilized as an in-vitro model for simultaneously monitoring extracellular-DA secreted from living cells and sensing mono-bioactive molecules in biological samples.

Low Incidence of Osteonecrosis in Childhood Acute Lymphoblastic Leukemia Treated With ALL-97 and ALL-02 Study of Japan Association of Childhood Leukemia Study Group.

Purpose Osteonecrosis (ON) is a serious complication of the treatment of childhood acute lymphoblastic leukemia (ALL); however, data relating to ON in Asian pediatric patients with ALL are scarce. Therefore, we performed a retrospective analysis of cohorts of Japanese patients with ALL to clarify the incidence, clinical characteristics, and risk factors of ON. Patients and Methods The incidence and characteristics of ON were determined in patients with ALL (n = 1,662) enrolled in two studies from the Japan Association of Childhood Leukemia Study (JACLS) group (n = 635 and n = 1,027 patients treated with the ALL-97 and ALL-02 protocols, respectively). Results In total, 24 of 1,662 patients suffered from ON, of which 12 of 635 and 12 of 1,027 patients were treated with the ALL-97 and the ALL-02 protocol, respectively. Of the 24 patients, 23 were older than 10 years. In multivariate analysis, age (≥ 10 years) was the sole significant risk factor for ON ( P < .001). Separate evaluation of patients ≥ 10 years of age indicated a 5-year cumulative incidence of ON of 7.2% (95% CI, 4.0% to 12.6%) and 5.9% (95% CI, 3.3% to 10.4%) in the ALL-97 and the ALL-02 protocol, respectively, which was lower than reported previously, despite an administration of dexamethasone (DEX) similar to that in comparable studies; however, concomitant administration of DEX and l-asparaginase was reduced in the JACLS protocols. Conclusion We identified a low frequency of ON in the JACLS ALL-97 and ALL-02 studies. Although the sole risk factor for ON was age (≥ 10 years), even among high-risk patients, ON incidence was significantly lower than that reported in previous studies. These results suggest that, not only the total amount of DEX, but also how DEX and l-asparaginase are administered, which affects the clearance of DEX, may be associated with ON incidence in patients with ALL.

Pulmonary and pleural metastasis of intracranial anaplastic meningioma in a 3-year-old boy: A case report.

In adults, meningiomas occasionally display aggressive behavior and may occasionally metastasize. By contrast, pediatric meningiomas are rare, and there is limited information regarding their clinical characteristics, treatment and prognosis. We herein report the case of a 3-year-old boy with anaplastic meningioma with a history of local recurrence and late pulmonary metastasis. At diagnosis, a 70-mm mass lesion in was identified in the right frontal lobe, with intratumoral hemorrhage. The tumor was attached to the falx cerebri and was completely resected. The histological diagnosis was anaplastic meningioma, World Health Organization grade III. Two months after the surgery, the meningioma recurred at the same site. Although the patient received radiotherapy after a second operation, the tumor metastasized to the lung and pleura 8 months after the initial operation. The metastasis was resistant to treatment, even after gross total resection, and the effectiveness of further radiotherapy was limited. The patient succumbed to the disease 1 year and 4 months after the initial diagnosis. The findings of the present case and a review of the relevant literature suggest that recurrence and metastasis of meningiomas are difficult to predict. Therefore, such patients should be carefully monitored throughout the follow-up period.

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.

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.

Calcium ions rescue human lung epithelial cells from the toxicity of zinc oxide nanoparticles.

Contradictory results have been reported for in vitro evaluations of whether zinc oxide nanoparticles (ZnO NPs) are cytotoxic. Though there have been reports of ZnO NPs cytotoxicity due to Zn ions released from the nanoparticles, there have also been reports concluding that Zn ions are not cytotoxic. This inconsistency is mostly attributed to the types of cells used. In this research, we investigated the difference in the level of ZnO NPs cytotoxicity due to culturing conditions. The sensitivity of human lung epithelial cells to ZnO NPs cytotoxicity differed depending on the dispersing medium, physiological state of the cells resulting from their growth stage, and composition of the medium. Further, with regard to the toxicity of ZnO NPs, NPs internalized into cells had a greater cytotoxic effect than Zn ions released from ZnO NPs. Instead of inducing cell death, ZnO NPs internalized into cells slowed the rate of cell proliferation. Furthermore, the cytotoxicity of ZnO NPs depended greatly on the concentration of calcium ions (Ca2+) in the medium. When the concentration of Ca2+ was low, the cytotoxicity of ZnO NPs increased markedly. However, the toxicity of ZnO NPs was mitigated by the addition of CaCl2 to the medium. Global gene expression analysis revealed that Ca2+ -induced upregulation of cell cycle functions could be attributable to the mitigation of ZnO NP toxicity by Ca2+.

Vortex-aligned fullerene nanowhiskers as a scaffold for orienting cell growth.

A versatile method for the rapid fabrication of aligned fullerene C60 nanowhiskers (C60NWs) at the air-water interface is presented. This method is based on the vortex motion of a subphase (water), which directs floating C60NWs to align on the water surface according to the direction of rotational flow. Aligned C60NWs could be transferred onto many different flat substrates, and, in this case, aligned C60NWs on glass substrates were employed as a scaffold for cell culture. Bone forming human osteoblast MG63 cells adhered well to the C60NWs, and their growth was found to be oriented with the axis of the aligned C60NWs. Cells grown on aligned C60NWs were more highly oriented with the axis of alignment than when grown on randomly oriented nanowhiskers. A study of cell proliferation on the C60NWs revealed their low toxicity, indicating their potential for use in biomedical applications.

Feature integration in the mapping of multi-attribute visual stimuli to responses.

In the human visual system, different attributes of an object, such as shape and color, are separately processed in different modules and then integrated to elicit a specific response. In this process, different attributes are thought to be temporarily "bound" together by focusing attention on the object; however, how such binding contributes to stimulus-response mapping remains unclear. Here we report that learning and performance of stimulus-response tasks was more difficult when three attributes of the stimulus determined the correct response than when two attributes did. We also found that spatially separated presentations of attributes considerably complicated the task, although they did not markedly affect target detection. These results are consistent with a paired-attribute model in which bound feature pairs, rather than object representations, are associated with responses by learning. This suggests that attention does not bind three or more attributes into a unitary object representation, and long-term learning is required for their integration.