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A great number of free radicals have a negative impact on the human body, and an increased interest in the identification of new natural molecules with antioxidant properties has emerged due to concerns about synthetic antioxidants. Here, the antioxidant effect of four exo-polysaccharides (EPS) extracts obtained from submerged cultivation of Nothophellinus andinopatagonicus and Pseudoinonotus crustosus (N and P, respectively) in two culture media (M1 and M2) at 2 concentrations (100 and 250 µg/ml) was studied; then, its relation with the chemical composition of the EPS was evaluated. To assess the antioxidant activities of the extracts, several in vitro assays were performed: DPPH and ABTS radical scavenging, ferric-reducing antioxidant power, chelating ability on ferrous ions, and inhibition of the lipid peroxidation. The concentrations tested here were much lower than those reported in previous works. Despite variations in chemical composition and monosaccharide profiles among the extracts, all demonstrated antioxidant activity, although the type of activity differed; only P-M1 exhibited a good antioxidant activity across all assays. This extract contained the highest proportion of phenolic compounds, and also displayed the highest radical scavenging activity. Although the utilization of polysaccharides as functional food ingredients remains limited, we propose P-M1 as a promising candidate for a nutraceutical product. Additionally, a formulation could be made with a combination of extracts to create an antioxidant-rich supplement. Additional research is needed to confirm our findings in a cellular environment and to elucidate the mechanisms that drive their antioxidant activities, ultimately facilitating their development and utilization as nutraceutical products., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Microbial exopolysaccharides (EPSs) have attracted extensive attention for their biological functions in antioxidant activities. In this study, we characterized a novel EPS produced by Bifidobacterium pseudocatenulatum Bi-OTA128 which exhibited the highest antioxidant capacity compared to nine other ropy bacterial strains, achieving 76.50 % and 93.84 % in DPPH· and ABTS· + scavenging activity, and ferric reducing power of 134.34 μM Fe 2+ . Complete genomic analysis identified an eps gene cluster involved in the EPS biosynthesis of Bi-OTA128 strain, which might be responsible for its ropy phenotype. The EPS was then isolated and purified by a DEAE-Sepharose Fast Flow column. A single elution part EPS 128 was obtained with a recovery rate of 43.5 ± 1.78 % and a total carbohydrate content of 93.6 ± 0.76 %. Structural characterization showed that EPS 128 comprised glucose, galactose, and rhamnose (molar ratio 4.0:1.2:1.1), featuring a putative complex backbone structure with four branched chains and an unusual acetyl group at O-2 of terminal rhamnose. Antioxidant assay in vitro indicated that EPS 128 exhibited antioxidant potential with 50.52 % DPPH· and 65.40 % ABTS· + scavenging activities, reaching 54.3 % and 70.44 % of the efficacy of standard Vitamin C at 2.0 mg/L. Furthermore, EPS 128 showed protective effects against H 2 O 2 -induced oxidative stress in HepG2 cells by reducing cellular reactive oxygen species (ROS) and increasing cell viability. These findings present the first comprehensive report of an antioxidant EPS from B. pseudocatenulatum, highlighting its potential as a natural antioxidant for applications in the food industry and clinical settings., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier GmbH. All rights reserved.)

Dye-decolorizing peroxidases (DyPs) belong to a novel superfamily of heme peroxidases that can oxidize recalcitrant compounds. In the current study, the GlDyP2 gene from Ganoderma lucidum was heterologously expressed in Escherichia coli, and the enzymatic properties of the recombinant GlDyP2 protein were investigated. The GlDyP2 protein could oxidize not only the typical peroxidase substrate ABTS but also two lignin substrates, namely guaiacol and 2,6-dimethoxy phenol (DMP). For the ABTS substrate, the optimum pH and temperature of GlDyP2 were 4.0 and 35 °C, respectively. The pH stability and thermal stability of GlDyP2 were also measured; the results showed that GlDyP2 could function normally in the acidic environment, with a T 50 value of 51 °C. Moreover, compared to untreated controls, the activity of GlDyP2 was inhibited by 1.60 mM of Mg 2+ , Ni 2+ , Mn 2+ , and ethanol; 0.16 mM of Cu 2+ , Zn 2+ , methanol, isopropyl alcohol, and Na 2 EDTA·2H 2 O; and 0.016 mM of Fe 2+ and SDS. The kinetic constants of recombinant GlDyP2 for oxidizing ABTS, Reactive Blue 19, guaiacol, and DMP were determined; the results showed that the recombination GlDyP2 exhibited the strongest affinity and the most remarkable catalytic efficiency towards guaiacol in the selected substrates. GlDyP2 also exhibited decolorization and detoxification capabilities towards several dyes, including Reactive Blue 19, Reactive Brilliant Blue X-BR, Reactive Black 5, Methyl Orange, Trypan Blue, and Malachite Green. In conclusion, GlDyP2 has good application potential for treating dye wastewater., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Coprinus comatus is an edible and medicinal fungus. In this study, the antioxidant activity of the fermentation product of C. comatus was investigated through optimization of fermentation process. The results indicated that the fermentation product of C. comatus had obvious scavenging ability for 2,2'-Azino-bis(3-ethylbenzothiazoline)-6-sulphonic acid (ABTS) free radical. The EC 50 of the n -butanol extract from the fermentation product on ABTS· + was 0.65 ± 0.02 mg/mL. On this basis, the liquid fermentation conditions of C. comatus were optimized through single factor and response surface optimization experiments according to the scavenging ability of ABTS· + to improve the antioxidant capacity of the fermentation product. The results showed that when the 14% of C. comatus was fermented in a culture medium with a C/N ratio of 48:1 for 6 days, the ABTS· + scavenging ability was the strongest, and the EC 50 of n -butanol extract was 0.57 ± 0.01 mg/mL, which was 12.31% higher than the initial activity. This study laid the foundation for the development of C. comatus .

Sulfur-containing heterocyclic derivatives have been disclosed for binding with a wide range of cancer-specific protein targets. Various interesting derivatives of sulfur-containing heterocyclics such as benzothiazole, thiazole, thiophene, thiazolidinedione, benzothiophene, and phenothiazine, etc have been shown to inhibit diverse signaling pathways implicated in cancer. Significant progress has also been made in molecular targeted therapy against specific enzymes such as kinase receptors due to potential binding interactions inside the ATP pocket. Sulfur-containing heterocyclic ring metal complexes i. e., benzothiazole, thiazole, thiophene, benzothiophene and phenothiazines are among the most promising active anticancer compounds. However, sulfur heteroaromatic rings, particularly thiophene, are of high structural alert due to their metabolism to reactive metabolites. The mere presence of a structural alert itself does not determine compound toxicity therefore, this review focuses on some specific findings that shed light on factors influencing the toxicity. In the current review, synthetic strategies of introducing the sulfur core ring in the synthesized derivatives are discussed with their structure-activity relationships to enhance our understanding of toxicity mechanisms and develop safer therapeutic options. The sulfur-containing marketed anticancer drugs included in this review direct the synthesis of novel compounds and will help in the development of potent, safer sulfur-based anticancer drugs in near future., (© 2024 Wiley-VHCA AG, Zurich, Switzerland.)

G-triplexes are G-rich oligonucleotides composed of three G-tracts and have absorbed much attention due to their potential biological functions and attractive performance in biosensing. Through the optimization of loop compositions, DNA lengths, and 5'-flanking bases of G-rich sequences, a new stable G-triplex sequence with 14 bases (G3-F15) was discovered to dramatically activate the fluorescence of Thioflavin T (ThT), a water-soluble fluorogenic dye. The fluorescence enhancement of ThT after binding with G3-F15 reached 3200 times, which was the strongest one by far among all of the G-rich sequences. The conformations of G3-F15 and G3-F15/ThT were studied by circular dichroism. The thermal stability measurements indicated that G3-F15 was a highly stable G-triplex structure. The conformations of G3-F15 and G3-F15/ThT in the presence of different metal cations were studied thoroughly by fluorescent spectroscopy, circular dichroism, and nuclear magnetic resonance. Furthermore, using the G3-F15/ThT complex as a fluorescent probe, a robust and simple turn-on fluorescent sensor for uracil-DNA glycosylase activity was developed. This study proposes a new systematic strategy to explore new functional G-rich sequences and their ligands, which will promote their applications in diagnosis, therapy, and biosensing.

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system that results from destruction of the myelin sheath. Due to heterogeneity of the symptoms and course of MS, periodic monitoring of disease activity is important for diagnosis and treatment. In the present study, we synthesized four radioiodinated benzoxazole (BO) and benzothiazole (BT) derivatives, and evaluated their utility as novel myelin imaging probes for single photon emission computed tomography (SPECT). In a biodistribution study using normal mice, three compounds ([ 125 I]BO-1, [ 125 I]BO-2, and [ 125 I]BT-2) displayed moderate brain uptake (2.7, 2.9, and 2.8% ID/g, respectively) at 2 min postinjection. On ex vivo autoradiography using normal mice, [ 125 I]BO-2 showed the most preferable ratio of radioactivity accumulation in white matter (myelin-rich region) versus gray matter (myelin-deficient region). In addition, the radioactivity of [ 125 I]BO-2 was reduced in the lysophosphatidylcholine-induced demyelination region. In conclusion, [ 123 I]BO-2 demonstrated the fundamental characteristics of a myelin imaging probe for SPECT., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Wilt disease caused by Fusarium solani, a soil-borne plant pathogenic fungus, is a serious disease in tomato causing economic losses. In the present study, among the nine isolates of Chaetomium globosum Kunze (Chg1-Chg9), screened against F. solani No. Fs-1, Chg2 exhibited the maximum inhibition (49.2 %), followed by the isolates Chg6 (47.4%) and Chg1 (46.3%) in dual culture. Further, the crude secondary metabolites from these three isolates showed maximum reduction of the mycelial growth of Fs-1 compared to control. Gas Chromatography-Mass Spectrometry (GC-MS) analysis of the metabolites from Chg2 revealed the presence of major compounds, viz., benzothiazole, 2-(2-hydroxyethylthio) (7.51%); 9,12,15- octadecatrienoic acid, 2,3-bis[(trimethylsilyl)oxy] propyl ester (3.13%); and hexadecanoic acid, 1- (hydroxymethyl)-1,2-ethanediyl ester (2.69%). Fourier transform infrared (FTIR) spectroscopy revealed the presence of alcohol, secondary amine, aliphatic primary amine, carboxylic acid, allene, conjugated ketone, imine/oxime, sulphate, and halo compound groups with a weak to strong range of intensity. Pure compound of benzothiazole @ 5000 ppm exhibited higher antagonistic activity of Fs-1which decreased at lower concentration. In pot culture experiment, dipping tomato seedlings cv. PKM1 in 2% ethyl acetate-fractionated biomolecules of Chg2 recorded a minimum disease incidence of 20.0%, whereas seedlings dipped in the culture filtrate of Chg2 showed a disease incidence of 26.6% compared to control (86.6%). Besides, these seedlings treated with the culture filtrate showed the highest plant height of 37.68 cm and the maximum number of leaves and flowers (39.6 and 5.48, respectively), followed by those treated with the biomolecules of Chg2 (C. globosum) and the culture filtrate of T. asperellum. The results revealed that the antifungal efficacy of C. globosum Chg2 is due to the presence of antimicrobial metabolites including benzothiazole. Exploring the use of this novel antifungal compound in the management of plant diseases is highly warranted., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: Neospora caninum is an apicomplexan parasite that is particularly responsible for abortions in cattle and neuromuscular disease in dogs. Due to the limited effectiveness of currently available drugs, there is an urgent need for new therapeutic approaches to control neosporosis. Luciferase-based assays are potentially powerful tools in the search for antiprotozoal compounds, permitting the development of faster and more automated assays. The aim of this study was to construct a luciferase-expressing N. caninum and evaluate anti-N. caninum drugs., Methods: Luciferase-expressing N. caninum (Nc1-Luc) was constructed using clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (CRISPR/Cas9). After testing the luciferase expression and phenotype of the Nc1-Luc strains, the drug sensitivity of Nc1-Luc strains was determined by treating them with known positive or negative drugs and calculating the half-maximal inhibitory concentration (IC 50 ). The selective pan-rapidly accelerated fibrosarcoma (pan-RAF) inhibitor TAK-632 was then evaluated for anti-N. caninum effects using Nc1-Luc by luciferase activity reduction assay and other in vitro and in vivo studies., Results: The phenotypes and drug sensitivity of Nc1-Luc strains were consistent with those of the parental strains Nc1, and Nc1-Luc strains can be used to determine the IC 50 for anti-N. caninum drugs. Using the Nc1-Luc strains, TAK-632 showed promising activity against N. caninum, with an IC 50 of 0.6131 μM and a selectivity index (SI) of 62.53. In vitro studies demonstrated that TAK-632 inhibited the invasion, proliferation, and division of N. caninum tachyzoites. In vivo studies showed that TAK-632 attenuated the virulence of N. caninum in mice and significantly reduced the parasite burden in the brain., Conclusions: In conclusion, a luciferase-expressing N. caninum strain was successfully constructed, which provides an effective tool for drug screening and related research on N. caninum. In addition, TAK-632 was found to inhibit the growth of N. caninum, which could be considered as a candidate lead compound for new therapeutics for neosporosis., (© 2024. The Author(s).)

Tozadenant (4-hydroxy- N -(4-methoxy-7-morpholinobenzo[ d ]thiazol-2-yl)-4-methylpiperidine-1-carboxamide) is a highly selective adenosine A 2A receptor (A 2A R) antagonist and a promising lead structure for the development of A 2A R-selective positron emission tomography (PET) probes. Although several 18 F-labelled tozadenant derivatives showed favorable in vitro properties, recent in vivo PET studies observed poor brain penetration and lower specific binding than anticipated from the in vitro data. While these findings might be attributable to the structural modification associated with 18 F-labelling, they could also reflect inherent properties of the parent compound. However, PET studies with radioisotopologues of tozadenant to evaluate its cerebral pharmacokinetics and brain distribution are still lacking. In the present work, we applied N -Boc- O -desmethyltozadenant as a suitable precursor for the preparation of [ O -methyl- 11 C]tozadenant ([ 11 C]tozadenant) by O -methylation with [ 11 C]methyl iodide followed by acidic deprotection. This approach afforded [ 11 C]tozadenant in radiochemical yields of 18 ± 2%, with molar activities of 50-60 GBq/µmol (1300-1600 mCi/µmol) and radiochemical purities of 95 ± 3%. In addition, in vitro autoradiography in pig and rat brain slices demonstrated the expected striatal accumulation pattern and confirmed the A 2A R specificity of the radioligand, making it a promising tool for in vivo PET studies on the cerebral pharmacokinetics and brain distribution of tozadenant.

A novel mononuclear platinum(II) complex, [Pt(L-H)Cl] (1, where L= N-(4-(benzo[d]thiazol-2-yl)phenyl)-2-((2-pyridylmethyl)(2-hydroxyethyl)-amino)acetamide), was obtained by covalently tethering a benzothiazole derivative 2-(4-aminophenyl)benzothiazole to the 2-pyridylmethyl-2-hydroxyethylamine chelating Pt II center. In vitro tests indicated that complex 1 displayed excellent antiproliferative activity against the tested cancer cell lines, especially liver cancer HepG-2 and SMMC-7221 cells. Importantly, the complex possessed 4.33-fold higher antiproliferative activity as compared with cisplatin against HepG-2 cells, but was less toxic to the normal cell line L02 with the selectivity index (SI = IC 50 (L02)/IC 50 (HepG-2)) value of 8.36 compared to cisplatin (SI, 1.40). The results suggested that 1 might have the potential to act as a candidate for the treatment of hepatocellular carcinoma (HCC). Cellular uptake and distribution studies showed that 1 could effectively pass through the membrane of cells, enter the nuclei and mitochondria, induce the platination of cellular DNA. The interaction of 1 with CT-DNA demonstrated that 1 could effectively bind to DNA in a dual binding mode, i.e., the intercalation of the 2-(4-aminophenyl)benzothiazole unit plus monofunctional platination of the platinum(II) moiety. In addition, Hoechst 33342 staining and flow cytometry analysis illustrated that 1 arrested the cell cycle in HepG-2 cancer cells at G2/M phases, induced mitochondrial membrane depolarization, increased ROS generation, and caused obvious cell apoptosis. Further cellular mechanism studies elucidated that 1 triggered HepG-2 cell apoptosis via the mitochondrial-mediated pathway by upregulating the gene and protein expression levels of Bax, downregulating the gene and protein expression levels of Bcl-2, and activating the caspase cascade., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier Inc.)

Cysteine (Cys), one of essential small-molecule-based biothiols in the human body, contributes to the regulation of redox reactions and is closely associated with many physiological and pathological metabolic processes. Herein, a novel fluorescent probe, hydroxyphenyl-conjugated benzothiazole (HBT-Cys) capable of detecting Cys was constructed, where acrylate served as the recognition group and hydroxyphenyl-linked benzothiazole acted as the fluorophore. The fluorescence of the probe was negligible in the absence of Cys, and an intense blue fluorescence was observed upon addition of Cys. The Cys-sensing mechanism could be ascribed to the Cys-involved hydrolysis reaction with acrylate, leading to light up the emission at 430 nm with about 80-fold enhancement. In addition, HBT-Cys exhibited a fast response time, remarkable selectivity and low detection limit. HBT-Cys also worked well in real-time monitoring of Cys in three different food samples (wolfberry, hawthorn, and red dates). Importantly, our probe had an excellent lysosomes-targeted ability, which was successfully employed to real-time visualize the fluctuation of both exogenous and endogenous Cys in living cells and zebrafish under lipopolysaccharide (LPS)-induced oxidative stress. Hopefully, the work shown here provides a potent candidate for the real-time tracking of Cys fluctuations in various biological samples., Competing Interests: Declaration of competing interest The authors declared that they have no conflicts of interest to this work and we do not have any commercial or associative interest that represents a conflict of interest in connection with the work submitted., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Background: [ 18 F]flutemetamol is a PET radioligand used to image brain amyloid, but its detection of myocardial amyloid is not well-characterized. This histological study characterized binding of fluorescently labeled flutemetamol (cyano-flutemetamol) to amyloid deposits in myocardium., Methods: Myocardial tissue was obtained post-mortem from 29 subjects with cardiac amyloidosis including transthyretin wild-type (ATTRwt), hereditary/variant transthyretin (ATTRv) and immunoglobulin light-chain (AL) types, and from 10 cardiac amyloid-free controls. Most subjects had antemortem electrocardiography, echocardiography, SPECT and cardiac MRI. Cyano-flutemetamol labeling patterns and integrated density values were evaluated relative to fluorescent derivatives of Congo red (X-34) and Pittsburgh compound-B (cyano-PiB)., Results: Cyano-flutemetamol labeling was not detectable in control subjects. In subjects with cardiac amyloidosis, cyano-flutemetamol labeling matched X-34- and cyano-PiB-labeled, and transthyretin- or lambda light chain-immunoreactive, amyloid deposits and was prevented by formic acid pre-treatment of myocardial sections. Cyano-flutemetamol mean fluorescence intensity, when adjusted for X-34 signal, was higher in the ATTRwt than the AL group. Cyano-flutemetamol integrated density correlated strongly with echocardiography measures of ventricular septal thickness and posterior wall thickness, and with heart mass., Conclusion: The high selectivity of cyano-flutemetamol binding to myocardial amyloid supports the diagnostic utility of [ 18 F]flutemetamol PET imaging in patients with ATTR and AL types of cardiac amyloidosis.

Thioflavin T, a cationic benzothiazole dye, is typically used to detect amyloid fibrils. In this study, we analyzed the staining properties of Bacillus subtilis cells using several fluorescent dyes, including thioflavin T analogs, 2-(4'-methylaminophenyl) benzothiazole (BTA-1), and 2-(4-aminophenyl) benzothiazole (APBT). Thioflavin T stained vegetative cells in the early log phase and outer layer structures of forespores and mature spores. The inner parts of forespores and heat-killed mature spores were also stained with thioflavin T. Congo red, auramine O, and rhodamine B stained forespores and mature spores similar to thioflavin T. In contrast, APBT and BTA-1 fluorescence was detected in the outer layers of vegetative cells, mother cells, forespores, and mature spores, indicating that they bind to the cell membrane and/or cell wall. The combination of the fluorescent dyes used in this study will help analyze morphogenetic processes during the sporulation and the damage mechanisms of vegetative cells and spores., (© The Author(s) 2022. Published by Oxford University Press on behalf of Japan Society for Bioscience, Biotechnology, and Agrochemistry.)

The misfolding of amyloid beta (Aβ) peptides into Aβ fibrillary aggregates is a major hallmark of Alzheimer's disease (AD), which responsible for the excess production of hydrogen peroxide (H 2 O 2 ), a prominent reactive oxygen species (ROS) from the molecular oxygen (O 2 ) by the reduction of the Aβ-Cu(I) complex. The excessive production of H 2 O 2 causes oxidative stress and inflammation in the AD brain. Here, we have designed and developed a dual functionalized molecule VBD by using π-conjugation (C═C) in the backbone structure. In the presence of H 2 O 2 , the VBD can turn into fluorescent probe VBD-1 by cleaving of the selective boronate ester group. The fluorescent probe VBD-1 can undergo intramolecular charge transfer transition (ICT) by a π-conjugative system, and as a result, its emission increases from the yellow (532 nm) to red (590 nm) region. The fluorescence intensity of VBD-1 increases by 3.5-fold upon binding with Aβ fibrillary aggregates with a high affinity ( K d = 143 ± 12 nM). Finally, the VBD reduces the cellular toxic H 2 O 2 as proven by the CCA assay and DCFDA assay and the binding affinity of VBD-1 was confirmed by using in vitro histological staining in 8- and 18-month-old triple transgenic AD (3xTg-AD) mice brain slices.

Odorant-binding proteins (OBPs) are believed to play critical roles in host-seeking behavior. However, little attention was paid to its different functions in male and female. The antenna-specific OBP gene from Bactrocera dorsalis, BdorOBP13, was cloned and its expression profile was examined. The results showed that BdorOBP13 was exclusively expressed in male and female adults, which exhibited a high transcript level in antennae. After injection of BdorOBP13 dsRNA, its transcript level in males and females decreased significantly. Electrophysiological responses of RNAi-injected flies to, methyl eugenol (male attractant) and γ-octalactone (female attractant) decreased significantly. However, no significant changes in the electrophysiological response were observed in RNAi-injected flies to benzothiazole, (+),dipentene, and ethyl tiglate. The behavioral bioassay showed that males treated with RNAi significantly reduced their preference to methyl eugenol, while RNAi-injected females showed a significantly lower preference to γ-octalactone, suggesting that BdorOBP13 may have different functions between males and females: it may be involved in the detection of methyl eugenol in males but is involved in the detection of γ-octalactone in females. These findings improve our understanding of insect OBPs and their roles in insect chemosensation, which may provide us with new molecular targets in the management of B. dorsalis., (Crown Copyright © 2022. Published by Elsevier B.V. All rights reserved.)

The formation and accumulation of amyloid beta (Aβ) peptide are considered the crucial events that are responsible for the progression of Alzheimer's disease (AD). Herein, we have designed and synthesized a series of fluorescent probes by using electron acceptor-donor end groups interacting with a π-conjugating system for the detection of Aβ aggregates. The chemical structure of these probes denoted as RMs, having a conjugated π-system (C═C), showed a maximum emission in PBS (>600 nm), which is the best range for a fluorescent imaging probe. Among all these probes, RM-28 showed an excellent fluorescence property with an emission maximum of >598 nm upon binding to Aβ aggregates. RM-28 also showed high sensitivity (7.5-fold) and high affinities toward Aβ aggregates ( K d = 175.69 ± 4.8 nM; K a = 0.5 × 10 7 M -1 ). It can cross the blood-brain barrier of mice efficiently. The affinity of RM-28 toward Aβ aggregates was observed in 3xTg-AD brain sections of the hippocampus and cortex region using a fluorescent imaging technique, as well as an in vitro fluorescence-based binding assay with Aβ aggregates. Moreover, RM-28 is highly specific to Aβ aggregates and does not bind with intracellular proteins like bovine serum albumin (BSA) and α-synuclein (α-Syn) aggregates. The results indicate that the probe RM-28 emerges as an efficient and veritable highly specific fluorescent probe for the detection of Aβ aggregates in both in vitro and in vivo model systems.

PET imaging with β-amyloid ligands is emerging as a molecular imaging technique targeting white matter integrity and demyelination. β-amyloid PET ligands such as 11 C-Pittsburgh compound B ( 11 C-PiB) have been considered for quantitative measurement of myelin content changes in multiple sclerosis, but 11 C-PiB is not commercially available given its short half-life. A 18 F PET ligand such as flutemetamol with a longer half-life may be an alternative, but its ability to differentiate white matter hyperintensities (WMH) from normal-appearing white matter (NAWM) and its relationship with age remains to be investigated. Methods: Cognitively unimpaired (CU) older and younger adults ( n = 61) were recruited from the community responding to a study advertisement for β-amyloid PET. Participants prospectively underwent MRI, 11 C-PiB, and 18 F-flutemetamol PET scans. MRI fluid-attenuated inversion recovery images were segmented into WMH and NAWM and registered to the T1-weighted MRI. 11 C-PiB and 18 F-flutemetamol PET images were also registered to the T1-weighted MRI. 11 C-PiB and 18 F-flutemetamol SUV ratios (SUVrs) from the WMH and NAWM were calculated using cerebellar crus uptake as a reference for both 11 C-PiB and 18 F-flutemetamol. Results: The median age was 38 y (range, 30-48 y) in younger adults and 67 y (range, 61-83 y) in older adults. WMH and NAWM SUVrs were higher with 18 F-flutemetamol than with 11 C-PiB in both older ( P < 0.001) and younger ( P < 0.001) CU adults. 11 C-PiB and 18 F-flutemetamol SUVrs were higher in older than in younger CU adults in both WMH ( P < 0.001) and NAWM ( P < 0.001). 11 C-PiB and 18 F-flutemetamol SUVrs were higher in NAWM than WMH in both older ( P < 0.001) and younger ( P < 0.001) CU adults. There was no apparent difference between 11 C-PiB and 18 F-flutemetamol SUVrs in differentiating WMH from NAWM in older and in younger adults. Conclusion: 11 C-PiB and 18 F-flutemetamol show a similar topographic pattern of uptake in white matter with a similar association with age in WMH and NAWM. 11 C-PiB and 18 F-flutemetamol can also effectively distinguish between WMH and NAWM. However, given its longer half-life, commercial availability, and higher binding potential, 18 F-flutemetamol can be an alternative to 11 C-PiB in molecular imaging studies specifically targeting multiple sclerosis to evaluate white matter integrity., (© 2022 by the Society of Nuclear Medicine and Molecular Imaging.)

Purpose: The recent developments of tau-positron emission tomography (tau-PET) enable in vivo assessment of neuropathological tau aggregates. Among the tau-specific tracers, the application of 11C-pyridinyl-butadienyl-benzothiazole 3 (11C-PBB3) in PET shows high sensitivity to Alzheimer disease (AD)-related tau deposition. The current study investigates the regional tau load in patients within the AD continuum, biomarker-negative individuals (BN) and patients with suspected non-AD pathophysiology (SNAP) using 11C-PBB3-PET., Materials and Methods: A total of 23 memory clinic outpatients with recent decline of episodic memory were examined using 11C-PBB3-PET. Pittsburg compound B (11C-PIB) PET was available for 17, 18F-flurodeoxyglucose (18F-FDG) PET for 16, and cerebrospinal fluid (CSF) protein levels for 11 patients. CSF biomarkers were considered abnormal based on Aβ42 (< 600 ng/L) and t-tau (> 450 ng/L). The PET biomarkers were classified as positive or negative using statistical parametric mapping (SPM) analysis and visual assessment. Using the amyloid/tau/neurodegeneration (A/T/N) scheme, patients were grouped as within the AD continuum, SNAP, and BN based on amyloid and neurodegeneration status. The 11C-PBB3 load detected by PET was compared among the groups using both atlas-based and voxel-wise analyses., Results: Seven patients were identified as within the AD continuum, 10 SNAP and 6 BN. In voxel-wise analysis, significantly higher 11C-PBB3 binding was observed in the AD continuum group compared to the BN patients in the cingulate gyrus, tempo-parieto-occipital junction and frontal lobe. Compared to the SNAP group, patients within the AD continuum had a considerably increased 11C-PBB3 uptake in the posterior cingulate cortex. There was no significant difference between SNAP and BN groups. The atlas-based analysis supported the outcome of the voxel-wise quantification analysis., Conclusion: Our results suggest that 11C-PBB3-PET can effectively analyze regional tau load and has the potential to differentiate patients in the AD continuum group from the BN and SNAP group., Competing Interests: Christine A.F. von Arnim received honoraria from serving on the scientific advisory board of Nutricia GmbH (2014) and Honkong University Research council (2014) and has received funding for travel and speaker honoraria from Nutricia GmbH (2014–2015), Lilly Deutschland GmbH (2013–2016), Desitin Arzneimittel GmbH (2014), Biogen (2016-2018), Roche (2017-2018) and Dr. Willmar Schwabe GmbH & Co. KG (2014-2015). This does not alter our adherence to PLOS ONE policies on sharing data and materials. Makoto Higuchi and co-workers at National Institutes for Quantum and Radiological Science and Technology (QST) hold a patent on tau imaging agents (JP 5422782/EP 12 884 742.3), and QST made a license agreement with APRINOIA Therapeutics Inc. regarding this patent. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

The comprehension of pathogenetic mechanisms in tauopathy-associated neurodegenerative diseases can be improved by the knowledge of the biochemical and biophysical features of mutated tau proteins. Here, we used the full-length, wild-type tau, the V363A and V363I mutated species, associated with pathology, and the P301L mutated tau as a benchmark. Using several techniques, including small-angle X-ray scattering, atomic force microscopy, thioflavin T binding, and electrophoretic separation, we compared their course from intrinsically disordered monomers in solution to early-stage recruitment in complexes and then aggregates of increasing size over long periods up to the asymptotic aggregative behavior of full-length tau proteins. We showed that diversity in the kinetics of recruitment and aggregate structure occurs from the beginning and spreads all over their pathway to very large objects. The different extents of conformational changes and types of molecular assemblies among the proteins were also reflected in their in vitro toxicity; this variation could correlate with physiopathology in humans, considering that the P301L mutation is more aggressive than V363A, especially V363I. This study identified the presence of aggregation intermediates and corroborated the oligomeric hypothesis of tauopathies., (Copyright © 2022. Published by Elsevier B.V.)

Henipaviruses are severe human pathogens within the Paramyxoviridae family. Beyond the P protein, the Henipavirus P gene also encodes the V and W proteins which share with P their N-terminal, intrinsically disordered domain (NTD) and possess a unique C-terminal domain. Henipavirus W proteins antagonize interferon (IFN) signaling through NTD-mediated binding to STAT1 and STAT4, and prevent type I IFN expression and production of chemokines. Structural and molecular information on Henipavirus W proteins is lacking. By combining various bioinformatic approaches, we herein show that the Henipaviruses W proteins are predicted to be prevalently disordered and yet to contain short order-prone segments. Using limited proteolysis, differential scanning fluorimetry, analytical size exclusion chromatography, far-UV circular dichroism and small-angle X-ray scattering, we experimentally confirmed their overall disordered nature. In addition, using Congo red and Thioflavin T binding assays and negative-staining transmission electron microscopy, we show that the W proteins phase separate to form amyloid-like fibrils. The present study provides an additional example, among the few reported so far, of a viral protein forming amyloid-like fibrils, therefore significantly contributing to enlarge our currently limited knowledge of viral amyloids. In light of the critical role of the Henipavirus W proteins in evading the host innate immune response and of the functional role of phase separation in biology, these studies provide a conceptual asset to further investigate the functional impact of the phase separation abilities of the W proteins.

Further clinical development of PF74, a lead compound targeting HIV-1 capsid, is impeded by low antiviral activity and inferior metabolic stability. By modifying the benzene (region I) and indole of PF74, we identified two potent compounds (7m and 7u) with significantly improved metabolic stability. Compared to PF74, 7u displayed greater metabolic stability in human liver microsomes (HLMs) with half-life (t 1/2 ) 109-fold that of PF74. Moreover, mechanism of action (MOA) studies demonstrated that 7m and 7u effectively mirrored the MOA of compounds that interact within the PF74 interprotomer pocket, showing direct and robust interactions with recombinant CA, and 7u displaying antiviral effects in both the early and late stages of HIV-1 replication. Furthermore, MD simulation corroborated that 7u was bound to the PF74 binding site, and the results of the online molinspiration software predicted that 7m and 7u had desirable physicochemical properties. Unexpectedly, this series of compounds exhibited better antiviral activity than PF74 against HIV-2, represented by compound 7m whose anti-HIV-2 activity was almost 5 times increased potency over PF74. Therefore, we have rationally redesigned the PF74 chemotype to inhibitors with novel structures and enhanced metabolic stability in this study. We hope that these new compounds can serve as a blueprint for developing a new generation of HIV treatment regimens., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021. Published by Elsevier Masson SAS.)

A hallmark of Alzheimer's disease (AD) is the accumulation of tau protein in the brain. Compelling evidence indicates that the presence of tau aggregates causes irreversible neuronal destruction, eventually leading to synaptic loss. So far, the inhibition of tau aggregation has been recognized as one of the most effective therapeutic strategies. Cannabidiol (CBD), a major component found in Cannabis sativa L., has antioxidant activities as well as numerous neuroprotective features. Therefore, we hypothesize that CBD may serve as a potent substance to hamper tau aggregation in AD. In this study, we aim to investigate the CBD effect on the aggregation of recombinant human tau protein 1N/4R isoform using biochemical methods in vitro and in silico. Using Thioflavin T (ThT) assay, circular dichroism (CD), and atomic force microscopy (AFM), we demonstrated that CBD can suppress tau fibrils formation. Moreover, by quenching assay, docking, and job's plot, we further demonstrated that one molecule of CBD interacts with one molecule of tau protein through a spontaneous binding. Experiments performed by quenching assay, docking, and Thioflavin T assay further established that the main forces are hydrogen Van der Waals and some non-negligible hydrophobic forces, affecting the lag phase of tau protein kinetics. Taken together, this study provides new insights about a natural substance, CBD, for tau therapy which may offer new hope for the treatment of AD.

Newly synthesised benzimidazole/benzotiazole derivatives bearing amidino, namely 3,4,5,6-tetrahydropyrimidin-1-ium chloride, substituents have been evaluated for their potential antitumor activity in vitro . Compounds and standard drugs (doxorubicin, staurosporine and vandetanib) were tested on three human lung cancer cell lines A549, HCC827 and NCI-H358. We tested compounds in MTS citotoxicity assay and in BrdU proliferative assay performed on 2 D and 3 D assay format. Because benzmidazole scaffold is similar to natural purines, we tested the most active compounds for ability to induce cell apoptosis of A549 by binding to DNA in comparison with doxorubicin and saturosporine. Additionally, the ADME properties of the most active benzothiazole/benzimidazole and non-active compounds were determined to see if the different ADME properties are the cause of different activity in 2 D and 3 D assays, as well as to see if the tested active compounds have drug like properties and potency for further profilation. ADME characterisation included solubility, lipophilicity, permeability, metabolic stability and binding to plasma proteins. In general, the benzothiazole derivatives were more active in comparison to their benzimidazole analogues. The exception was 2-phenyl substituted benzimidazole 6a being active with very pronounced activity especially towards HCC827 cells. All active compounds have similar mode of action on A549 cell line as standard compound doxorubicin, which binds to nucleic acids with the DNA double helix. Tested active benzothiazole compounds were characterised by moderate to good solubility, good metabolic stability, low permeability and high binding to plasma proteins. One tested active benzimidazole derivative showed ADME properties, but lower lipophilicity resulted in low PPB and higher metabolic instability. In addition, no significant difference was observed in ADME profile between active and non-active compounds.

Bilin-binding fluorescent proteins like UnaG-bilirubin are noncovalent ligand-dependent reporters for oxygen-free microscopy but are restricted to blue and far-red fluorescence. Here we describe a high-throughput screening approach to provide a new UnaG-ligand pair that can be excited in the 532 nm green excitation microscopy channel. We identified a novel orange UnaG-ligand pair that maximally emits at 581 nm. Whereas the benzothiazole-based ligand itself is nominally fluorescent, the compound binds UnaG with high affinity ( K d = 3 nM) to induce a 2.5-fold fluorescence intensity enhancement and a 10 nm red shift. We demonstrated this pair in the anaerobic fluorescence microscopy of the prevalent gut bacterium Bacteroides thetaiotaomicron and in Escherichia coli . This UnaG-ligand pair can also be coupled to IFP2.0-biliverdin to differentiate cells in mixed-species two-color imaging. Our results demonstrate the versatility of the UnaG ligand-binding pocket and extend the ability to image cells at longer wavelengths in anoxic environments.

Dotinurad, a novel selective urate reabsorption inhibitor, is used to treat hyperuricemia. In humans, orally administered dotinurad is excreted mainly as glucuronide and sulfate conjugates in urine. To identify the isoforms of UDP-glucuronosyltransferase (UGT) and sulfotransferase (SULT) involved in dotinurad glucuronidation and sulfation, microsome and cytosol fractions of liver, intestine, kidney, and lung tissues (cytosol only) were analyzed along with recombinant human UGT and SULT isoforms. Dotinurad was mainly metabolized to its glucuronide conjugate by human liver microsomes (HLMs), and the glucuronidation followed the two-enzyme Michaelis-Menten equation. Among the recombinant human UGT isoforms expressed in the liver, UGT1A1, UGT1A3, UGT1A9, and UGT2B7 catalyzed dotinurad glucuronidation. Based on inhibition analysis using HLMs, bilirubin, imipramine, and diflunisal decreased glucuronosyltransferase activities by 45.5%, 22.3%, and 22.2%, respectively. Diflunisal and 3'-azido-3'-deoxythymidine, in the presence of 1% bovine serum albumin, decreased glucuronosyltransferase activities by 21.1% and 13.4%, respectively. Dotinurad was metabolized to its sulfate conjugate by human liver cytosol (HLC) and human intestinal cytosol (HIC) samples, with the sulfation reaction in HLC samples following the two-enzyme Michaelis-Menten equation and that in HIC samples following the Michaelis-Menten equation. All eight recombinant human SULT isoforms used herein catalyzed dotinurad sulfation. Gavestinel decreased sulfotransferase activity by 15.3% in HLC samples, and salbutamol decreased sulfotransferase activity by 68.4% in HIC samples. These results suggest that dotinurad glucuronidation is catalyzed mainly by UGT1A1, UGT1A3, UGT1A9, and UGT2B7, whereas its sulfation is catalyzed by many SULT isoforms, including SULT1B1 and SULT1A3. SIGNIFICANCE STATEMENT: The identification of enzymes involved in drug metabolism is important to predicting drug-drug interactions (DDIs) and interindividual variability for safe drug use. The present study revealed that dotinurad glucuronidation is catalyzed mainly by UGT1A1, UGT1A3, UGT1A9, and UGT2B7 and that its sulfation is catalyzed by many SULT isoforms, including SULT1B1 and SULT1A3. Therefore, dotinurad, a selective urate reabsorption inhibitor, is considered safe for use with a small risk of DDIs and low interindividual variability., (Copyright © 2021 The Author(s).)

Lung cancer caused one-quarter of all cancer deaths that was more than other cancers. Chemoprevention is a potential strategy to reducing lung cancer incidence and death, and the effective chemopreventive agents are needed. We investigated the efficacy and mechanism of garlic oil (GO), the garlic product, in the chemoprevention of tobacco carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced lung cancer in A/J mice and MRC-5 cell models in the present study. As a result, it was demonstrated that GO significantly inhibited the NNK-induced lung cancer in vivo and protected MRC-5 cells from NNK-induced cell damage. GO could induce the expressions of the phase II drug-metabolizing enzymes, including NAD(P)H: quinone oxidoreductase 1 (NQO-1), glutathione S-transferase alpha 1 (GSTA1), and antioxidative enzymes heme oxygenase-1 (HO-1). These results supported the potential of GO as a novel candidate agent for the chemoprevention of tobacco carcinogens induced lung cancer., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

d-ribose, a reducing sugar, in diabetic hyperglycemia provokes non-enzymatic glycoxidation of hemoglobin (Hb), an abundant protein of red blood cells (RBCs). Different types of intermediates adduct formation occur during glycoxidation, such as advanced glycation end-products (AGEs) which lead to amyloid formation due to structural and conformational alterations in protein. Therefore, the study of these intermediate adducts plays a pivotal role to discern their relationship with diabetes mellitus and related disorders. Here, we investigated the interaction mechanism of d-ribose with Hb, and Hb prebound phytochemical thymoquinone (TQ). Our investigation reveals that the interaction of TQ with histidine residues of Hb interferes with the interaction of d-ribose with glycine residues at the glycation-site. Based on that, we had performed a time-based (21-days) in-vitro glycoxidation study at 37 °C to investigate the structural perturbation mechanism of Hb at different time-intervals in absence/presence of TQ. We found that prolonged glycoxidation induces amyloid formation in absence of TQ but in its presence, the process was prohibited. In summary, this study examined and characterized biophysically different intermediate-states of protein carrying glycoxidation-modification. Our findings suggested that TQ potentially affects interaction of d-ribose with Hb that prevents glycoxidation and protofibril formation, which establishes TQ as a potential therapeutic agent., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Thioflavin T (ThT) is a commercially available fluorescent dye that is commonly used in biomedical research for over five decades. It was first reported as an extrinsic fluorescent probe for the detection of amyloid fibrils and related processes and it has also been used extensively for assessing protein binding in fluorescence-based assays. Although the nucleic acid binding of ThT was reported half of a century ago in the 1970s, it was not widely explored until the start of this decade. In recent years, Thioflavin T has become a major tool in the recognition of many types of non-canonical nucleic acid conformations including duplexes, triplexes, and G-quadruplexes. The propensity of ThT binding is more towards base aberrations, bulges, and mismatches highlighting its importance in serving as a diagnostic tool in a variety of ailments/disease conditions. In this review, we cover major advancements in nucleic acid detection/binding by ThT to a variety of nucleic acid structures., Competing Interests: Declaration of competing interest None declared., (Copyright © 2021 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

The interactions of ligands with nucleic acids are central to numerous reactions in the biological cell. How such reactions are affected by harsh environmental conditions such as low temperatures, high pressures, and high concentrations of destructive ions is still largely unknown. To elucidate the ions' role in shaping habitability in extraterrestrial environments and the deep subsurface of Earth with respect to fundamental biochemical processes, we investigated the effect of selected salts (MgCl 2 , MgSO 4 , and Mg(ClO 4 ) 2 ) and high hydrostatic pressure (relevant for the subsurface of that planet) on the complex formation between tRNA and the ligand ThT. The results show that Mg 2+ salts reduce the binding tendency of ThT to tRNA. This effect is largely due to the interaction of ThT with the salt anions, which leads to a strong decrease in the activity of the ligand. However, at mM concentrations, binding is still favored. The ions alter the thermodynamics of binding, rendering complex formation that is more entropy driven. Remarkably, the pressure favors ligand binding regardless of the type of salt. Although the binding constant is reduced, the harsh conditions in the subsurface of Earth, Mars, and icy moons do not necessarily preclude nucleic acid-ligand interactions of the type studied here.

In this work we present a computational analysis together with experimental studies, focusing on the interaction between a benzothiazole (BTS) and lysozyme. Results obtained from isothermal titration calorimetry, UV-vis, and fluorescence were contrasted and complemented with molecular docking and machine learning techniques. The free energy values obtained both experimentally and theoretically showed excellent similarity. Calorimetry, UV-vis, and 3D/2D-lig-plot analysis revealed that the most relevant interactions between BTS and lysozyme are based on a predominance of aromatic, hydrophobic Van der Waals interactions, mainly aromatic edge-to-face (T-shaped) π-π stacking interactions between the benzene ring belonging to the 2-(methylthio)-benzothiazole moiety of BTS and the aromatic amino acid residue TRP108 of the lysozyme receptor. Next, conventional hydrogen bonding interactions contribute to the stability of the BTS-lysozyme coupling complex. In addition, mechanistic approaches performed using elastic network models revealed that the BTS ligand theoretically induces propagation of allosteric signals, suggesting non-physiological conformational flexing in large blocks of lysozyme affecting α-helices. Likewise, the BTS ligand interacts directly with allosteric residues, inducing perturbations in the conformational dynamics expressed as a moderate conformational softening in the α-helices H1, H2, and their corresponding β-loop in the lysozyme receptor, in contrast to the unbound state of lysozyme.

Protein misfolding and amyloid deposition are associated with numerous diseases. The detailed characterization of the proteospecies mediating cell death remains elusive owing to the (supra)structural polymorphism and transient nature of the assemblies populating the amyloid pathway. Here we describe the identification of toxic amyloid fibrils with oligomer-like characteristics, which were assembled from an islet amyloid polypeptide (IAPP) derivative containing an Asn-to-Gln substitution (N21Q). While N21Q filaments share structural properties with cytocompatible fibrils, including the 4.7 Å inter-strand distance and β-sheet-rich conformation, they concurrently display characteristics of oligomers, such as low thioflavin-T binding, high surface hydrophobicity and recognition by the A11 antibody, leading to high potency to disrupt membranes and cause cellular dysfunction. The toxic oligomer-like conformation of N21Q fibrils, which is preserved upon elongation, is transmissible to naïve IAPP. These stable fibrils expanding the conformational diversity of amyloid assemblies represent an opportunity to elucidate the structural basis of amyloid disorders., (© 2021. The Author(s).)

Abstract: A 76-year-old woman underwent 18F-florapronol (18F-FC119S, an amyloid β imaging PET agent) PET, owing to cognitive impairment. 18F-florapronol PET images revealed an incidental 18F-florapronol uptake in the right frontal lobe. A well-enhancing extra-axial mass in the right frontal lobe was observed on MRI scans, suggesting a meningioma. After excision of the tumor, the biopsy results confirmed it as a meningothelial meningioma., Competing Interests: Conflicts of interest and sources of funding: This project has been funded in part with a grant from the Korea Institute of Radiological and Medical Sciences, funded by the Ministry of Science, ICT (MSIT), Republic of Korea (no. 50547-2020), and in part with grants from the National Research Foundation of Korea funded by the Ministry of Science, ICT (MSIT), Republic of Korea (no. 2020R1A2C2102492). None declared to all authors., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

Amyloid beta (Aβ) 42 peptide accumulated in Alzheimer disease (AD) patients' brain, often colocalized with serine protease inhibitor family A member 3 (SERPINA3). Being a chaperon, SERPINA3 accelerated Aβ42 fibrillization. While analyzing chaperon activity of human SERPINA3 polymorphisms, we found SERPINA3-R124C played a role in protecting cells from Aβ42 cytotoxicity. SH-SY5Y cells exposed to Aβ42 preincubated with wild-type SERPINA3 (SERPINA3-WT) resulted in extended toxicity leading cell death whereas Aβ42 with SERPINA3-R124C resulted in less cytotoxicity. Transmission electron microscope and thioflavin T assay revealed that SERPINA3-R124C shortened lifetime of small soluble oligomer and maintained β-sheet rich protofibril-like aggregates for longer time compared to that of with SERPINA3-WT. Western blot assay confirmed that SERPINA3-R124C converted Aβ42 mostly into high molecular aggregates. Here, we demonstrate first time that polymorphic SERPINA3 acts as a benign chaperon by modulating the transition states of Aβ42, which may contribute to the reduction of AD risk., (© The Author(s) 2021. Published by Oxford University Press on behalf of Japan Society for Bioscience, Biotechnology, and Agrochemistry.)

The discovery of small molecules that exhibit turn-on far-red or near-infrared (NIR) fluorescence upon DNA binding and understanding how they bind DNA are important for imaging and bioanalytical applications. Here we report the DNA-bound structure and the DNA binding mechanism of quinone cyanine dithiazole (QCy-DT), a recently reported AT-specific turn-on NIR fluorescent probe for double-stranded DNA. The nuclear magnetic resonance (NMR)-derived structure showed minor groove binding but no specific ligand-DNA interactions, consistent with an endothermic and entropy-driven binding mechanism deduced from isothermal titration calorimetry. Minor groove binding is typically fast because it minimally perturbs the DNA structure. However, QCy-DT exhibited unusually slow DNA binding. The cyanine-based probe is capable of cis - trans isomerization due to overlapping methine bridges, with 16 possible slowly interconverting cis / trans isomers. Using NMR, density functional theory, and free energy calculations, we show that the DNA-free and DNA-bound environments of QCy-DT prefer distinctly different isomers, indicating that the origin of the slow kinetics is a cis - trans isomerization and that the minor groove preferentially selects an otherwise unstable cis / trans isomer of QCy-DT. Flux analysis showed the conformational selection pathway to be the dominating DNA binding mechanism at low DNA concentrations, which switches to the induced fit pathway at high DNA concentrations. This report of cis / trans isomerization of a ligand, upon binding the DNA minor groove, expands the prevailing understanding of unique discriminatory powers of the minor groove and has an important bearing on using polymethine cyanine dyes to probe the kinetics of molecular interactions.

Optimal pharmacokinetic models for quantifying amyloid beta (Aβ) burden using both [ 18 F]flutemetamol and [ 18 F]florbetaben scans have previously been identified at a region of interest (ROI) level. The purpose of this study was to determine optimal quantitative methods for parametric analyses of [ 18 F]flutemetamol and [ 18 F]florbetaben scans. Forty-six participants were scanned on a PET/MR scanner using a dual-time window protocol and either [ 18 F]flutemetamol (N=24) or [ 18 F]florbetaben (N=22). The following parametric approaches were used to derive DVR estimates: reference Logan (RLogan), receptor parametric mapping (RPM), two-step simplified reference tissue model (SRTM2) and multilinear reference tissue models (MRTM0, MRTM1, MRTM2), all with cerebellar grey matter as reference tissue. In addition, a standardized uptake value ratio (SUVR) was calculated for the 90-110 min post injection interval. All parametric images were assessed visually. Regional outcome measures were compared with those from a validated ROI method, i.e. DVR derived using RLogan. Visually, RPM, and SRTM2 performed best across tracers and, in addition to SUVR, provided highest AUC values for differentiating between Aβ-positive vs Aβ-negative scans ([ 18 F]flutemetamol: range AUC=0.96-0.97 [ 18 F]florbetaben: range AUC=0.83-0.85). Outcome parameters of most methods were highly correlated with the reference method (R 2 ≥0.87), while lowest correlation were observed for MRTM2 (R 2 =0.71-0.80). Furthermore, bias was low (≤5%) and independent of underlying amyloid burden for MRTM0 and MRTM1. The optimal parametric method differed per evaluated aspect; however, the best compromise across aspects was found for MRTM0 followed by SRTM2, for both tracers. SRTM2 is the preferred method for parametric imaging because, in addition to its good performance, it has the advantage of providing a measure of relative perfusion (R 1 ), which is useful for measuring disease progression., Competing Interests: Declaration of Competing Interest None., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

The functional amyloid Orb2 belongs to the cytoplasmic polyadenylation element binding (CPEB) protein family and plays an important role in long-term memory formation in Drosophila. The Orb2 domain structure combines RNA recognition motifs with low-complexity sequences similar to many RNA-binding proteins shown to form protein droplets via liquid-liquid phase separation (LLPS) in vivo and in vitro. This similarity suggests that Orb2 might also undergo LLPS. However, cellular Orb2 puncta have very little internal protein mobility, and Orb2 forms fibrils in Drosophila brains that are functionally active indicating that LLPS might not play a role for Orb2. In the present work, we reconcile these two views on Orb2 droplet formation. Using fluorescence microscopy, we show that soluble Orb2 can indeed phase separate into protein droplets. However, fluorescence recovery after photobleaching (FRAP) data shows that these droplets have either no or only an extremely short-lived liquid phase and appear maturated right after formation. Orb2 fragments that lack the C-terminal RNA-binding domain (RBD) form fibrils out of these droplets. Solid-state NMR shows that these fibrils have well-ordered static domains in addition to the Gln/His-rich fibril core. Further, we find that full-length Orb2B, which is by far the major component of Orb2 fibrils in vivo, does not transition into fibrils but remains in the droplet phase. Together, our data suggest that phase separation might play a role in initiating the formation of functional Orb2 fibrils., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

The low depth of tissue penetration by therapeutic light sources severely restricts photodynamic therapy (PDT) in treating deep-seated tumors. Using a luciferase/d-luciferin bioluminescence system to artificially create internal light sources in cells instead of external light sources is an effective means of solving the above problems. However, high-efficiency bioluminescence requires a higher concentration of luciferase in the cell, which poses a considerable challenge to the existing system of enzyme loading, delivery, activity and retention of drugs, and dramatically increases the cost of treatment. We loaded the substrate D-luciferin, and the photosensitizer hypericin into a polyethyleneimine (PEI)-modified nano-calcium phosphate (CaP) to solve this problem. Subsequently, the plasmid DNA containing the luciferase gene was loaded onto it using the high-density positive charge characteristic of PEI from the nanodrug (denoted DHDC). After the DHDC enters the tumor cell, it collapses and releases the plasmid DNA, which uses the intracellular protein synthesis system to continuously and massively express luciferase. Using endogenous ATP, Mg 2+ , and O 2 in cells, luciferase oxidizes d-luciferin and produces luminescence. The luminescence triggers hypericin excitation to generate ROS and kill cancer cells. This study provides a new strategy for the application of bioluminescence in PDT treatment., (© 2021 Wiley-VCH GmbH.)

Telomerase RNA (TR) carries the template for synthesis of telomere DNA and provides a scaffold for telomerase assembly. Fungal TRs are long and have been compared to higher eukaryotes, where they show considerable diversity within phylogenetically close groups. TRs of several Saccharomycetaceae were recently identified, however, many of these remained uncharacterised in the template region. Here we show that this is mainly due to high variability in telomere sequence. We predicted the telomere sequences using Tandem Repeats Finder and then we identified corresponding putative template regions in TR candidates. Remarkably long telomere units and the corresponding putative TRs were found in Tetrapisispora species. Notably, variable lengths of the annealing sequence of the template region (1-10 nt) were found. Consequently, species with the same telomere sequence may not harbour identical TR templates. Thus, TR sequence alone can be used to predict a template region and telomere sequence, but not to determine these exactly. A conserved feature of telomere sequences, tracts of adjacent Gs, led us to test the propensity of individual telomere sequences to form G4. The results show highly diverse values of G4-propensity, indicating the lack of ubiquitous conservation of this feature across Saccharomycetaceae.

This work describes a single-stranded degradable modular grafting probe for analyzing microRNA-21. In the system, the exonuclease activity of phi29 polymerase restrains the SYBR Green I/ssDNA induced background. The palindrome activation caused remarkable target fluorescence. The detection limit was achieved as 0.26 fM, showing potential in biochemical analysis.

The binding mechanism of thioflavin T (ThT) to DNA was studied using polarized light spectroscopy and fluorescence-based techniques in solutions and in solid films. Linear dichroism measurements showed that ThT binds to DNA duplex by intercalation. Time-resolved fluorescence studies revealed a second binding mode which is the external binding to the DNA phosphate groups. Both binding modes represent the nonspecific type of interactions. The studies were complemented with the analysis of short oligonucleotides having DNA cavities. The results indicate that the interplay between three binding modes-intercalation, external binding, and binding inside DNA cavities-determines the effective fluorescence quantum yield of the dye in the DNA structures. External binding was found to be responsible for fluorescence quenching because of energy transfer between intercalated and externally bound molecules. Finally, amplified spontaneous emission (ASE) was successfully generated in the ThT-stained films and used for detecting different DNA structures. ASE measurements show that ThT-stained DNA structures can be used for designing bioderived microlasers.

Recently, irreversible inhibitors have attracted great interest in antitumors due to their advantages of forming covalent bonds to target proteins. Herein, some benzothiazepinone compounds (BTZs) have been designed and synthesized as novel covalent GSK-3β inhibitors with high selectivity for the kinase panel. The irreversible covalent binding mode was identified by kinetics and mass spectrometry, and the main labeled residue was confirmed to be the unique Cys14 that exists only in GSK-3β. The candidate 4 - 3 (IC 50 = 6.6 μM) showed good proliferation inhibition and apoptosis-inducing ability to leukemia cell lines, low cytotoxicity on normal cell lines, and no hERG inhibition, which hinted the potential efficacy and safety. Furthermore, 4 - 3 exhibited decent pharmacokinetic properties in vivo and remarkably inhibited tumor growth in the acute promyelocytic leukemia (APL) mouse model. All the results suggest that these newly irreversible BTZ compounds might be useful in the treatment of cancer such as APL.

Several cell-surface receptors for neurotoxic forms of amyloid-β (Aβ) have been described, but their molecular interactions with Aβ assemblies and their relative contributions to mediating Alzheimer's disease pathology have remained uncertain. Here, we used super-resolution microscopy to directly visualize Aβ-receptor interactions at the nanometer scale. We report that one documented Aβ receptor, PrP C , specifically inhibits the polymerization of Aβ fibrils by binding to the rapidly growing end of each fibril, thereby blocking polarized elongation at that end. PrP C binds neurotoxic oligomers and protofibrils in a similar fashion, suggesting that it may recognize a common, end-specific, structural motif on all of these assemblies. Finally, two other Aβ receptors, FcγRIIb and LilrB2, affect Aβ fibril growth in a manner similar to PrP C . Our results suggest that receptors may trap Aβ oligomers and protofibrils on the neuronal surface by binding to a common molecular determinant on these assemblies, thereby initiating a neurotoxic signal.

A novel bioluminescent Monoacylglycerol lipase (MAGL) substrate 6-O-arachidonoylluciferin, a D-luciferin derivative, was synthesized, physico-chemically characterized, and used as highly sensitive substrate for MAGL in an assay developed for this purpose. We present here a new method based on the enzymatic cleavage of arachidonic acid with luciferin release using human Monoacylglycerol lipase ( h MAGL) followed by its reaction with a chimeric luciferase, PLG2, to produce bioluminescence. Enzymatic cleavage of the new substrate by MAGL was demonstrated, and kinetic constants Km and Vmax were determined. 6-O-arachidonoylluciferin has proved to be a highly sensitive substrate for MAGL. The bioluminescence assay (LOD 90 pM, LOQ 300 pM) is much more sensitive and should suffer fewer biological interferences in cells lysate applications than typical fluorometric methods. The assay was validated for the identification and characterization of MAGL modulators using the well-known MAGL inhibitor JZL184. The use of PLG2 displaying distinct bioluminescence color and kinetics may offer a highly desirable opportunity to extend the range of applications to cell-based assays.

The interaction of the recently reported quinazoline derivative (E)-4-(2-(pyridin-2-ylmethylene)hydrazinyl)quinazoline (L) with a series of metal(II) (= copper(II), nickel(II), cobalt(II) and cadmium(II)) chlorides or nitrates resulted in the formation of mononuclear complexes which were characterized by spectroscopic techniques and single-crystal X-ray crystallography, i.e. [Cu(L) 2 ]Cl 2 ·4H 2 O (1·4H 2 O), [Ni(L) 2 ]Cl 2 ·4H 2 O (2·4H 2 O), [Ni(L) 2 ](NO 3 ) 2 ·MeOH (3·MeOH), [Co(L) 2 ]Cl 2 ·4H 2 O (4·4H 2 O), [Co(L) 2 ](NO 3 ) 2 ·H 2 O (5·H 2 O), [Co(L) 2 ](NO 3 ) 3 ·2.5H 2 O (6·2.5H 2 O), [Cd(L)(Cl) 2 ]·H 2 O (7·H 2 O) and [Cd(L)(CH 3 OH)(H 2 O)(NO 3 )](NO 3 ) (8). The biological profile of the complexes was further assessed in regard to their binding affinity with calf-thymus DNA, their cleavage ability towards pBluescript II KS plasmid DNA in the absence or presence of irradiation of various wavelengths, their interaction with bovine serum albumin and finally, their ability to scavenge 1,1-diphenyl-picrylhydrazyl and 2,2΄-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) radicals and to reduce H 2 O 2 ., (Copyright © 2021 Elsevier Inc. All rights reserved.)

An amphipathic α-helical peptide, Hp1404, was isolated from the venomous gland of the scorpion Heterometrus petersii . Hp1404 exhibits antimicrobial activity against methicillin-resistant Staphylococcus aureus but is cytotoxic. In this study, we designed antimicrobial peptides by substituting amino acids at the 14 C-terminal residues of Hp1404 to reduce toxicity and improve antibacterial activity. The analog peptides, which had an amphipathic α-helical structure, were active against gram-positive and gram-negative bacteria, particularly multidrug-resistant Acinetobacter baumannii , and showed lower cytotoxicity than Hp1404. N -phenyl-1-naphthylamine uptake and DisC 3 -5 assays demonstrated that the peptides kill bacteria by effectively permeating the outer and cytoplasmic membranes. Additionally, the analog peptides inhibited biofilm formation largely than Hp1404 at low concentrations. These results suggest that the analog peptides of Hp1404 can be used as therapeutic agents against A. baumannii infection.

Protein detection plays an important role in biological and biomedical sciences. The immunoassay based on fluorescence labeling has good specificity but a high labeling cost. Herein, on the basis of G-triplex molecular beacon (G3MB) and thioflavin T (ThT), we developed a simple and label-free biosensor for protein detection. The biotin and streptavidin were used as model enzymes. In the presence of target streptavidin (SA), the streptavidin hybridized with G3MB-b (biotin-linked-G-triplex molecular beacon) perfectly and formed larger steric hindrance, which hindered the hydrolysis of probes by exonuclease III (Exo III). In the absence of target streptavidin, the exonuclease III successively cleaved the stem of G3MB-b and released the G-rich sequences which self-assembled into a G-triplex and subsequently activated the fluorescence signal of thioflavin T. Compared with the traditional G-quadruplex molecular beacon (G4MB), the G3MB only needed a lower dosage of exonuclease III and a shorter reaction time to reach the optimal detection performance, because the concise sequence of G-triplex was good for the molecular beacon design. Moreover, fluorescence experiment results exhibited that the G3MB-b had good sensitivity and specificity for streptavidin detection. The developed label-free biosensor provides a valuable and general platform for protein detection.

Transient receptor potential cation channel subfamily M member 5 (TRPM5) is a nonselective monovalent cation channel activated by intracellular Ca 2+ increase. Within the gastrointestinal system, TRPM5 is expressed in the stoma, small intestine, and colon. In the search for a selective agonist of TRPM5 possessing in vivo gastrointestinal prokinetic activity, a high-throughput screening was performed and compound 1 was identified as a promising hit. Hit validation and hit to lead activities led to the discovery of a series of benzo[ d ]isothiazole derivatives. Among these, compounds 61 and 64 showed nanomolar activity and excellent selectivity (>100-fold) versus related cation channels. The in vivo drug metabolism and pharmacokinetic profile of compound 64 was found to be ideal for a compound acting locally at the intestinal level, with minimal absorption into systemic circulation. Compound 64 was tested in vivo in a mouse motility assay at 100 mg/kg, and demonstrated increased prokinetic activity.

Amyloid fibril formation of α-synuclein (αS) is associated with multiple neurodegenerative diseases, including Parkinson's disease (PD). Growing evidence suggests that progression of PD is linked to cell-to-cell propagation of αS fibrils, which leads to seeding of endogenous intrinsically disordered monomer via templated elongation and secondary nucleation. A molecular understanding of the seeding mechanism and driving interactions is crucial to inhibit progression of amyloid formation. Here, using relaxation-based solution NMR experiments designed to probe large complexes, we probe weak interactions of intrinsically disordered acetylated-αS (Ac-αS) monomers with seeding-competent Ac-αS fibrils and seeding-incompetent off-pathway oligomers to identify Ac-αS monomer residues at the binding interface. Under conditions that favor fibril elongation, we determine that the first 11 N-terminal residues on the monomer form a common binding site for both fibrils and off-pathway oligomers. Additionally, the presence of off-pathway oligomers within a fibril seeding environment suppresses seeded amyloid formation, as observed through thioflavin-T fluorescence experiments. This highlights that off-pathway αS oligomers can act as an auto-inhibitor against αS fibril elongation. Based on these data taken together with previous results, we propose a model in which Ac-αS monomer recruitment to the fibril is driven by interactions between the intrinsically disordered monomer N terminus and the intrinsically disordered flanking regions (IDR) on the fibril surface. We suggest that this monomer recruitment may play a role in the elongation of amyloid fibrils and highlight the potential of the IDRs of the fibril as important therapeutic targets against seeded amyloid formation., Competing Interests: The authors declare no competing interest.

Tau and Aβ assemblies of Alzheimer's disease (AD) can be visualized in living subjects using positron emission tomography (PET). Tau assemblies comprise paired helical and straight filaments (PHFs and SFs). APN-1607 (PM-PBB3) is a recently described PET ligand for AD and other tau proteinopathies. Since it is not known where in the tau folds PET ligands bind, we used electron cryo-microscopy (cryo-EM) to determine the binding sites of APN-1607 in the Alzheimer fold. We identified two major sites in the β-helix of PHFs and SFs and a third major site in the C-shaped cavity of SFs. In addition, we report that tau filaments from posterior cortical atrophy (PCA) and primary age-related tauopathy (PART) are identical to those from AD. In support, fluorescence labelling showed binding of APN-1607 to intraneuronal inclusions in AD, PART and PCA. Knowledge of the binding modes of APN-1607 to tau filaments may lead to the development of new ligands with increased specificity and binding activity. We show that cryo-EM can be used to identify the binding sites of small molecules in amyloid filaments.