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We designed 6-dimethylamino 3-methyleneisoindolin-1-one as an environment-sensitive fluorophore, examining its applications for protein labeling. Synthesized 3-methyleneisoindolin-1-one exhibits solvatochromic fluorescence ( λ emmax ; 472 nm in 2-PrOH, 512 nm in H 2 O). A positive linear dependence between λ emmax and solvent dielectric constant (DC), as well as between Stokes shift and DC, and a negative correlation between fluorescence quantum yield and DC are observed in protic solvents. These properties are similar to those of the oxygen isosteric fluorophore, 4-dimethylaminophthalimide, a slovatochromic fluorophore utilized for labeling oligodeoxynucleotides (ODNs) and peptides. Notably, fluorescence intensity of 3-methyleneisoindolin-1-one is higher than the phthalimide in protic solvents used in this study. The 3-methyleneisoindolin-1-one demonstrated the higher stability in pH 8 solution than in pH 6 solution in contrast to the stability profile of the phthalimide, which was stable at pH 6 but was hydrolyzed at pH 8. We also synthesized an o -keto benzaldehyde derivative that converts a primary amine to 6-dimethylamino 3-methyleneisoindolin-1-one under biocompatible conditions and introduced it into ODNs for turn-on fluorescent protein labeling. The synthesized ODN with a protein-binding sequence of Escherichia coli DnaA was employed to modify the DNA-binding domain of DnaA, and the fluorescent properties of the modified protein were investigated.

In middle to late 2023, a sublineage of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron XBB, EG.5.1 (a progeny of XBB.1.9.2), is spreading rapidly around the world. We performed multiscale investigations, including phylogenetic analysis, epidemic dynamics modeling, infection experiments using pseudoviruses, clinical isolates, and recombinant viruses in cell cultures and experimental animals, and the use of human sera and antiviral compounds, to reveal the virological features of the newly emerging EG.5.1 variant. Our phylogenetic analysis and epidemic dynamics modeling suggested that two hallmark substitutions of EG.5.1, S:F456L and ORF9b:I5T are critical to its increased viral fitness. Experimental investigations on the growth kinetics, sensitivity to clinically available antivirals, fusogenicity, and pathogenicity of EG.5.1 suggested that the virological features of EG.5.1 are comparable to those of XBB.1.5. However, cryo-electron microscopy revealed structural differences between the spike proteins of EG.5.1 and XBB.1.5. We further assessed the impact of ORF9b:I5T on viral features, but it was almost negligible in our experimental setup. Our multiscale investigations provide knowledge for understanding the evolutionary traits of newly emerging pathogenic viruses, including EG.5.1, in the human population., (© 2024 The Author(s). Microbiology and Immunology published by The Societies and John Wiley & Sons Australia, Ltd.)

Circulation of SARS-CoV-2 Omicron XBB has resulted in the emergence of XBB.1.5, a new Variant of Interest. Our phylogenetic analysis suggests that XBB.1.5 evolved from XBB.1 by acquiring the S486P spike (S) mutation, subsequent to the acquisition of a nonsense mutation in ORF8. Neutralization assays showed similar abilities of immune escape between XBB.1.5 and XBB.1. We determine the structural basis for the interaction between human ACE2 and the S protein of XBB.1.5, showing similar overall structures between the S proteins of XBB.1 and XBB.1.5. We provide the intrinsic pathogenicity of XBB.1 and XBB.1.5 in hamsters. Importantly, we find that the ORF8 nonsense mutation of XBB.1.5 resulted in impairment of MHC suppression. In vivo experiments using recombinant viruses reveal that the XBB.1.5 mutations are involved with reduced virulence of XBB.1.5. Together, our study identifies the two viral functions defined the difference between XBB.1 and XBB.1.5., (© 2024. The Author(s).)

In late 2022, SARS-CoV-2 Omicron subvariants have become highly diversified, and XBB is spreading rapidly around the world. Our phylogenetic analyses suggested that XBB emerged through the recombination of two cocirculating BA.2 lineages, BJ.1 and BM.1.1.1 (a progeny of BA.2.75), during the summer of 2022. XBB.1 is the variant most profoundly resistant to BA.2/5 breakthrough infection sera to date and is more fusogenic than BA.2.75. The recombination breakpoint is located in the receptor-binding domain of spike, and each region of the recombinant spike confers immune evasion and increases fusogenicity. We further provide the structural basis for the interaction between XBB.1 spike and human ACE2. Finally, the intrinsic pathogenicity of XBB.1 in male hamsters is comparable to or even lower than that of BA.2.75. Our multiscale investigation provides evidence suggesting that XBB is the first observed SARS-CoV-2 variant to increase its fitness through recombination rather than substitutions., (© 2023. The Author(s).)

In late 2022, various Omicron subvariants emerged and cocirculated worldwide. These variants convergently acquired amino acid substitutions at critical residues in the spike protein, including residues R346, K444, L452, N460, and F486. Here, we characterize the convergent evolution of Omicron subvariants and the properties of one recent lineage of concern, BQ.1.1. Our phylogenetic analysis suggests that these five substitutions are recurrently acquired, particularly in younger Omicron lineages. Epidemic dynamics modelling suggests that the five substitutions increase viral fitness, and a large proportion of the fitness variation within Omicron lineages can be explained by these substitutions. Compared to BA.5, BQ.1.1 evades breakthrough BA.2 and BA.5 infection sera more efficiently, as demonstrated by neutralization assays. The pathogenicity of BQ.1.1 in hamsters is lower than that of BA.5. Our multiscale investigations illuminate the evolutionary rules governing the convergent evolution for known Omicron lineages as of 2022., (© 2023. The Author(s).)

The SARS-CoV-2 Omicron BA.2.75 variant emerged in May 2022. BA.2.75 is a BA.2 descendant but is phylogenetically distinct from BA.5, the currently predominant BA.2 descendant. Here, we show that BA.2.75 has a greater effective reproduction number and different immunogenicity profile than BA.5. We determined the sensitivity of BA.2.75 to vaccinee and convalescent sera as well as a panel of clinically available antiviral drugs and antibodies. Antiviral drugs largely retained potency, but antibody sensitivity varied depending on several key BA.2.75-specific substitutions. The BA.2.75 spike exhibited a profoundly higher affinity for its human receptor, ACE2. Additionally, the fusogenicity, growth efficiency in human alveolar epithelial cells, and intrinsic pathogenicity in hamsters of BA.2.75 were greater than those of BA.2. Our multilevel investigations suggest that BA.2.75 acquired virological properties independent of BA.5, and the potential risk of BA.2.75 to global health is greater than that of BA.5., Competing Interests: Declaration of interests Y.Y. and T.N. are founders and shareholders of HiLung, Inc. Y.Y. is a co-inventor of patents (PCT/JP2016/057254; “method for inducing differentiation of alveolar epithelial cells,” PCT/JP2016/059786, “method of producing airway epithelial cells”)., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

After the global spread of the SARS-CoV-2 Omicron BA.2, some BA.2 subvariants, including BA.2.9.1, BA.2.11, BA.2.12.1, BA.4, and BA.5, emerged in multiple countries. Our statistical analysis showed that the effective reproduction numbers of these BA.2 subvariants are greater than that of the original BA.2. Neutralization experiments revealed that the immunity induced by BA.1/2 infections is less effective against BA.4/5. Cell culture experiments showed that BA.2.12.1 and BA.4/5 replicate more efficiently in human alveolar epithelial cells than BA.2, and particularly, BA.4/5 is more fusogenic than BA.2. We further provided the structure of the BA.4/5 spike receptor-binding domain that binds to human ACE2 and considered how the substitutions in the BA.4/5 spike play roles in ACE2 binding and immune evasion. Moreover, experiments using hamsters suggested that BA.4/5 is more pathogenic than BA.2. Our multiscale investigations suggest that the risk of BA.2 subvariants, particularly BA.4/5, to global health is greater than that of original BA.2., Competing Interests: Declaration of interests Y.Y. and T.N. are founders and shareholders of HiLung, Inc. J.K. is an employee of HiLung, Inc. Y.Y. is a co-inventor of patents (PCT/JP2016/057254; “Method for inducing differentiation of alveolar epithelial cells,” PCT/JP2016/059786, “Method of producing airway epithelial cells”)., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

We have prepared a monoclonal antibody against notoginsenoside R1, a primary active constituent of Sanqi ginseng (roots of Panax notoginseng). The monoclonal antibody was raised by immunizing BALB/c male mice with notoginsenoside R1-bovine albumin conjugates following cell fusion via electroporation. This method has been shown to be very effective for producing hybridomas with excellent antibody prevalence following cell fusion of their splenocytes with cells of the myeloma cell line SP2/0. Of all the hybridomas secreting a monoclonal antibody against notoginsenoside R1, only the 1A1P cell line produces a highly specific antibody to this compound. Surprisingly, the cross-reactivity of this monoclonal antibody for ginsenoside Rg1 and ginsenoside Re, derivatives of protopanaxatriol, was below 1.02% in a competitive immunoassay. Based on this characteristic of the monoclonal antibody, an indirect competitive ELISA (range of measurement 0.56-9 µg/mL) was established and applied as a quality control method. In conclusion, the developed immunoassay was easy to handle and reliable for the analysis of notoginsenoside R1 in Sanqi ginseng products without requiring a pretreatment., (Georg Thieme Verlag KG Stuttgart · New York.)

Salvinorin A (1), the main active constituent in Salvia divinorum, is a highly selective kappa-opioid receptor agonist with hallucinogenic effects, which is regulated in several countries. In the present study, a monoclonal antibody (mAb) against 1 was prepared, and an indirect competitive enzyme-linked immunosorbent assay (icELISA) system was developed for the detection of salvinorins. To raise mAbs against 1, salvinorin B (2) hemisuccinate was synthesized and used to prepare the immunogen 2-bovine serum albumin conjugate. This technique was used to prepare a hybridoma cell line, 3D5, which secreted a mAb that recognized 1. The mAb was shown to have specificity for 1 and other salvinorins in cross-reactivity tests. The intra-assay calibration range by icELISA using the mAb against 1 was 0.0195-0.625 μg/mL. After validating the icELISA using intra- and interassays, a recovery experiment and analysis of several plants in the family Lamiaceae, including S. divinorum, confirmed that the analytical method based on ELISA is not only simple but also precise, accurate, sensitive, and sufficiently reliable. The results indicate that icELISA is a useful tool in the identification of S. divinorum.

Paclitaxel, the major active component of the yew tree, is used as an important anti-cancer agent. To obtain the monoclonal antibody (MAb) against paclitaxel for paclitaxel determination using immunoassay, 7-xylosyltaxol was conjugated to the carrier protein bovine serum albumin (BSA) to construct the immunogen, and the ratio of hapten in XylTax-BSA conjugate was determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. After immunization of mice with this conjugate, hybridomas secreting MAbs against paclitaxel were obtained by fusing the splenocytes with the mouse myeloma cell line SP2/0. After hybridoma screening, the anti-paclitaxel MAb 3A3 was obtained, which showed a relatively high specificity to paclitaxel (cross-reactivities against other naturally occurred taxanes: 7-xylosyltaxol, 31.8%; cephalomannine, 6.17%; baccatin III, 10-deacetyl-baccatin III, 1-hydroxybaccatin I, 13-acetyl-9-dihydrobaccatin III and 1-acetoxyl-5-deacetyl-baccatin I, <0.11%). Using the MAb 3A3, we established an indirect competitive enzyme-linked immunosorbent assay (icELISA) for paclitaxel determination with a detection range of 0.098-312.5 μg ml(-1). Determination of paclitaxel contents in various yew tree samples with this icELISA resulted in recovery rates ranging from 92 to 94.8%, and intra- and inter-assay variations of 3.6 and 4.7%, respectively. This icELISA provides a valuable method of paclitaxel determination for various purposes.

Two different recombinant antibodies, a single-chain variable fragment (scFv) and an antigen-binding fragment (Fab), were prepared against artemisinin (AM) and artesunate (AS) and were developed for use in an enzyme-linked immunosorbent assay (ELISA). The recombinant antibodies, which were derived from a single monoclonal antibody against AM and AS (mAb 1C1) prepared by us, were expressed by Escherichia coli cells and their reactivity and specificity were characterized. As a result, to obtain sufficient signal in indirect ELISA, a much greater amount of a first antibody was needed in the use of scFv due to the differences of the secondary antibody and conformational stability. Therefore, we focused on the development of the recombinant Fab antibodies and applied it to indirect competitive ELISA. The specificity of the Fab was similar to that of mAb 1C1 in that it showed specific reactivity toward AM and AS only. The sensitivity of the icELISA (0.16 μg/mL to 40 μg/mL for AM and 8.0 ng/mL to 60 ng/mL for AS) was sufficient for analysis of antimalarial drugs, and its utility for quality control of analysis of Artemisia spp. was validated. The Fab expression and refolding systems provided a good yield of high-quality antibodies. The recombinant antibody against AM and AS provides an essential component of an economically attractive immunoassay and will be useful in other immunochemical applications for the analysis and purification of antimalarial drugs.

A single-chain variable fragment antibody against herbicide, 2,4-dichlorophenoxyacetic acid (2,4-D-scFv) has been successfully expressed in the hemolymph of silkworm larvae using a rapid Bombyx mori nucleopolyhedrovirus (BmNPV) bacmid DNA system. Variable heavy- and light-chain domains were cloned directly from the cDNA of the hybridoma cell line 2C4 and assembled together with flexible peptide linker (Gly(4)Ser)(3) between two domains. The yield of functional 2,4-D-scFv after purification was 640 μg per 30 ml of hemolymph, which is equivalent to 21.3 mg per liter of hemolymph. The characterization of 2,4-D-scFv using an indirect competitive enzyme-linked immunosorbent assay (icELISA) revealed that it has wide cross-reactivities against 2,4,5-trichlorophenoxyacetic acid (65.5%), 2,4-dichlorophenol (47.9%), and 2,4-dichlorobenzoic acid (26.0%), making it possible to apply 2,4-D-scFv to icELISA for detecting/determining 2,4-D and its metabolites. Judging from its cost and time requirements and its ease of handling, this BmNPV bacmid DNA expression system is more useful for expressing functional scFv than bacterial systems, which frequently require costly and time-consuming refolding.

A fluorescent single-domain antibody (fluobody), a chimera of a green fluorescent protein (AcGFP) with a single chain variable fragment antibody (scFv), against plumbagin (5-hydorxy-2-methyl-1,4-naphthoquinone; PL) was successfully expressed in the hemolymph of silkworm larvae using a Bombyx mori nucleopolyhedrovirus (BmNPV) bacmid DNA system to develop a rapid, simple, and sensitive fluorescence-linked immunosorbent assay (FLISA). In this study, two kinds of fluobody, in which the PL-scFv was fused at the N-terminus (N-fluobody) or C-terminus of AcGFP (C-fluobody), were expressed in silkworm larvae for comparative purposes. Interestingly, both fluobodies expressed in the BmNPV bacmid DNA system retained both of their original functions as an AcGFP and a PL-scFv, although the functions of the N-fluobody were found to be inferior to those of C-fluobody when they were expressed in Escherichia coli. Moreover, an improvement in the limit of quantification for PL measurement was observed in FLISA (24 ng mL(-1)) compared with conventional ELISA (0.2 µg mL(-1)). Since both the C-fluobody and N-fluobody are useful probes for FLISA and the time-, cost-consuming refolding step required in the conventional bacterial expression system can be avoided when they are expressed in the BmNPV bacmid DNA system, the silkworm expression system is useful for expressing fluobodies when developing FLISA.