Cu(II) Specifically Disassembles Insulin Amyloid Nanostructures via Direct Interaction with Cross-ß Fibrils.

In this work, we demonstrate direct evidence of the antiamyloid potential of Cu(II) ions against amyloid formation of insulin. The Cu(II) ions were found to efficiently disassemble the preformed amyloid nanostructures into soluble species and suppress monomer fibrillation under aggregation-prone conditions. The direct interaction of Cu(II) ions with the cross-ß structure of amyloid fibrils causes substantial disruption of both the interchain and intrachain interactions, predominantly the H-bonds and hydrophobic contacts. Further, the Cu(II) ions show a strong affinity for the aggregation-prone conformers of the protein and inhibit their spontaneous self-assembly. These results reveal the possible molecular mechanism for the antiamyloidogenic potential of Cu(II) which could be important for the development of metal-ion specific therapeutic strategies against amyloid linked complications.

Key contributions of a glycolipid to membrane protein integration.

Regulation of membrane protein integration involves molecular devices such as Sec-translocons or the insertase YidC. We have identified an integration-promoting factor in the inner membrane of Escherichia coli called membrane protein integrase (MPIase). Structural analysis revealed that, despite its enzyme-like name, MPIase is a glycolipid with a long glycan comprising N-acetyl amino sugars, a pyrophosphate linker, and a diacylglycerol (DAG) anchor. Additionally, we found that DAG, a minor membrane component, blocks spontaneous integration. In this review, we demonstrate how they contribute to Sec-independent membrane protein integration in bacteria using a comprehensive approach including synthetic chemistry and biophysical analyses. DAG blocks unfavorable spontaneous integrations by suppressing mobility in the membrane core, whereas MPIase compensates for this. Moreover, MPIase plays critical roles in capturing a substrate protein to prevent its aggregation, attracting it to the membrane surface, facilitating its insertion into the membrane, and delivering it to other factors. The combination of DAG and MPIase efficiently regulates the integration of membrane proteins.

Sensitive SERS assay for L-cysteine based on functionalized silver nanoparticles.

L-cysteine, an indispensable amino acid present in natural proteins, plays pivotal roles in various biological processes. Consequently, precise and selective monitoring of its concentrations is imperative. Herein, we propose a Surface-enhanced Raman Scattering (SERS) sensor for detecting L-cysteine based on the anti-aggregation of 4-mercaptobenzoic acid (4-MBA) and histidine (His) functionalized silver nanoparticles (Ag NPs). The presence of Hg2+ ions can induce the aggregation of Ag NPs@His@4-MBA due to the unique nanostructures of Ag NPs@His@4-MBA, resulting in a robust SERS intensity of 4-MBA. However, in the presence of L-cysteine, the stronger affinity between L-cysteine and Hg2+ reduces the concentration of free Hg2+, causing the dispersion of the aggregated functionalized Ag NPs and the reduction of the SERS signal intensity of 4-MBA. The developed SERS platform demonstrates excellent performance with a low detection limit of 5 nM (S/N = 3) and linear detection capabilities within the range of 0.01-100 µM for L-cysteine. Additionally, the method was successfully employed for the determination of L-cysteine in spiked serum samples, yielding recoveries ranging from 95.0 % to 108.1 % with relative standard deviations of less than 3.3 %. This study not only presents a novel approach for fabricating highly sensitive and specific SERS biosensors for biomolecule detection but also offers a significant strategy for the development and construction of SERS substrates using anti-aggregation design.

2-Butoxytetrahydrofuran, Isolated from Holothuria scabra, Attenuates Aggregative and Oxidative Properties of α-Synuclein and Alleviates Its Toxicity in a Transgenic Caenorhabditis elegans Model of Parkinson's Disease.

Aggregative α-synuclein and incurring oxidative stress are pivotal cascading events, leading to dopaminergic (DAergic) neuronal loss and contributing to clinical manifestations of Parkinson's disease (PD). Our previous study demonstrated that 2-butoxytetrahydrofuran (2-BTHF), isolated from Holothuria scabra (H. scabra), could inhibit amyloid-ß aggregation and its ensuing toxicity, which leads to Alzheimer's disease. In the present study, we found that 2-BTHF also attenuated the aggregative and oxidative activities of α-synuclein and lessened its toxicity in a transgenic Caenorhabditis elegans (C. elegans) PD model. Such worms treated with 100 µM of 2-BTHF showed substantial reductions in α-synuclein accumulation and DAergic neurodegeneration. Mechanistically, 2-BTHF, at this concentration, significantly decreased aggregation of monomeric α-synuclein and restored locomotion and dopamine-dependent behaviors. Molecular docking exhibited potential bindings of 2-BTHF to HSF-1 and DAF-16 transcription factors. Additionally, 2-BTHF significantly increased the mRNA transcripts of genes encoding proteins involved in proteostasis, including the molecular chaperones hsp-16.2 and hsp-16.49, the ubiquitination/SUMOylation-related ubc-9 gene, and the autophagy-related genes atg-7 and lgg-1. Transcriptomic profiling revealed an additional mechanism of 2-BTHF in α-synuclein-expressing worms, which showed upregulation of PPAR signaling cascades that mediated fatty acid metabolism. 2-BTHF significantly restored lipid deposition, upregulated the fat-7 gene, and enhanced gcs-1-mediated glutathione synthesis in the C. elegans PD model. Taken together, this study demonstrated that 2-BTHF could abrogate aggregative and oxidative properties of α-synuclein and attenuate its toxicity, thus providing a possible therapeutic application for the treatment of α-synuclein-induced PD.

Curcumin Inhibits α-Synuclein Aggregation by Acting on Liquid-Liquid Phase Transition.

Parkinson's disease (PD), the second most common neurodegenerative disorder, is linked to α-synuclein (α-Syn) aggregation. Despite no specific drug being available for its treatment, curcumin, from the spice turmeric, shows promise. However, its application in PD is limited by a lack of understanding of its anti-amyloidogenic mechanisms. In this study, we first reconstructed the liquid-liquid phase separation (LLPS) of α-Syn in vitro under different conditions, which may be an initial step in entraining the pathogenic aggregation. Subsequently, we evaluated the effects of curcumin on the formation of droplets, oligomers, and aggregated fibers during the LLPS of α-synuclein, as well as its impact on the toxicity of aggregated α-synuclein to cultured cells. Importantly, we found that curcumin can inhibit amyloid formation by inhibiting the occurrence of LLPS and the subsequent formation of oligomers of α-Syn in the early stages of aggregation. Finally, the molecular dynamic simulations of interactions between α-Syn decamer fibrils and curcumin showed that van der Waal's interactions make the largest contribution to the anti-aggregation effect of curcumin. These results may help to clarify the mechanism by which curcumin inhibits the formation of α-Syn aggregates during the development of PD.

Verbenone Affects the Behavior of Insect Predators and Other Saproxylic Beetles Differently: Trials Using Pheromone-Baited Bark Beetle Traps.

In our study, we assessed the effects of verbenone, the most widely studied bark beetle aggregation inhibitor, on saproxylic beetles in a Mediterranean pine forest in Tuscany. Verbenone pouches were devised in the laboratory and then applied to Ips sexdentatus pheromone traps so that their catches could be compared to those of traps containing just the pheromone. The trial was carried out in spring-summer 2023, and insect catches were collected every two weeks. A total of 9440 beetles were collected that belonged to 32 different families and 57 species. About 80% of the captures were bark beetles, mainly Orthotomicus erosus. Beetle predators accounted for about 17% of the captures, with a total of 12 species. Some of these predator species had not yet been studied in relation to verbenone effects, like other saproxylic beetles recorded in this study. A significant reduction in captures was recorded for some beetles (e.g., I. sexdentatus and O. erosus), while for other species, no differences emerged, and in some cases, captures increased significantly when verbenone was present in the traps (i.e., Hylurgus ligniperda, Corticeus pini, and Aulonium ruficorne). The diversity of caught saproxylic beetles increased significantly in the verbenone traps, highlighting possible implications of the use of verbenone when managing bark beetle outbreaks.

Riesgo de hemorragia cerebral en el traumatismo craneal leve y tratamiento antitrombótico / Risk of cerebral hemorrhage in mild traumatic brain injury and antithrombotic treatment

Objetivo El tiempo de observación en el traumatismo craneoencefálico leve (TCEL) es controvertido. Nuestro objetivo se basó en evaluar el riesgo de complicaciones neurológicas en el TCEL con y sin tratamiento antitrombótico. Método Evaluamos retrospectivamente los pacientes con TCEL atendidos en urgencias durante 3 años. Consideramos TCEL aquellos con Glasgow ≥13 al ingreso. Se realizó una TC craneal en todos los casos con >1 factor de riesgo al ingreso y a las 24h en aquellos con deterioro neurológico o TC craneal inicial patológica. Se revisó retrospectivamente las complicaciones en los siguientes 3 meses. Resultados Evaluamos 907 pacientes con una edad media de 73±19 años. El 91% presentaron factores de riesgo, con un 60% en tratamiento antitrombótico. Detectamos un 11% de hemorragia cerebral inicial, 0,4% a las 24h y ningún caso a los 3 meses. El tratamiento antitrombótico no se asoció con incremento de riesgo de hemorragia cerebral (9,9 con vs. 11,9% sin tratamiento; p=0,3). El 39% de las hemorragias presentaron síntomas neurológicos (18% amnesia postraumática, 12% cefalea, 8% vómitos, 1% convulsiones), siendo en un 78,4% síntomas leves. De las 4 hemorragias detectadas a las 24h, 3 fueron asintomáticas y un caso emporó la cefalea inicial. Ningún paciente asintomático sin lesión en la TC craneal inicial presentó clínica a las 24h. Conclusiones Nuestro estudio sugiere que los pacientes con TCEL asintomáticos, sin lesión en la TC craneal inicial no precisarían periodo de observación ni TC craneal de control, independientemente del tratamiento antitrombótico o nivel de INR (AU)

Introduction The observation time in mild traumatic brain injury (mTBI) is controversial. Our aim was to assess the risk of neurological complications in mTBI with and without antithrombotic treatment. Method We retrospectively evaluated patients with mTBI seen in the emergency room for 3 years. We considered MTBI those with Glasgow ≥13 at admission. A cranial CT was performed in all cases with >1 risk factor at admission and at 24h in those with neurological impairment or initial pathological cranial CT. Complications in the following 3 months were retrospectively reviewed. Results We evaluated 907 patients with a mean age of 73±19 years. Ninety-one percent presented risk factors, with 60% on antithrombotic treatment. We detected 11% of initial brain hemorrhage, 0.4% at 24h, and no cases at 3 months. Antithrombotic treatment was not associated with an increased risk of brain hemorrhage (9.9% with vs. 11.9% without treatment, P=.3). 39% of the hemorrhages presented neurological symptoms (18% post-traumatic amnesia, 12% headache, 8% vomiting, 1% seizures), with 78.4% having mild symptoms. Of the 4 hemorrhages detected at 24h, 3 were asymptomatic and one case that worsened the initial headache. No asymptomatic patient without lesion on initial clinical cranial CT presented at 24h. Conclusions Our study suggests that patients with asymptomatic mTBI, without a lesion on the initial cranial CT, would not require the observation period or CT control regardless of antithrombotic treatment or INR level (AU)

Hesperidin Methyl Chalcone reduces extracellular Aß(25-35) peptide aggregation and fibrillation and also protects Neuro 2a cells from Aß(25-35) induced neuronal dysfunction.

In the present study, we evaluated the neuroprotective potential of Hesperidin Methyl Chalcone (HMC) against the neurotoxicity induced by Aß(25-35) peptide. HMC demonstrated higher free-radical scavenging activity than Hesperidin in initial cell-free studies. Investigations using the fluorescent dye thioflavin T with Aß(25-35) peptide showed that HMC has the ability to combat extracellular amyloid aggregation by possessing anti-aggregation property against oligomers and by disaggregating mature fibrils. Also, the results of the molecular simulation studies show that HMC ameliorated oligomer formation. Further, the anti-Alzheimer's property of HMC was investigated in in vitro cell conditions by pre-treating the neuro 2a (N2a) cells with HMC before inducing Aß(25-35) toxicity. The findings demonstrate that HMC increased cell viability, reduced oxidative stress, prevented macromolecular damage, allayed mitochondrial dysfunction, and exhibited anticholinesterase activity. HMC also reduced Aß induced neuronal cell death by modulating caspase-3 activity, Bax expression and Bcl2 overexpression, demonstrating that HMC pre-treatment reduced mitochondrial damage and intrinsic apoptosis induced by Aß(25-35).In silico evaluation against potential AD targets reveal that HMC could be a potent inhibitor of BACE-1, inhibiting the formation of toxic Aß peptides. Overall, the findings imply that the neuroprotective efficacy of HMC has high prospects for addressing a variety of pathogenic consequences caused by amyloid beta in AD situations and alleviating cognitive impairments.

Risk of cerebral hemorrhage in mild traumatic brain injury and antithrombotic treatment.

INTRODUCTION: The observation time in mild traumatic brain injury (mTBI) is controversial. Our aim was to assess the risk of neurological complications in mTBI with and without antithrombotic treatment. METHOD: We retrospectively evaluated patients with mTBI seen in the emergency room for 3 years. We considered MTBI those with Glasgow ≥13 at admission. A cranial CT was performed in all cases with ≥1 risk factor at admission and at 24 h in those with neurological impairment or initial pathological cranial CT. Complications in the following 3 months were retrospectively reviewed. RESULTS: We evaluated 907 patients with a mean age of 73 ±â€¯19 years. Ninety-one percent presented risk factors, with 60% on antithrombotic treatment. We detected 11% of initial brain hemorrhage, 0.4% at 24 h, and no cases at 3 months. Antithrombotic treatment was not associated with an increased risk of brain hemorrhage (9.9% with vs 11.9% without treatment, p = 0.3). 39% of the hemorrhages presented neurological symptoms (18% post-traumatic amnesia, 12% headache, 8% vomiting, 1% seizures), with 78.4% having mild symptoms. Of the 4 hemorrhages detected at 24 h, 3 were asymptomatic and one case that worsened the initial headache. No asymptomatic patient without lesion on initial clinical cranial CT presented at 24 h. CONCLUSIONS: Our study suggests that patients with asymptomatic mTBI, without a lesion on the initial cranial CT, would not require the observation period or CT control regardless of antithrombotic treatment or INR level.

Evaluation of N- and O-Linked Indole Triazines for a Dual Effect on α-Synuclein and Tau Aggregation.

Alzheimer's disease (AD) is the most prevalent neurodegenerative disorder underlying dementia in the geriatric population. AD manifests by two pathological hallmarks: extracellular amyloid-ß (Aß) peptide-containing senile plaques and intraneuronal neurofibrillary tangles comprised of aggregated hyperphosphorylated tau protein (p-tau). However, more than half of AD cases also display the presence of aggregated α-synuclein (α-syn)-containing Lewy bodies. Conversely, Lewy bodies disorders have been reported to have concomitant Aß plaques and neurofibrillary tangles. Our drug discovery program focuses on the synthesis of multitarget-directed ligands to abrogate aberrant α-syn, tau (2N4R), and p-tau (1N4R) aggregation and to slow the progression of AD and related dementias. To this end, we synthesized 11 compounds with a triazine-linker and evaluated their effectiveness in reducing α-syn, tau isoform 2N4R, and p-tau isoform 1N4R aggregation. We utilized biophysical methods such as thioflavin T (ThT) fluorescence assays, transmission electron microscopy (TEM), photoinduced cross-linking of unmodified proteins (PICUP), and M17D intracellular inclusion cell-based assays to evaluate the antiaggregation properties and cellular protection of our best compounds. We also performed disaggregation assays with isolated Aß-plaques from human AD brains. Our results demonstrated that compound 10 was effective in reducing both oligomerization and fibril formation of α-syn and tau isoform 2N4R in a dose-dependent manner via ThT and PICUP assays. Compound 10 was also effective at reducing the formation of recombinant α-syn, tau 2N4R, and p-tau 1N4R fibrils by TEM. Compound 10 reduced the development of α-syn inclusions in M17D neuroblastoma cells and stopped the seeding of tau P301S using biosensor cells. Disaggregation experiments showed smaller Aß-plaques and less paired helical filaments with compound 10. Compound 10 may provide molecular scaffolds for further optimization and preclinical studies for neurodegenerative proteinopathies.

Antiplatelet Effects of Selected Xanthine-Based Adenosine A2A and A2B Receptor Antagonists Determined in Rat Blood.

The platelet aggregation inhibitory activity of selected xanthine-based adenosine A2A and A2B receptor antagonists was investigated, and attempts were made to explain the observed effects. The selective A2B receptor antagonist PSB-603 and the A2A receptor antagonist TB-42 inhibited platelet aggregation induced by collagen or ADP. In addition to adenosine receptor blockade, the compounds were found to act as moderately potent non-selective inhibitors of phosphodiesterases (PDEs). TB-42 showed the highest inhibitory activity against PDE3A along with moderate activity against PDE2A and PDE5A. The antiplatelet activity of PSB-603 and TB-42 may be due to inhibition of PDEs, which induces an increase in cAMP and/or cGMP concentrations in platelets. The xanthine-based adenosine receptor antagonists were found to be non-cytotoxic for platelets. Some of the compounds showed anti-oxidative properties reducing lipid peroxidation. These results may provide a basis for the future development of multi-target xanthine derivatives for the treatment of inflammation and atherosclerosis and the prevention of heart infarction and stroke.

Exploring the impact of nucleic acids on protein stability in bacterial cell lysate.

- Addition of salt enhanced thermal stability of model substrate proteins by reducing electrostatic repulsion between protein molecules. - However, the opposite effect was observed with bacterial cell lysate, indicating that certain molecules within the lysate could enhance protein stability via electrostatic interactions. - Such molecules present in cell lysate were found to be nucleic acids known to have a potent anti-aggregation activity toward proteins involving electrostatic interactions. - Nucleic acids showed chaperone activity in physiological salt concentration within cells and in buffer or medium commonly used in experiments. - The chaperone activity of nucleic acids should be taken into account when performing various in vitro assays using cell lysate or samples containing nucleic acids.

Oxymatrine-mediated prevention of amyloid ß-peptide-induced apoptosis on Alzheimer's model PC12 cells: in vitro cell culture studies and in vivo cognitive assessment in rats.

Alzheimer's disease (AD) is a major neurological disease affecting elderly individuals worldwide. Existing drugs only reduce the symptoms of the disease without addressing the underlying causes. Commonly, Aß25-35 peptide aggregation is the main reason for AD development. Recently, the discovery of multiple protein-targeting molecules has provided a new strategy for treating AD. This study demonstrates the neuroprotective potential of oxymatrine against multiple mechanisms, such as acetylcholinesterase, mitochondrial damage, and ß-amyloid-induced cell toxicity. The in vitro cell culture studies showed that oxymatrine possesses significant potential to inhibit acetylcholine esterase and promotes antioxidant, antiapoptotic effects while preventing Aß25-35 peptide aggregation in PC12 cells. Furthermore, oxymatrine protects PC12 cells against Aß25-35-induced cytotoxicity and down-regulates the reactive oxygen species generation. The in vivo acute toxicological studies confirm the safety of oxymatrine without causing organ damage or death in animals. Overall, this study provided evidence that oxymatrine is an efficient neuroprotective agent, with a potential to be a multifunctional drug for Alzheimer's disease treatment. These findings present a reliable and synergistic approach for treating AD.

Plasmonic nanoparticle's anti-aggregation application in sensor development for water and wastewater analysis.

Colorimetric sensors have emerged as a powerful tool in the detection of water pollutants. Plasmonic nanoparticles use localized surface plasmon resonance (LSPR)-based colorimetric sensing. LSPR-based sensing can be accomplished through different strategies such as etching, growth, aggregation, and anti-aggregation. Based on these strategies, various sensors have been developed. This review focuses on the newly developed anti-aggregation-based strategy of plasmonic nanoparticles. Sensors based on this strategy have attracted increasing interest because of their exciting properties of high sensitivity, selectivity, and applicability. This review highlights LSPR-based anti-aggregation sensors, their classification, and role of plasmonic nanoparticles in these sensors for the detection of water pollutants. The anti-aggregation based sensing of major water pollutants such as heavy metal ions, anions, and small organic molecules has been summarized herein. This review also provides some personal insights into current challenges associated with anti-aggregation strategy of LSPR-based colorimetric sensors and proposes future research directions.

Soy protein particles with enhanced anti-aggregation behaviors under various heating temperatures, pH, and ionic strengths.

Protein-containing food products are frequently heated during processing to passivate anti-nutritional components. However, heating also contributes to protein aggregation and gelation, which limits its application in protein-based aqueous systems. In this study, heat-stable soy protein particles (SPPs) were fabricated by preheating at 120 °C for 30 min and at 0.5% (w/v) protein concentration. Compared to untreated soy proteins (SPs), SPPs exhibited a higher denaturation ratio, stronger conformational rigidity, compacter colloidal structure, and higher surface charge. The aggregation state of SPs and SPPs at various heating conditions (temperatures, pH, ionic strength, and types) was analyzed by dynamic light scattering, atomic force microscopy, and cryo-scanning electron microscopy. SPPs showed less increase in particle size and greater anti-aggregation ability than SPs. When heated in the presence of salt ions (Na+, Ca2+) or at acidic conditions, both SPs and SPPs formed larger spherical particles, but the size increase rate of SPPs was significantly lower than SPs. These findings provide theoretical information for preparing heat-stable SPPs. Furthermore, the development of SPPs is conducive to designing protein-enriched ingredients for producing innovative foods.

Aß8-20 Fragment as an Anti-Fibrillogenic and Neuroprotective Agent: Advancing toward Efficient Alzheimer's Disease Treatment.

Alzheimer's disease (AD) is the most common cause of dementia, characterized by a spectrum of symptoms associated with memory loss and cognitive decline with deleterious consequences in everyday life. The lack of specific drugs for the treatment and/or prevention of this pathology makes AD an ever-increasing economic and social emergency. Oligomeric species of amyloid-beta (Aß) are recognized as the primary cause responsible for synaptic dysfunction and neuronal degeneration, playing a crucial role in the onset of the pathology. Several studies have been focusing on the use of small molecules and peptides targeting oligomeric species to prevent Aß aggregation and toxicity. Among them, peptide fragments derived from the primary sequence of Aß have also been used to exploit any eventual recognition abilities toward the full-length Aß parent peptide. Here, we test the Aß8-20 fragment which contains the self-recognizing Lys-Leu-Val-Phe-Phe sequence and lacks Arg 5 and Asp 7 and the main part of the C-terminus, key points involved in the aggregation pathway and stabilization of the fibrillary structure of Aß. In particular, by combining chemical and biological techniques, we show that Aß8-20 does not undergo random coil to ß sheet conformational transition, does not form amyloid fibrils by itself, and is not toxic for neuronal cells. Moreover, we demonstrate that Aß8-20 mainly interacts with the 4-11 region of Aß1-42 and inhibits the formation of toxic oligomeric species and Aß fibrils. Finally, our data show that Aß8-20 protects neuron-like cells from Aß1-42 oligomer toxicity. We propose Aß8-20 as a promising drug candidate for the treatment of AD.

Bioflavonoids ameliorate crowding induced hemoglobin aggregation: a spectroscopic and molecular docking approach.

The cellular environment is densely crowded, confining biomacromolecules including proteins to less available space. This macromolecular confinement may affect the physiological conformation of proteins in long-term processes like ageing. Changes in physiological protein structure can lead to protein conformational disorders including neurodegeneration. An intervention approach using food and plant derived bioflavonoids offered a way to find a treatment for these enervating pathological conditions as there is no remedy available. The bioflavonoids NAR (naringenin), 7HD (7 hydroxyflavanone) and CHR (chrysin) were tested for their ability to protect Hb (hemoglobin) against crowding-induced aggregation. Morphological and secondary structural transitions were studied using microscopic and circular dichroism experiments, respectively. The kinetic study was carried out using the relative thioflavin T assay. Molecular docking, AmylPred2, admetSAR and FRET were applied to understand the binding parameters of bioflavonoids with Hb and their drug likeliness. Isolated human lymphocytes were used as a cellular system to study the toxic effects of Hb aggregates. Redox perturbation and cytotoxicity were evaluated by DCFH-DA and MTT assays, respectively. This study suggests that bioflavonoids bind to Hb in the vicinity of aggregation prone amino acid sequences. Binding of the bioflavonoids stabilizes the Hb against crowding-induced structural alterations. Therefore, this study signifies the potential of bioflavonoids for future treatment of many proteopathies including neurodegeneration.Communicated by Ramaswamy H. Sarma.

Computational screening for new neuroprotective ingredients against Alzheimer's disease from bilberry by cheminformatics approaches.

Bioactive ingredients from natural products have always been an important resource for the discovery of drugs for Alzheimer's disease (AD). Senile plaques, which are formed with amyloid-beta (Aß) peptides and excess metal ions, are found in AD brains and have been suggested to play an important role in AD pathogenesis. Here, we attempted to design an effective and smart screening method based on cheminformatics approaches to find new ingredients against AD from Vaccinium myrtillus (bilberry) and verified the bioactivity of expected ingredients through experiments. This method integrated advanced artificial intelligence models and target prediction methods to realize the stepwise analysis and filtering of all ingredients. Finally, we obtained the expected new compound malvidin-3-O-galactoside (Ma-3-gal-Cl). The in vitro experiments showed that Ma-3-gal-Cl could reduce the OH· generation and intracellular ROS from the Aß/Cu2+/AA mixture and maintain the mitochondrial membrane potential of SH-SY5Y cells. Molecular docking and Western blot results indicated that Ma-3-gal-Cl could reduce the amount of activated caspase-3 via binding with unactivated caspase-3 and reduce the expression of phosphorylated p38 via binding with mitogen-activated protein kinase kinases-6 (MKK6). Moreover, Ma-3-gal-Cl could inhibit the Aß aggregation via binding with Aß monomer and fibers. Thus, Ma-3-gal-Cl showed significant effects on protecting SH-SY5Y cells from Aß/Cu2+/AA induced damage via antioxidation effect and inhibition effect to the Aß aggregation.

A bacterial glycolipid essential for membrane protein integration.

The proper conformation and orientation of membrane protein integration in cells is an important biological event. Interestingly, a new factor named MPIase (membrane protein integrase) was proven essential in this process in Escherichia coli, besides proteinaceous factors, such as Sec translocons and an insertase YidC. A combination of spectroscopic analyses and synthetic work has revealed that MPIase is a glycolipid despite its enzyme-like activity. MPIase has a long glycan chain comprised of repeating trisaccharide units, a pyrophosphate linker, and a diacylglycerol anchor. In order to determine the mechanism of its activity, we synthesized a trisaccharyl pyrophospholipid termed mini-MPIase-3, a minimal unit of MPIase, and its derivatives. A significant activity of mini-MPIase-3 indicated that it involves an essential structure for membrane protein integration. We also analyzed intermolecular interactions of MPIase or its synthetic analogs with a model substrate protein using physicochemical methods. The structure-activity relationship studies demonstrated that the glycan part of MPIase prevents the aggregation of substrate proteins, and the 6-O-acetyl group on glucosamine and the phosphate of MPIase play important roles for interactions with substrate proteins. MPIase serves at an initial step in the Sec-independent integration, whereas YidC, proton motive force, and/or SecYEG cooperatively function(s) with MPIase at the following step in vivo. Furthermore, depletion of the biosynthetic enzyme demonstrated that MPIase is crucial for membrane protein integration and cell growth. Thus, we elucidated new biological functions of glycolipids using a combination of synthetic chemistry, biochemistry, physicochemical measurements, and molecular-biological approaches.

Direct determination of ethanol in alcoholic beverages based on its anti-aggregation of melamine-silver nanoparticle assembly.

We report a new method for determination of ethanol based on anti-aggregation of silver nanoparticles (AgNPs) in the presence of melamine. In the system, ethanol and melamine act as protecting and aggregating agents, respectively. Melamine can induce citrate-stabilized AgNPs to aggregate, leading to a color change from yellow to green. However, if the AgNPs are pre-incubated in ethanol, ethanol readily surrounds the particles by forming hydrogen bonds with the citrate stabilizer. An external nanoshell of ethanol hinders particle aggregation caused by melamine. Minor aggregation of AgNPs was observed, the solution color maintained its yellow-orange color. Higher ethanol concentrations result in a lower degree of particle aggregation. The colorimetric response of AgNPs was monitored using a UV-vis spectrophotometer at 390 nm. The current method could determine ethanol concentrations over a wide dynamic range of 5-80% (v/v), with a detection limit of 3.1% (v/v) (3SD of blank/slope). This method was applied for direct quantification of ethanol in alcoholic drinks without sample pretreatment and the results are well correlated with those of gas chromatography. Our method is convenient and cost effective, making it auspicious for ethanol monitoring in alcoholic drink manufacture and control.