Antioxidant Activities of Exopolysaccharides Extracts from Two Endemic Fungi from Patagonia.

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.

Heterologous expression and characterization of a dye-decolorizing peroxidase from Ganoderma lucidum, and its application in decolorization and detoxifization of different types of dyes.

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 T50 value of 51 °C. Moreover, compared to untreated controls, the activity of GlDyP2 was inhibited by 1.60 mM of Mg2+, Ni2+, Mn2+, and ethanol; 0.16 mM of Cu2+, Zn2+, methanol, isopropyl alcohol, and Na2EDTA·2H2O; and 0.016 mM of Fe2+ 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.

New G-Triplex DNA Dramatically Activates the Fluorescence of Thioflavin T and Acts as a Turn-On Fluorescent Sensor for Uracil-DNA Glycosylase Activity Detection.

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.

Anticancer Potential of the S-Heterocyclic Ring Containing Drugs and its Bioactivation to Reactive Metabolites.

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.

Synthesis and biological evaluation of radioiodinated benzoxazole and benzothiazole derivatives for imaging myelin in multiple sclerosis.

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 ([125I]BO-1, [125I]BO-2, and [125I]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, [125I]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 [125I]BO-2 was reduced in the lysophosphatidylcholine-induced demyelination region. In conclusion, [123I]BO-2 demonstrated the fundamental characteristics of a myelin imaging probe for SPECT.

Construction of luciferase-expressing Neospora caninum and drug screening.

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 (IC50). 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 IC50 for anti-N. caninum drugs. Using the Nc1-Luc strains, TAK-632 showed promising activity against N. caninum, with an IC50 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.

Radiosynthesis and In Vitro Evaluation of [11C]tozadenant as Adenosine A2A Receptor Radioligand.

Tozadenant (4-hydroxy-N-(4-methoxy-7-morpholinobenzo[d]thiazol-2-yl)-4-methylpiperidine-1-carboxamide) is a highly selective adenosine A2A receptor (A2AR) antagonist and a promising lead structure for the development of A2AR-selective positron emission tomography (PET) probes. Although several 18F-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 18F-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-11C]tozadenant ([11C]tozadenant) by O-methylation with [11C]methyl iodide followed by acidic deprotection. This approach afforded [11C]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 A2AR 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 benzothiazole-based mononuclear platinum(II) complex displaying potent antiproliferative activity in HepG-2 cells via mitochondrial-mediated apoptosis.

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 PtII 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 = IC50(L02)/IC50(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.

Optimization of liquid fermentation conditions for Coprinus comatus to enhance antioxidant activity.

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 EC50 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 EC50 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.

Real-time monitoring of endogenous cysteine in LPS-induced oxidative stress process with a novel lysosome-targeted fluorescent probe.

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.

Vanillin Benzothiazole Derivative Reduces Cellular Reactive Oxygen Species and Detects Amyloid Fibrillar Aggregates in Alzheimer's Disease Brain.

The misfolding of amyloid beta (Aß) peptides into Aß fibrillary aggregates is a major hallmark of Alzheimer's disease (AD), which responsible for the excess production of hydrogen peroxide (H2O2), a prominent reactive oxygen species (ROS) from the molecular oxygen (O2) by the reduction of the Aß-Cu(I) complex. The excessive production of H2O2 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 H2O2, 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 Aß fibrillary aggregates with a high affinity (Kd = 143 ± 12 nM). Finally, the VBD reduces the cellular toxic H2O2 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.

The flutemetamol analogue cyano-flutemetamol detects myocardial AL and ATTR amyloid deposits: a post-mortem histofluorescence analysis.

BACKGROUND: [18F]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 [18F]flutemetamol PET imaging in patients with ATTR and AL types of cardiac amyloidosis.

Comparative analysis of thioflavin T and other fluorescent dyes for fluorescent staining of Bacillus subtilis vegetative cell, sporulating cell, and mature spore.

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.

Design and Development of Benzothiazole-Based Fluorescent Probes for Selective Detection of Aß Aggregates in Alzheimer's Disease.

The formation and accumulation of amyloid beta (Aß) 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 Aß 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 Aß aggregates. RM-28 also showed high sensitivity (7.5-fold) and high affinities toward Aß aggregates (Kd = 175.69 ± 4.8 nM; Ka = 0.5 × 107 M-1). It can cross the blood-brain barrier of mice efficiently. The affinity of RM-28 toward Aß 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 Aß aggregates. Moreover, RM-28 is highly specific to Aß 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 Aß aggregates in both in vitro and in vivo model systems.

An odorant binding protein mediates Bactrocera dorsalis olfactory sensitivity to host plant volatiles and male attractant compounds.

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.

Quantitative analysis of regional distribution of tau pathology with 11C-PBB3-PET in a clinical setting.

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 Aß42 (< 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.

Biochemical and biophysical features of disease-associated tau mutants V363A and V363I.

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.

Experimental Evidence of Intrinsic Disorder and Amyloid Formation by the Henipavirus W Proteins.

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.

Comparison of 11C-Pittsburgh Compound B and 18F-Flutemetamol White Matter Binding in PET.

PET imaging with ß-amyloid ligands is emerging as a molecular imaging technique targeting white matter integrity and demyelination. ß-amyloid PET ligands such as 11C-Pittsburgh compound B (11C-PiB) have been considered for quantitative measurement of myelin content changes in multiple sclerosis, but 11C-PiB is not commercially available given its short half-life. A 18F 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 ß-amyloid PET. Participants prospectively underwent MRI, 11C-PiB, and 18F-flutemetamol PET scans. MRI fluid-attenuated inversion recovery images were segmented into WMH and NAWM and registered to the T1-weighted MRI. 11C-PiB and 18F-flutemetamol PET images were also registered to the T1-weighted MRI. 11C-PiB and 18F-flutemetamol SUV ratios (SUVrs) from the WMH and NAWM were calculated using cerebellar crus uptake as a reference for both 11C-PiB and 18F-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 18F-flutemetamol than with 11C-PiB in both older (P < 0.001) and younger (P < 0.001) CU adults. 11C-PiB and 18F-flutemetamol SUVrs were higher in older than in younger CU adults in both WMH (P < 0.001) and NAWM (P < 0.001). 11C-PiB and 18F-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 11C-PiB and 18F-flutemetamol SUVrs in differentiating WMH from NAWM in older and in younger adults. Conclusion:11C-PiB and 18F-flutemetamol show a similar topographic pattern of uptake in white matter with a similar association with age in WMH and NAWM. 11C-PiB and 18F-flutemetamol can also effectively distinguish between WMH and NAWM. However, given its longer half-life, commercial availability, and higher binding potential, 18F-flutemetamol can be an alternative to 11C-PiB in molecular imaging studies specifically targeting multiple sclerosis to evaluate white matter integrity.

Design, synthesis, and mechanistic investigations of phenylalanine derivatives containing a benzothiazole moiety as HIV-1 capsid inhibitors with improved metabolic stability.

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 (t1/2) 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.