Design and Escherichia coli Expression of a Natively Folded Multi-Disulfide Bonded Influenza H1N1-PR8 Receptor-Binding Domain (RBD).

Refolding multi-disulfide bonded proteins expressed in E. coli into their native structure is challenging. Nevertheless, because of its cost-effectiveness, handiness, and versatility, the E. coli expression of viral envelope proteins, such as the RBD (Receptor-Binding Domain) of the influenza Hemagglutinin protein, could significantly advance research on viral infections. Here, we show that H1N1-PR8-RBD (27 kDa, containing four cysteines forming two disulfide bonds) expressed in E. coli and was purified with nickel affinity chromatography, and reversed-phase HPLC was successfully refolded into its native structure, as assessed with several biophysical and biochemical techniques. Analytical ultracentrifugation indicated that H1N1-PR8-RBD was monomeric with a hydrodynamic radius of 2.5 nm. Thermal denaturation, monitored with DSC and CD at a wavelength of 222 nm, was cooperative with a midpoint temperature around 55 °C, strongly indicating a natively folded protein. In addition, the 15N-HSQC NMR spectrum exhibited several 1H-15N resonances indicative of a beta-sheeted protein. Our results indicate that a significant amount (40 mg/L) of pure and native H1N1-PR8-RBD can be produced using an E. coli expression system with our refolding procedure, offering potential insights into the molecular characterization of influenza virus infection.

Cellular Penetration and Intracellular Dynamics of Perfluorocarbon-Conjugated DNA/RNA as a Potential Means of Conditional Nucleic Acid Delivery.

Nucleic acid-based therapeutics represent a novel approach for controlling gene expression. However, a practical delivery system is required that overcomes the poor cellular permeability and intercellular instability of nucleic acids. Perfluorocarbons (PFCs) are highly stable structures that can readily traverse the lipid membrane of cells. Thus, PFC-DNA/RNA conjugates have properties that offer a potential means of delivering nucleic acid therapeutics, although the cellular dynamics of the conjugates remain unknown. Here, we performed systematic analysis of the cellular permeability of sequence-controlled PFC-DNA conjugates (N[PFC]n-DNA, n = 1,2,3,4,5) that can be synthesized by conventional phosphoramidite chemistry. We showed that DNA conjugates with two or more PFC-containing units (N[PFC]n≥2-DNA) penetrated HeLa cells without causing cellular damage. Imaging analysis along with quantitative flow cytometry analysis revealed that N[PFC]2-DNA rapidly passes through the cell membrane and is evenly distributed within the cytoplasm. Moreover, N[PFC]2-modified cyclin B1-targeting siRNA promoted gene knockdown efficacy of 30% compared with naked siRNA. A similar cell penetration without associated toxicity was consistent among the seven different human cell lines tested. These unique cellular environmental properties make N[PFC]2-DNA/RNA a potential nucleic acid delivery platform that can meet a wide range of applications.

Optimizing Exosome Preparation Based on Size and Morphology: Insights From Electron Microscopy.

Extracellular vesicles (EVs), including exosomes, are crucial in intercellular communication, but differentiating between exosomes and microvesicles is challenging due to their similar morphology and size. This study focuses on multivesicular bodies (MVBs), where exosomes mature, and optimizes exosome isolation using transmission electron microscopy (TEM) for size information. Considering that EVs are nanocolloidal particles, a salt-free Bis-Tris buffer is found to maintain EV integrity better than phosphate-buffered saline (PBS). Dynamic light scattering (DLS) and TEM analysis confirm that intact exosome fractions under the salt-free Bis-Tris buffer condition exhibit polydispersity, including a unique population of <50 nm vesicles resembling intraluminal membrane vesicles (ILVs) in MVBs, alongside larger populations. This <50 nm population disappears in PBS or Bis-Tris buffer with 140 mM NaCl, transforming into a monodisperse population >100 nm. Immunoelectron microscopy also validates the presence of CD63, an exosome biomarker, on approximately 50 nm EVs. These findings provide valuable insights into exosome characterization and isolation, essential for future biomedical applications in diagnostics and drug delivery.

Production of desmethyl-gregatin A, a possible causative toxin of brown stem rot in adzuki bean, by Phialophora gregata f. sp. adzukicola.

To elucidate the cause of brown stem rot in the adzuki bean, we re-evaluated the phytotoxins produced in cultures of the causative agent, Phialophora gregata f. sp. adzukicola. The ethyl acetate-soluble acidic fraction of the culture, as well as the neutral fraction, inhibited the growth of alfalfa seedlings. In the neutral fraction, known phytotoxins gregatin A, B, and C or D and penicilliol A were present. Although the phytotoxins in the acidic fraction were unstable, liquid chromatography-mass spectrometry analysis of the partially purified material suggested that one phytotoxin present was the non-methylated gregatin desmethyl-gregatin A (gregatinic acid A).

The Synthesis and Properties of Ladder-Type π-Conjugated Compounds with Pyrrole and Phosphole Rings.

The phosphole ring is known as a useful building block for constructing π-conjugated organic materials. Here, we report ladder-type benzophospholo[3,2-b]indole (BPI) derivatives where the phosphole and the pyrrole rings are directly fused. Compounds 8a-8d with different aryl groups on the phosphorous center were successfully synthesized, and the solid-state structure of 8a was confirmed using X-ray crystallographic analysis. The BPIs exhibit relatively high fluorescence quantum yield (Φ 0.50-0.72) and demonstrate a larger Stokes shift compared with a series of benzophospholo[3,2-b]benzoheteroles. The benzophospholo[3,2-b]carbazole derivative 9, which possesses a benzene ring between the phosphole and the pyrrole rings of the BPI, was also synthesized, and its solid-state structure was confirmed using X-ray crystallographic analysis. Compound 9 was found to show a smaller Stokes shift compared with the BPI.

Furan-Containing Chiral Spiro-Fused Polycyclic Aromatic Compounds: Synthesis and Photophysical Properties.

Spiro-fused polycyclic aromatic compounds (PACs) have received growing interest as rigid chiral scaffolds. However, furan-containing spiro-fused PACs have been quite limited. Here, we design spiro[indeno[1,2-b][1]benzofuran-10,10'-indeno[1,2-b][1]benzothiophene] as a new family of spiro-fused PACs that contains a furan unit. The compound was successfully synthesized in enantiopure form and also transformed to its S,S-dioxide derivative and the pyrrole-containing analog via aromatic metamorphosis. The absorption and emission properties of the obtained furan-containing chiral spiro-fused PACs are apparently different from those of their thiophene analogs that have been reported, owing to the increased electron-richness of furan compared to thiophene. All of the furan-containing chiral spiro-fused PACs were found to be circularly polarized luminescent materials.

Transformation of Thia[7]helicene to Aza[7]helicenes and [7]Helicene-like Compounds via Aromatic Metamorphosis.

[n]Helicenes with helically twisted structures have attracted increasing interest owing to their unique properties. Therefore, it has been an important issue to develop facile synthetic methodologies which allow access to a variety of [n]helicenes. Here we report the synthesis of [7]helicenes and [7]helicene-like compounds from the thia[7]helicene as a common starting material. Desulfurative dilithiation of the thia[7]helicene and the subsequent reaction with silicon and phosphorus electrophiles afforded the silole- and phosphole-fused [7]helicene-like compounds, respectively. The cyclopentadiene-fused [7]helicene-like compound and the pyrrole-fused aza[7]helicenes were also successfully synthesized via twofold SNAr reactions of the thia[7]helicene S,S-dioxide with the carbon and nitrogen nucleophiles, respectively. The thia[7]helicene S,S-dioxide showed a slightly red-shifted absorption spectrum than the parent thia[7]helicene, which was well demonstrated by the theoretical calculations. The substituents on the silicon atom of silole-fused [7]helicene-like compounds have little impact on the longest absorption maximum. Such little effect of the substituents on absorption properties was also observed for cyclopentadiene-fused [7]helicene-like compounds and aza[7]helicenes and was well demonstrated by the theoretical calculations. The thia[7]helicene S,S-dioxide and the silole-fused [7]helicene-like compound exhibited bright blue emission, and the cyclopentadiene-fused [7]helicene-like compound and the aza[7]helicenes showed strong violet emission. Each single enantiomer of the aza[7]helicenes showed circularly-polarized luminescence with the dissymmetry factors of 4.2~4.4 × 10-3.

Blocking PSD95-PDZ3's amyloidogenesis through point mutations that inhibit high-temperature reversible oligomerization (RO).

The third PDZ domain of the postsynaptic density protein 95 (PSD95-PDZ3; 11 kDa, 103 residues) has a propensity to form amyloid fibrils at high temperatures. At neutral pH, PDZ3 is natively folded, but it exhibits a peculiar three-state thermal unfolding with a reversible oligomerization (RO) equilibrium at high temperatures, which is uncharacteristic in the unfolding of a small globular protein as PDZ3 is. Here, we examined the RO's role in PDZ3's amyloidogenesis at high-temperature using two variants (F340A and L342A) that suppress the high-temperature RO and five single-alanine-mutated variants, where we mutated surface-exposed hydrophobic residues to alanine. Circular Dichroism (CD), Analytical Ultracentrifuge (AUC), and other spectroscopic measurements confirmed the retention of the native structure at ambient temperature. Differential Scanning Calorimetry (DSC) was used to assess the presence or absence of the high-temperature RO, and the amyloidogenicity of the variants was measured by Thioflavin T (ThT) fluorescence and Transmission Electron Microscopy (TEM). By comparing the fraction of RO and the ThT signal, we found that mutations that suppressed the high-temperature RO strongly inhibited amyloidogenesis. On the other hand, all variants forming RO also formed amyloids under the same conditions as the wild-type PDZ3.

Oligomeric Structural Transition of HspB1 from Chinese Hamster.

HspB1 is a mammalian sHsp that is ubiquitously expressed in almost all tissues and involved in regulating many vital functions. Although the recent crystal structure of human HspB1 showed that 24 monomers form the oligomeric complex of human HspB1 in a spherical configuration, the molecular architecture of HspB1 is still controversial. In this study, we examined the oligomeric structural change of CgHspB1 by sedimentation velocity analytical ultracentrifugation. At the low temperature of 4 °C, CgHspB1 exists as an 18-mer, probably a trimeric complex of hexamers. It is relatively unstable and partially dissociates into small oligomers, hexamers, and dodecamers. At elevated temperatures, the 24-mer was more stable than the 18-mer. The 24-mer is also in dynamic equilibrium with the dissociated oligomers in the hexameric unit. The hexamer further dissociates to dimers. The disulfide bond between conserved cysteine residues seems to be partly responsible for the stabilization of hexamers. The N-terminal domain is involved in the assembly of dimers and the interaction between hexamers. It is plausible that CgHspB1 expresses a chaperone function in the 24-mer structure.

Split conformation of Chaetomium thermophilum Hsp104 disaggregase.

Hsp104 and its bacterial homolog ClpB form hexameric ring structures and mediate protein disaggregation. The disaggregated polypeptide is thought to thread through the central channel of the ring. However, the dynamic behavior of Hsp104 during disaggregation remains unclear. Here, we reported the stochastic conformational dynamics and a split conformation of Hsp104 disaggregase from Chaetomium thermophilum (CtHsp104) in the presence of ADP by X-ray crystallography, cryo-electron microscopy (EM), and high-speed atomic force microscopy (AFM). ADP-bound CtHsp104 assembles into a 65 left-handed spiral filament in the crystal structure at a resolution of 2.7 Å. The unit of the filament is a hexamer of the split spiral structure. In the cryo-EM images, staggered and split hexameric rings were observed. Further, high-speed AFM observations showed that a substrate addition enhanced the conformational change and increased the split structure's frequency. Our data suggest that split conformation is an off-pathway state of CtHsp104 during disaggregation.

Chiral Benzo[b]silole-Fused 9,9'-Spirobi[fluorene]: Synthesis, Chiroptical Properties, and Transformation to π-Extended Polycyclic Arene.

Chiral spiro π-conjugated compounds have emerged as a new class of circularly polarized luminescent organic materials. Here we report the synthesis and (chir)optical properties of a chiral benzo[b]silole-fused 9,9'-spirobi[fluorene] (SBF) and π-extended spiro polycyclic arene. The benzo[b]silole-fused SBF was successfully synthesized by a rhodium-catalyzed intramolecular silylative cyclization. It was further transformed to the chiral π-extended spiro polycyclic arene by an annulative π-extension reaction. Less effective spiroconjugation was observed for these spiro compounds through UV-Vis absorption spectroscopy and theoretical calculations. They exhibit circularly polarized luminescence with the dissymmetry factors of up to 0.76×10-3 . Theoretical calculations demonstrate that emission of the benzo[b]silole-fused SBF occurs from one subunit, the structure of which is slightly different from that in the Frank-Condon state.

Hot Spot Mutagenesis Improves the Functional Expression of Unique Mammalian Odorant Receptors.

Vertebrate animals detect odors through olfactory receptors (ORs), members of the G protein-coupled receptor (GPCR) family. Due to the difficulty in the heterologous expression of ORs, studies of their odor molecule recognition mechanisms have progressed poorly. Functional expression of most ORs in heterologous cells requires the co-expression of their chaperone proteins, receptor transporting proteins (RTPs). Yet, some ORs were found to be functionally expressed without the support of RTP (RTP-independent ORs). In this study, we investigated whether amino acid residues highly conserved among RTP-independent ORs improve the functional expression of ORs in heterologous cells. We found that a single amino acid substitution at one of two sites (NBW3.39 and 3.43) in their conserved residues (E and L, respectively) significantly improved the functional expression of ORs in heterologous cells. E3.39 and L3.43 also enhanced the membrane expression of RTP-dependent ORs in the absence of RTP. These changes did not alter the odorant responsiveness of the tested ORs. Our results showed that specific sites within transmembrane domains regulate the membrane expression of some ORs.

PV1 Protein from Plasmodium falciparum Exhibits Chaperone-Like Functions and Cooperates with Hsp100s.

Plasmodium falciparum parasitophorous vacuolar protein 1 (PfPV1), a protein unique to malaria parasites, is localized in the parasitophorous vacuolar (PV) and is essential for parasite growth. Previous studies suggested that PfPV1 cooperates with the Plasmodium translocon of exported proteins (PTEX) complex to export various proteins from the PV. However, the structure and function of PfPV1 have not been determined in detail. In this study, we undertook the expression, purification, and characterization of PfPV1. The tetramer appears to be the structural unit of PfPV1. The activity of PfPV1 appears to be similar to that of molecular chaperones, and it may interact with various proteins. PfPV1 could substitute CtHsp40 in the CtHsp104, CtHsp70, and CtHsp40 protein disaggregation systems. Based on these results, we propose a model in which PfPV1 captures various PV proteins and delivers them to PTEX through a specific interaction with HSP101.

Solvent-sensitive circularly polarized luminescent compounds bearing a 9,9'-spirobi[fluorene] skeleton.

A series of chiral 9,9'-spirobi[fluorene] (SBF) derivatives with diphenylamino donor and cyano acceptor units were designed as a new family of circularly polarized luminescent materials. The designed SBF derivatives were successfully synthesized from 7,7'-dibromo-9,9'-spirobi[fluorene]-2,2'-diol. No racemization occurred in the synthetic sequence. Therefore, each enantiomer of the SBF derivatives can be prepared from the enantiomerically pure starting material. Absorption and fluorescence spectroscopy and theoretical calculations revealed that the phenylene linker between the donor/acceptor units and the SBF core has a great impact on their photophysical properties. In particular, the phenylene linker was found to induce a red shift in their emission bands. The obtained chiral SBF derivatives exhibited solvent-dependent circularly polarized luminescence owing to their donor-π-acceptor structures.

BF3-Catalyzed Skeletal Rearrangement of 7-En-2-ynones to endo-Type Cyclic Dienes.

Transition-metal-catalyzed cycloisomerization reactions with skeletal rearrangement of 1,n-enynes to afford cyclic dienes (particularly exo-cyclization products) have been well studied. However, there are few reports on the nonmetal-catalyzed skeletal rearrangement of enynes and skeletal rearrangement of electron-deficient enynes such as n-en-2-ynones. Here, we describe BF3·MeCN-catalyzed synthesis of 3-alkylidenecyclohexenes from 7-en-2-ynones, representing the first nonmetal-catalyzed skeletal rearrangement of 1,6-enynes to endo-type cyclic dienes.

Computational and Experimental Analysis on the Conformational Preferences of Anticancer Saponin OSW-1.

The conformational properties of anticancer saponin OSW-1 were investigated by X-ray crystallography and by an integrated approach combining a conformational search and the evaluation of the computed conformational distribution by comparing the experimental and simulated spectroscopic data. Our results suggested that OSW-1 adopts two preferred conformations in solution at an approximately 2:1 ratio, of which the crystal structure is consistent with the major conformation. In the solution models, the arabinose residue of OSW-1 appears to serve as a molecular hinge by flipping from the standard 4C1 form in the major conformer to the unusual 1C4 form in the minor conformer. This results in different orientations of the biologically essential p-methoxybenzoyl group, thereby inducing a dramatic alteration of the three-dimensional shape and polarity of OSW-1.

Needle-shaped amyloid deposition in rat mammary gland: evidence of a novel amyloid fibril protein.

Amyloidosis is an extremely rare event in rats. In this study, we report that lipopolysaccharide binding protein (LBP) is the most likely amyloidogenic protein in rat mammary amyloidosis. Histologically, corpora amylacea (CA) and stromal amyloid (SA) were observed in rat mammary glands, and needle-shaped amyloid (NA) was also observed on the surface or gap of CA and SA. Following surveillance in aged rats, NA was observed in 62% of mammary tumours, 25% of male mammary glands and 83% of female mammary glands. Proteomic analysis showed that lactadherin was a major constitutive protein of CA and SA, and both were positive following immunohistochemistry with anti-lactadherin antibodies. In the same analysis, LBP was detected as a prime candidate protein in NA, and NA was positive following immunohistochemistry and immunoelectron microscopy with anti-LBP antibody. Furthermore, synthetic peptides derived from rat LBP formed amyloid fibrils in vitro. Overall, these results provide evidence that LBP is an amyloid precursor protein of NA in rat mammary glands.

Purification and characterization of proteins in multifloral honey from kelulut bee (stingless bee).

The kelulut bee (Meliponini) is a subfamily of stingless bees that produce honey. A total of 89 species out of a total of 500 species of kelulut bees are known to originate from the Indo-Australian region. Kelulut bees do not have quality standards so they still refer to the Codex and EU Directive which basically only applied for Apis honey. The Codex and EU Directive are formed by several psychochemical parameters, one of it is diastase activity. Diastase activity in kelulut honey is known not to meet existing standards or even undetectable. Therefore, this study aimed to explore proteins inside kelulut honey and investigate the possibility of using a specific protein as a biomarker to differentiate honey produced by kelulut bee from other honey. This research can also be considered as an initial step to optimize the exploration of protein in kelulut honey. This research is divided into two sections which are the preliminary research and the research expansion. From preliminary section, glucose dehydrogenase enzyme (GDH) was found to be present inside Tetragonula spp honey. A further examination of GDH enzyme was made in four kelulut bee honeys namely Tetragonula leaviceps, T. biroi, Heterotrigona itama, and Geniotrigona thoracica. The preliminary research has five stages that are exactly the as expansion research section except it didn't include GDH activity measurement. The research includes seven main stages. First honeys were dialyzed to remove the sugar content followed by centrifugation. The samples were then purified using liquid chromatography with anion exchanger column. The molecular weight of proteins was analysed by SDS-PAGE method. The GDH activity was measured using spectrophotometer followed by qualitative analysis using LC-MS/MS. The peptide sequences resulted from LC-MS/MS were then matched with Uniprot to identify the unknow protein. The results showed that only T. biroi and T. laeviceps had GDH enzyme activity of 0,1891 U/mL and 0,1652-1,579 U/mL, respectively. Bands from both species were also qualitatively identified as GDH. With these results, it can be concluded that the GDH enzyme cannot be used as a biomarker to distinguish the kelulut honey.

Crystal structure and Hirshfeld surface analysis of 2-hy-droxy-7-meth-oxy-1,8-bis-(2,4,6-tri-chloro-benzo-yl)naphthalene.

In the title compound, C25H12Cl6O4, the two carbonyl groups are oriented in a same direction with respect to the naphthalene ring system and are situated roughly parallel to each other, while the two 2,4,6-tri-chloro-benzene rings are orientated in opposite directions with respect to the naphthalene ring system: the carbonyl C-(C=O)-C planes subtend dihedral angles of 45.54 (15) and 30.02 (15)° to the naphthalene ring system are. The dihedral angles formed by the carbonyl groups and the benzene rings show larger differences, the C=O vectors being inclined to the benzene rings by 46.39 (16) and 79.78 (16)°. An intra-molecular O-H⋯O=C hydrogen bond forms an S(6) ring motif. In the crystal, no effective inter-molecular hydrogen bonds are found; instead, O⋯Cl and C⋯Cl close contacts are observed along the 21 helical-axis direction. The Hirshfeld surface analysis reveals several weak interactions, the major contributor being Cl⋯H/H⋯Cl contacts.