Selection and characterization of aptamers targeting the Vif-CBFß-ELOB-ELOC-CUL5 complex.

The viral infectivity factor (Vif) of human immunodeficiency virus 1 forms a complex with host proteins, designated as Vif-CBFß-ELOB-ELOC-CUL5 (VßBCC), initiating the ubiquitination and subsequent proteasomal degradation of the human antiviral protein APOBEC3G (A3G), thereby negating its antiviral function. While recent cryo-electron microscopy (cryo-EM) studies have implicated RNA molecules in the Vif-A3G interaction that leads to A3G ubiquitination, our findings indicated that the VßBCC complex can also directly impede A3G-mediated DNA deamination, bypassing the proteasomal degradation pathway. Employing the Systematic Evolution of Ligands by EXponential enrichment (SELEX) method, we have identified RNA aptamers with high affinity for the VßBCC complex. These aptamers not only bind to the VßBCC complex but also reinstate A3G's DNA deamination activity by inhibiting the complex's function. Moreover, we delineated the sequences and secondary structures of these aptamers, providing insights into the mechanistic aspects of A3G inhibition by the VßBCC complex. Analysis using selected aptamers will enhance our understanding of the inhibition of A3G by the VßBCC complex, offering potential avenues for therapeutic intervention.

Recombinant mycobacterial DNA-binding protein 1 with post-translational modifications boosts IFN-gamma production from BCG-vaccinated individuals' blood cells in combination with CpG-DNA.

Tuberculosis remains a large health threat, despite the availability of the tuberculosis vaccine, BCG. As BCG efficacy gradually decreases from adolescence, BCG-Prime and antigen-booster may be an efficient strategy to confer vaccine efficacy. Mycobacterial DNA-binding protein 1 (MDP1, namely Rv2986c, hupB or HU) is a major Mycobacterium tuberculosis protein that induces vaccine-efficacy by co-administration with CpG DNA. To produce MDP1 for booster-vaccine use, we have created recombinant MDP1 produced in both Escherichia coli (eMDP1) and Mycolicibacterium smegmatis (mMDP1), an avirulent rapid-growing mycobacteria. We tested their immunogenicity by checking interferon (IFN)-gamma production by stimulated peripheral blood cells derived from BCG-vaccinated individuals. Similar to native M. tuberculosis MDP1, we observed that most lysin resides in the C-terminal half of mMDP1 are highly methylated. In contrast, eMDP1 had less post-translational modifications and IFN-gamma stimulation. mMDP1 stimulated the highest amount of IFN-gamma production among the examined native M. tuberculosis proteins including immunodominant MPT32 and Antigen 85 complex. MDP1-mediated IFN-gamma production was more strongly enhanced when combined with a new type of CpG DNA G9.1 than any other tested CpG DNAs. Taken together, these results suggest that the combination of mMDP1 and G9.1 possess high potential use for human booster vaccine against tuberculosis.

Fluoride Ion in Alcohols: Isopropanol vs Hexafluoroisopropanol.

The utility of alcohol as a hydrogen bonding donor is considered a providential avenue for moderating the high basicity and reactivity of the fluoride ion, typically used with large cations. However, the practicality of alcohol-fluoride systems in reactions is hampered by the limited understanding of the pertinent interactions between the OH group and F-. Therefore, this study comparatively investigates the thermal, structural, and physical properties of the CsF-2-propanol and CsF-1,1,1,3,3,3-hexafluoro-2-propanol systems to explicate the effects of the fluoroalkyl group on the interaction of alcohols and F-. The two systems exhibit vastly different phase diagrams despite the similar saturated concentrations. A combination of spectroscopic analyses, alcohol activity coefficient measurements, and theoretical calculations reveal the fluorinated alcohol system harbors the stronger OH···F- interactions between the two systems. The diffusion coefficient and ionic conductivity measurements attribute the present results to disparate states of ion association in the two systems.

Enhancing Bioethanol Production from Rice Straw through Environmentally Friendly Delignification Using Versatile Peroxidase.

Rice straw (RS), an agricultural residue rich in carbohydrates, has substantial potential for bioethanol production. However, the presence of lignin impedes access to these carbohydrates, hindering efficient carbohydrate-to-bioethanol conversion. Here, we expressed versatile peroxidase (VP), a lignin-degrading enzyme, in Pichia pastoris and used it to delignify RS at 30 °C using a membrane bioreactor that continuously discarded the degraded lignin. Klason lignin analysis revealed that VP-treatment led to 35% delignification of RS. We then investigated the delignified RS by SEC, FTIR, and SEM. The results revealed the changes of RS caused by VP-mediated delignification. Additionally, we compared the saccharification and fermentation yields between RSs treated with and without VP, VP-RS, and Ctrl-RS, respectively. This examination unveiled an improvement in glucose and bioethanol production, VP-RS exhibiting up to 1.5-fold and 1.4-fold production, respectively. These findings underscore the potential of VP for delignifying RS and enhancing bioethanol production through an eco-friendly approach.

Direct inhibition of human APOBEC3 deaminases by HIV-1 Vif independent of the proteolysis pathway.

HIV-1 Vif is known to counteract the antiviral activity of human apolipoprotein B mRNA-editing catalytic polypeptide-like (A3), a cytidine deaminase, in various ways. However, the precise mechanism behind this interaction has remained elusive. Within infected cells, Vif forms a complex called VßBCC, comprising CBFß and the components of E3 ubiquitin ligase, Elongin B, Elongin C, and Cullin5. Together with the ubiquitin-conjugating enzyme, VßBCC induces ubiquitination-mediated proteasomal degradation of A3. However, Vif exhibits additional counteractive effects. In this study, we elucidate that VßBCC inhibits deamination by A3G, A3F, and A3B independently of proteasomal degradation. Surprisingly, we discovered that this inhibition for A3G is directly attributed to the interaction between VßBCC and the C-terminal domain of A3G. Previously, it was believed that Vif did not interact with the C-terminal domain. Our findings suggest that inhibiting the interaction between VßBCC and the C-terminal domain, as well as the N-terminal domain known to be targeted for ubiquitination, of A3G may be needed to prevent counteraction by Vif.

Structural insights into the molecular mechanisms of substrate recognition and hydrolysis by feruloyl esterase from Aspergillus sydowii.

The depolymerization of lignocellulosic biomass is facilitated by feruloyl esterases (FAEs), which hydrolyze ester bonds between lignin and polysaccharides. Fungal FAEs belonging to subfamily (SF) 6 release precursors such as ferulic acid derivatives, attractive for biochemical production. Among these, Aspergillus sydowii FAE (AsFaeE), an SF6 FAE, exhibits remarkable activity across various substrates. In this study, we conducted X-ray crystallography and kinetic analysis to unravel the molecular mechanisms governing substrate recognition and catalysis by AsFaeE. AsFaeE exhibits a typical α/ß-hydrolase fold, characterized by a catalytic triad of serine, aspartate, and histidine. Comparative analysis of substrate-free, ferulic acid-bound, and sinapic acid-bound forms of AsFaeE suggests a conformational change in the loop covering the substrate-binding pocket upon binding. Notably, Pro158 and Phe159 within this loop cover the phenolic part of the substrate, forming three layers of planar rings. Our structure-based functional mutagenesis clarifies the roles of the residues involved in substrate binding and catalytic activity. Furthermore, distinct substrate-binding mechanisms between AsFaeE and other studied FAEs are identified. This investigation offers the initial structural insights into substrate recognition by SF6 FAEs, equipping us with structural knowledge that might facilitate the design of FAE variants capable of efficiently processing a wider range of substrate sizes.

NMR characterization of the structure of the intrinsically disordered region of human origin recognition complex subunit 1, hORC1, and of its interaction with G-quadruplex DNAs.

Human origin recognition complex (hORC) binds to the DNA replication origin and then initiates DNA replication. However, hORC does not exhibit DNA sequence-specificity and how hORC recognizes the replication origin on genomic DNA remains elusive. Previously, we found that hORC recognizes G-quadruplex structures potentially formed near the replication origin. Then, we showed that hORC subunit 1 (hORC1) preferentially binds to G-quadruplex DNAs using a hORC1 construct comprising residues 413 to 511 (hORC1413-511). Here, we investigate the structural characteristics of hORC1413-511 in its free and complex forms with G-quadruplex DNAs. Circular dichroism and nuclear magnetic resonance (NMR) spectroscopic studies indicated that hORC1413-511 is disordered except for a short α-helical region in both the free and complex forms. NMR chemical shift perturbation (CSP) analysis suggested that basic residues, arginines and lysines, and polar residues, serines and threonines, are involved in the G-quadruplex DNA binding. Then, this was confirmed by mutation analysis. Interestingly, CSP analysis indicated that hORC1413-511 binds to both parallel- and (3 + 1)-type G-quadruplex DNAs using the same residues, and thereby in the same manner. Our study suggests that hORC1 uses its intrinsically disordered G-quadruplex binding region to recognize parallel-type and (3 + 1)-type G-quadruplex structures at replication origin.

Enzymatic degradation of glucosaminoglucan and cellulase resistance of cellulose nanofiber coated with glucosaminoglucan.

AIMS: Enzymatic degradation of ß-1,4-linked glucose and glucosamine (glucosaminoglucan, GG), which is prepared from Thiothrix nivea and can act as a cellulose-aminating agent with a strong affinity to cellulose, was attempted. METHODS AND RESULTS: A chitosanase-secreting fungal strain was isolated as a GG-degrading microbe. GG was found to be degraded by not only chitosanases but also cellulases. Based on nuclear magnetic resonance spectroscopy, both enzymes were found to produce GlcN-Glc from GG. The cellulases also produced GlcN-Glc-GlcN-Glc as an additional final digest. Furthermore, aminated (GG-coated) cellulose nanofibers exhibited cellulase resistance. The flexibility of GG adsorbed onto a cellulose crystal was almost identical to that of cellulose, as estimated via the molecular dynamics calculations. CONCLUSIONS: The chitosanase and cellulase hydrolyzed the ß-1,4-linkage from Glc to GlcN and were expected to recognize the tetramer and hexamer units of GG depending on their final products. The cellulose nanofibers acquired cellulase resistance via amination with GG, probably because of the lower activity of cellulase to GG than cellulose.

Complex Formation of an RNA Aptamer with a Part of HIV-1 Tat through Induction of Base Triples in Living Human Cells Proven by In-Cell NMR.

An RNA aptamer that strongly binds to a target molecule has the potential to be a nucleic acid drug inside living human cells. To investigate and improve this potential, it is critical to elucidate the structure and interaction of RNA aptamers inside living cells. We examined an RNA aptamer for HIV-1 Tat (TA), which had been found to trap Tat and repress its function in living human cells. We first used in vitro NMR to examine the interaction between TA and a part of Tat containing the binding site for trans-activation response element (TAR). It was revealed that two U-A∗U base triples are formed in TA upon binding of Tat. This was assumed to be critical for strong binding. Then, TA in complex with a part of Tat was incorporated into living human cells. The presence of two U-A∗U base triples was also revealed for the complex in living human cells by in-cell NMR. Thus, the activity of TA in living human cells was rationally elucidated by in-cell NMR.

A heterodimeric hyaluronate lyase secreted by the activated sludge bacterium Haliscomenobacter hydrossis.

Haliscomenobacter hydrossis is a filamentous bacterium common in activated sludge. The bacterium was found to utilize hyaluronic acid, and hyaluronate lyase activity was detected in its culture. However, no hyaluronate lyase gene was found in the genome, suggesting the bacterium secretes a novel hyaluronate lyase. The purified enzyme exhibited two bands on SDS-PAGE and a single peak on gel filtration chromatography, suggesting a heterodimeric composition. N-terminal amino acid sequence and mass spectrometric analyses suggested that the subunits are molybdopterin-binding and [2Fe-2S]-binding subunits of a xanthine oxidase family protein. The presence of the cofactors was confirmed using spectrometric analysis. Oxidase activity was not detected, revealing that the enzyme is not an oxidase but a hyaluronate lyase. Nuclear magnetic resonance analysis of the enzymatic digest revealed that the enzyme breaks hyaluronic acid to 3-(4-deoxy-ß-d-gluc-4-enuronosyl)-N-acetyl-d-glucosamine. As hyaluronate lyases (EC 4.2.2.1) are monomeric or trimeric, the enzyme is the first heterodimeric hyaluronate lyase.

Detection of interaction between an RNA aptamer and its target compound in living human cells using 2D in-cell NMR.

We introduced an isotopically labeled RNA aptamer for HIV-1 Tat prepared by E. coli transcription into HeLa cells. We successfully recorded the first heteronuclear 2D in-cell NMR spectra, which makes it possible to study the interaction of the RNA aptamer with argininamide in living human cells with higher resolution.

Shedding light on the base-pair opening dynamics of nucleic acids in living human cells.

Base-pair opening is a fundamental property of nucleic acids that plays important roles in biological functions. However, studying the base-pair opening dynamics inside living cells has remained challenging. Here, to determine the base-pair opening kinetics inside living human cells, the exchange rate constant ([Formula: see text]) of the imino proton with the proton of solvent water involved in hairpin and G-quadruplex (GQ) structures is determined by the in-cell NMR technique. It is deduced on determination of [Formula: see text] values that at least some G-C base pairs of the hairpin structure and all G-G base-pairs of the GQ structure open more frequently in living human cells than in vitro. It is suggested that interactions with endogenous proteins could be responsible for the increase in frequency of base-pair opening. Our studies demonstrate a difference in dynamics of nucleic acids between in-cell and in vitro conditions.

Biophysical Study of the Structure, Dynamics, and Function of Nucleic Acids.

Nucleic acids have essential roles in all biological processes related to genetic information, such as replication, transcription, translation, repair, and recombination [...].

Correction: Significance of a histone-like protein with its native structure for the diagnosis of asymptomatic tuberculosis.

[This corrects the article DOI: 10.1371/journal.pone.0204160.].

Detection of parallel and antiparallel DNA triplex structures in living human cells using in-cell NMR.

We introduced oligodeoxynucleotides (ODNs) that form parallel and antiparallel triplex structures in vitro into living human cells and recorded their in-cell NMR spectra. Observation of landmark signals for triplex structures proved for the first time that parallel and antiparallel triplex structures are formed in living human cells.

Non-coding RNA suppresses FUS aggregation caused by mechanistic shear stress on pipetting in a sequence-dependent manner.

Fused in sarcoma/translocated in liposarcoma (FUS/TLS) is a multitasking RNA/DNA binding protein. FUS aggregation is implicated in various neurodegenerative diseases. RNA was suggested to modulate phase transition of FUS. Here, we found that FUS transforms into the amorphous aggregation state as an instant response to the shear stress caused by usual pipetting even at a low FUS concentration, 100 nM. It was revealed that non-coding RNA can suppress the transformation of FUS into aggregates. The suppressive effect of RNA on FUS aggregation is sequence-dependent. These results suggested that the non-coding RNA could be a prospective suppressor of FUS aggregation caused by mechanistic stress in cells. Our finding might pave the way for more research on the role of RNAs as aggregation inhibitors, which could facilitate the development of therapies for neurodegenerative diseases.

Structural determination of the sheath-forming polysaccharide of Sphaerotilus montanus using thiopeptidoglycan lyase which recognizes the 1,4 linkage between α-d-GalN and ß-d-GlcA.

Sphaerotilus natans is a filamentous sheath-forming bacterium commonly found in activated sludge. Its sheath is assembled from a thiolic glycoconjugate called thiopeptidoglycan. S. montanus ATCC-BAA-2725 is a sheath-forming member of stream biofilms, and its sheath is morphologically similar to that of S. natans. However, it exhibits heat susceptibility, which distinguishes it from the S. natans sheath. In this study, chemical composition and solid-state NMR analyses suggest that the S. montanus sheath is free of cysteine, indicating that disulfide linkage is not mandatory for sheath formation. The S. montanus sheath was successfully solubilized by N-acetylation, allowing solution-state NMR analysis to determine the sugar sequence. The sheath was susceptible to thiopeptidoglycan lyase prepared from the thiopeptidoglycan-assimilating bacterium, Paenibacillus koleovorans. The reducing ends of the enzymatic digests were labeled with 4-aminobenzoic acid ethyl ester, followed by HPLC. Two derivatives were detected, and their structures were determined. We found that the sheath has no peptides and is assembled as follows: [→4)-ß-d-GlcA-(1→4)-ß-d-Glc-(1→3)-ß-d-GalNAc-(1→4)-α-d-GalNAc-(1→4)-α-d-GalN-(1→]n (ß-d-Glc and α-d-GalNAc are stoichiometrically and substoichiometrically 3-O-acetylated, respectively). Thiopeptidoglycan lyase was thus confirmed to cleave the 1,4 linkage between α-d-GalN and ß-d-GlcA, regardless of the peptide moiety. Furthermore, vital fluorescent staining of the sheath demonstrated that elongation takes place at the tips, as with the S. natans sheath.

[Successful Conservative Management of Bronchopleural Fistula after Intraoperative Bronchial Injury].

A 64-year-old woman diagnosed as primary lung cancer was admitted for surgery. Right lower lobectomy and ND2a-1 nodal dissection was performed under video-assisted thoracic surgery( VATS). The membranous portion of intermediate bronchus was injured about length of 5 mm while dissecting subcarinal lymph nodes. The fistula was closed by knotted suture using 4-0 polydioxanone (PDS) and covered with pericardial fat pad. Although the postoperative course was uneventful and discharged at postoperative day (POD) nine, bloody sputum appeared and right pneumothorax developed at POD 11. Bronchoscopy revealed a slit-like bronchopleural fistula at intermediate bronchus. By continuous thoracic drainage, the fistula successfully closed at POD 13.

Investigation of the Interaction of Human Origin Recognition Complex Subunit 1 with G-Quadruplex DNAs of Human c-myc Promoter and Telomere Regions.

Origin recognition complex (ORC) binds to replication origins in eukaryotic DNAs and plays an important role in replication. Although yeast ORC is known to sequence-specifically bind to a replication origin, how human ORC recognizes a replication origin remains unknown. Previous genome-wide studies revealed that guanine (G)-rich sequences, potentially forming G-quadruplex (G4) structures, are present in most replication origins in human cells. We previously suggested that the region comprising residues 413-511 of human ORC subunit 1, hORC1413-511, binds preferentially to G-rich DNAs, which form a G4 structure in the absence of hORC1413-511. Here, we investigated the interaction of hORC1413-511 with various G-rich DNAs derived from human c-myc promoter and telomere regions. Fluorescence anisotropy revealed that hORC1413-511 binds preferentially to DNAs that have G4 structures over ones having double-stranded structures. Importantly, circular dichroism (CD) and nuclear magnetic resonance (NMR) showed that those G-rich DNAs retain the G4 structures even after binding with hORC1413-511. NMR chemical shift perturbation analyses revealed that the external G-tetrad planes of the G4 structures are the primary binding sites for hORC1413-511. The present study suggests that human ORC1 may recognize replication origins through the G4 structure.

Proton conduction in hydronium solvate ionic liquids affected by ligand shape.

We investigated the ligand dependence of the proton conduction of hydronium solvate ionic liquids (ILs), consisting of a hydronium ion (H3O+), polyether ligands, and a bis[(trifluoromethyl)sulfonyl]amide anion (Tf2N-; Tf = CF3SO2). The ligands were changed from previously reported 18-crown-6 (18C6) to other cyclic or acyclic polyethers, namely, dicyclohexano-18-crown-6 (Dh18C6), benzo-18-crown-6 (B18C6) and pentaethylene glycol dimethyl ether (G5). Pulsed-field gradient spin echo nuclear magnetic resonance results revealed that the protons of H3O+ move faster than those of cyclic 18C6-based ligands but as fast as those of acyclic G5 ligands. Based on these results and density functional theory calculations, we propose that the coordination of a cyclic ether ligand to the H3O+ ion is essential for fast proton conduction in hydronium solvate ILs. Our results attract special interest for many electro- and bio-chemical applications such as electrolyte systems for fuel cells and artificial ion channels for biological cells.