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To realize highly sensitive immunoassays, high optical density probes conjugated with antibodies for target antigens have been demanded in order to increase the visibility of antigen–antibody complex formation. We herein demonstrate the development of an immunoassay system using magnetic and fluorescent Janus particles as probes in conjunction with an antibody-immobilized microfluidic device. The concentration of the detection limit at which there was a significant difference between SARS-CoV-2 and human coronavirus 229E antigens was 3.1 ng/mL, and the standard deviation of the signal was less than 5%. The immunofluorescent probe and immunoassay system developed in this study are expected to be applicable not only to SARS-CoV-2 but also to the quantitative measurement of various other disease marker proteins and biomolecules.

We observed a stellar occultation by (3200) Phaethon, which occurred in western Japan on 2021 October 3 (UTC). This observation was requested by the DESTINY+mission team, which plans to conduct a flyby of asteroid Phaethon in 2028. Overall, this research effort contributes towards a large-scale observation campaign with a total of 72 observers observing from western Japan to southern Korea. 36 stations were established, and stellar occultation by the asteroid Phaethon was detected in 18 of them. This is the first time that this many multiple chord observations have been made for such a small asteroid (it has a diameter of 5–6 km). Observational reductions show that the apparent cross-section of Phaethon at the time of the occultation could be approximated using an ellipse with a major diameter of 6.12 ± 0.07 km and a minor diameter of 4.14 ± 0.07 km, and a position angle of 117.°4 ± 1.°5. As can be seen from the small error bars of the fitted ellipse, we have succeeded in estimating the shape and size of the asteroid with an extremely high degree of accuracy. Our observation results, together with other observations, will be used to create a 3D model of Phaethon and to improve its orbit. The instruments that we used for this observation are commonly used by many amateur astronomers in Japan for occultation observations and are not difficult to obtain. This paper describes the method and results of our observations using a CMOS camera and a GPS module, so that many people can participate in occultation observations in the future.

We have designed, synthesized, and evaluated a new class of potent HIV-1 protease inhibitors with novel bicyclic oxazolidinone derivatives as the P2 ligand. We have developed an enantioselective synthesis of these bicyclic oxazolidinones utilizing a key o-iodoxybenzoic acid mediated cyclization. Several inhibitors displayed good to excellent activity toward HIV-1 protease and significant antiviral activity in MT-4 cells. Compound 4khas shown an enzyme Kiof 40 pM and antiviral IC50of 31 nM. Inhibitors 4kand 4lwere evaluated against a panel of highly resistant multidrug-resistant HIV-1 variants, and their fold-changes in antiviral activity were similar to those observed with darunavir. Additionally, two X-ray crystal structures of the related inhibitors 4aand 4ebound to HIV-1 protease were determined at 1.22 and 1.30 Å resolution, respectively, and revealed important interactions in the active site that have not yet been explored.

The change of the dielectric constants and thicknesses has been measured in-situ on alkyl-urethane-substituted polydiacetylene (PDA) films grown on a silver (Ag) film during a photopolymerization and photochromic change by using the attenuated total reflection geometry. Photopolymerization and photochromic change in the vacuum-deposited PDA films from the monomer to the blue, the red, and the bleached forms, have been readily achieved by ultraviolet light irradiation with a D2lamp. Surprisingly, the values of the dielectric constant of the thin PDA films deposited on the Ag film were found to be comparable with those of oriented films. The dielectric constants of PDA films grown on a BK7 glass have been measured by using a normal-reflection method in the wavelength region from 400 to 1400 nm at room temperature.

Human adenovirus type 19 (HAdV-19) is a major cause of the epidemic keratoconjunctivitis. Outbreaks of keratoconjunctivitis are problematic to human health, especially for infants, the elderly, and immunocompromised individuals. However, the development of anti-HAdV drugs has been hampered by inconvenient screening systems; therefore, development of a simple screening method is highly desirable. In this study, we identified that HAdV-19 can infect a human lymphoid cell line transformed with human T-cell leukemia virus (MT-2 cells). MT-2 cells supported HAdV-19 replication and showed apparent cytopathic effects within five days post-infection. Using a thiazolyl blue tetrazolium bromide (MTT)-based colorimetric assay on MT-2 cells, we were able to detect the anti-HAdV-19 activities of previously reported nucleoside/tide compounds, including (S)-1–(3-hydroxy-2-phosphonylmethoxypropyl)cytosine (cidofovir), 2′,3′-dideoxycytidine (zalcitabine) and 3′-deoxy-3′-fluorothymidine (trifluridine). Compared with previous methods, this system represents a more simple and rapid method to screen anti-HAdV-19 agents.

Background and Objectives: Although cholangiolocellular carcinoma is considered a combined hepatocellular and cholangiocarcinoma, we feel that this classification is not appropriate. Therefore, we compared the diagnostic imaging findings, surgical prognosis, and pathological features of cholangiolocellular carcinoma with those of other combined hepatocellular and cholangiocarcinoma subtypes, hepatocellular carcinoma, and cholangiocarcinoma.Methods: The study patients included 7 with classical type combined hepatocellular and cholangiocarcinoma; 8 with stem cell feature, intermediate type combined hepatocellular and cholangiocarcinoma; 13 with cholangiolocellular carcinoma; 58 with cholangiocarcinoma; and 359 with hepatocellular carcinoma. All patients underwent hepatectomy or living-related donor liver transplantation from 2001 to 2014.Results: cholangiolocellular carcinoma could be distinguished from hepatocellular carcinom, other combined hepatocellular and cholangiocarcinoma subtypes, and cholangiocarcinoma by the presence of intratumoral Glisson’s pedicle, hepatic vein penetration, and tumor-staining pattern on angiography-assisted CT. Cholangiolocellular carcinoma was associated with a significantly lower SUV-max than that of cholangiocarcinoma on FDG-PET. Hepatocellular carcinoma, classical type, and cholangiolocellular carcinoma had significantly better prognoses than stem cell feature, intermediate type and cholangiocarcinoma. A cholangiocarcinoma component was detected in cholangiolocellular carcinoma that progressed to the hepatic hilum, and the cholangiocarcinoma component was found in perineural invasion and lymph node metastases.Conclusions: From the viewpoint of surgeon, cholangiolocellular carcinoma should be classified as a good-prognosis subtype of biliary tract carcinoma because of its tendency to differentiate into cholangiocarcinoma during its progression, and its distinctive imaging and few recurrence rates different from other combined hepatocellular and cholangiocarcinoma subtypes.

Combination antiretroviral therapy has achieved dramatic reductions in the mortality and morbidity in people with HIV-1 infection. Darunavir (DRV) represents a most efficacious and well-tolerated protease inhibitor (PI) with a high genetic barrier to the emergence of drug-resistant HIV-1. However, highly DRV-resistant variants have been reported in patients receiving long-term DRV-containing regimens.

There is currently no specific therapeutics for the HIV-1-related central nervous system (CNS) complications. Here we report that three newly designed CNS-targeting HIV-1 protease inhibitors (PIs), GRL-083-13, GRL-084-13, and GRL-087-13, which contain a P1-3,5-bis-fluorophenyl or P1-para-monofluorophenyl ring, and P2-bis-tetrahydrofuran (bis-THF) or P2-tetrahydropyrano-tetrahydrofuran (Tp-THF), with a sulfonamide isostere, are highly active against wild-type HIV-1 strains and primary clinical isolates (50% effective concentration [EC50], 0.0002 to ∼0.003 μM), with minimal cytotoxicity.

We generated two novel nonpeptidic HIV-1 protease inhibitors (PIs), GRL-001-15 and GRL-003-15, which contain unique crown-like tetrahydropyranofuran (Crn-THF) and P2′-cyclopropyl-aminobenzothiazole (Cp-Abt) moieties as P2 and P2′ ligands, respectively. GRL-001-15 and GRL-003-15 have meta-monofluorophenyl and para-monofluorophenyl at the P1 site, respectively, exert highly potent activity against wild-type HIV-1 with 50% effective concentrations (EC50s) of 57 and 50 pM, respectively, and have favorable cytotoxicity profiles with 50% cytotoxic concentrations (CC50s) of 38 and 11 μM, respectively.

ABSTRACTDarunavir (DRV) has bimodal activity against HIV-1 protease, enzymatic inhibition and protease dimerization inhibition, and has an extremely high genetic barrier against development of drug resistance. We previously generated a highly DRV-resistant HIV-1 variant (HIVDRVRP51). We also reported that four amino acid substitutions (V32I, L33F, I54M, and I84V) identified in the protease of HIVDRVRP51are largely responsible for its high-level resistance to DRV. Here, we attempted to elucidate the role of each of the four amino acid substitutions in the development of DRV resistance. We found that V32I is a key substitution, which rarely occurs, but once it occurs, it predisposes HIV-1 to develop high-level DRV resistance. When two infectious recombinant HIV-1 clones carrying I54M and I84V (rHIVI54Mand rHIVI84V, respectively) were selected in the presence of DRV, V32I emerged, and the virus rapidly developed high-level DRV resistance. rHIVV32Ialso developed high-level DRV resistance. However, wild-type HIVNL4-3(rHIVWT) failed to acquire V32I and did not develop DRV resistance. Compared to rHIVWT, rHIVV32Iwas highly susceptible to DRV and had significantly reduced fitness, explaining why V32I did not emerge upon selection of rHIVWTwith DRV. When the only substitution is at residue 32, structural analysis revealed much stronger van der Waals interactions between DRV and I-32 than between DRV and V-32. These results suggest that V32I is a critical amino acid substitution in multiple pathways toward HIV-1’s DRV resistance development and elucidate, at least in part, a mechanism of DRV’s high genetic barrier to development of drug resistance. The results also show that attention should be paid to the initiation or continuation of DRV-containing regimens in people with HIV-1 containing the V32I substitution.IMPORTANCEDarunavir (DRV) is the only protease inhibitor (PI) recommended as a first-line therapeutic and represents the most widely used PI for treating HIV-1-infected individuals. DRV possesses a high genetic barrier to development of HIV-1’s drug resistance. However, the mechanism(s) of the DRV’s high genetic barrier remains unclear. Here, we show that the preexistence of certain single amino acid substitutions such as V32I, I54M, A71V, and I84V in HIV-1 protease facilitates the development of high-level DRV resistance. Interestingly, all in vitro-selected highly DRV-resistant HIV-1 variants acquired V32I but never emerged in wild-type HIV (HIVWT), and V32I itself rendered HIV-1 more sensitive to DRV and reduced viral fitness compared to HIVWT, strongly suggesting that the emergence of V32I plays a critical role in the development of HIV-1’s resistance to DRV. Our results would be of benefit in the treatment of HIV-1-infected patients receiving DRV-containing regimens.

ABSTRACTWe identified four novel nonpeptidic human immunodeficiency virus type 1 (HIV-1) protease inhibitors (PIs), GRL-078, -079, -077, and -058, containing an alkylamine at the C-5 position of P2 tetrahydropyrano-tetrahydrofuran (Tp-THF) and a P2′ cyclopropyl (Cp) (or isopropyl)-aminobenzothiazole (Abt) moiety. Their 50% effective concentrations (EC50s) were 2.5 to 30 nM against wild-type HIV-1NL4-3, 0.3 to 6.7 nM against HIV-2EHO, and 0.9 to 90 nM against laboratory-selected PI-resistant HIV-1 and clinical HIV-1 variants resistant to multiple FDA-approved PIs (HIVMDR). GRL-078, -079, -077, and -058 also effectively blocked the replication of HIV-1 variants highly resistant to darunavir (DRV) (HIVDRVrp51), with EC50s of 38, 62, 61, and 90 nM, respectively, while four FDA-approved PIs examined (amprenavir, atazanavir, lopinavir [LPV], and DRV) had virtually no activity (EC50s of >1,000 nM) against HIVDRVrp51. Structurally, GRL-078, -079, and -058 form strong hydrogen bond interactions between Tp-THF modified at C-5 and Asp29/Asp30/Gly48 of wild-type protease, while the P2′ Cp-Abt group forms strong hydrogen bonds with Asp30′. The Tp-THF and Cp-Abt moieties also have good nonpolar interactions with protease residues located in the flap region. For selection with LPV and DRV by use of a mixture of 11 HIVMDRstrains (HIV11MIX), HIV11MIXbecame highly resistant to LPV and DRV over 13 to 32 and 32 to 41 weeks, respectively. However, for selection with GRL-079 and GRL-058, HIV11MIXfailed to replicate at >0.08 μM and >0.2 μM, respectively. Thermal stability results supported the highly favorable anti-HIV-1 potency of GRL-079 as well as other PIs. The present data strongly suggest that the P2 Tp-THF group modified at C-5 and the P2′ Abt group contribute to the potent anti-HIV-1 profiles of the four PIs against HIV-1NL4-3and a wide spectrum of HIVMDRstrains.

Fluorescent biosensors are expected to be utilized for a wide range of applications, such as diagnostic and pharmacological tools. However, fluorescent biosensors synthesized by the direct coupling between the ligand bindings and optical signalling events are not always guaranteed to execute the expected optical signals. Our strategy of the stepwise molding using a framework of ribonucleopeptide (RNP) provides fluorescent biosensors with a variety of binding and signaltransducing characteristics for small molecules. RNPbased fluorescent sensors were created with a combination of in vitro selection method and a successive modification of the peptide of RNP with a fluorophore. The RNP containing the fluorophore labeled peptide subunit showed a remarkable fluorescence emission change upon binding to biologically active amines.

Fluorescent biosensors are crucial tools to facilitate sensitive detection of small molecules. However, construction of fluorescent biosensors with desired characteristics, that is, detection wavelengths and concentration ranges for ligand detection, from macromolecular receptors is not a straightforward task. Previously, we reported a modular strategy (Hagihara, et al., J. Am. Chem. Soc., 2006, 128, 12932) for constructing fluorescent ATP sensors from ribonucleopeptide (RNP) complexes. These RNP sensors had a variety of emission wavelengths and/or responding ligand concentration ranges. The modular strategy allows choosing a fluorescent RNP sensor with appropriate optical properties, but does not allow us to design optimal properties of fluorescent RNP sensors. Here we report a design strategy to optimize the response of fluorescent RNP sensors based on the secondary structural analyses of ATP-binding RNPs. We analyzed the RNA secondary structure of RNP sensors and examined the relationship between the secondary structure and fluorescent characteristics. We have discovered that fluorescent ATP sensors with high value of I/I0 ratio tend to contain interior loop and to record low fluorescence intensity in the absence of ATP. Indeed, ATP sensors, which was inserted sequence that construct an interior loop of ATP sensor with large fluorescence intensity change, were induced increase of fluorescent intensity change. This result suggests that use of the secondary structural elements would allow a rational functional design of RNP.

Biliary complications have great importance for liver transplant recipients because of affecting long-term prognosis. In rare situations, an internal hernia of the Roux-en-Y loop cause graft injury. A 42-year-old woman with a history of living donor liver transplantation 6 years ago presented with prolonged graft injury during the past 6 months. She suddenly developed ileus of the small bowel with internal hernia through the defect of the mesentery around the Roux-en-Y limb of the hepaticojejunostomy. Emergent surgery was performed to reduce the hernia and volvulus; also the mesenteric rent was closed with interrupted suture of silk. Internal hernia of the small bowel after liver transplantation is rare but causes graft injury due to associated biliary complications and rapid deterioration of patient’s condition.