DELTEX E3 ligases ubiquitylate ADP-ribosyl modification on nucleic acids.

Although ubiquitylation had traditionally been considered limited to proteins, the discovery of non-proteinaceous substrates (e.g. lipopolysaccharides and adenosine diphosphate ribose (ADPr)) challenged this perspective. Our recent study showed that DTX2 E3 ligase efficiently ubiquitylates ADPr. Here, we show that the ADPr ubiquitylation activity is also present in another DELTEX family member, DTX3L, analysed both as an isolated catalytic fragment and the full-length PARP9:DTX3L complex, suggesting that it is a general feature of the DELTEX family. Since structural predictions show that DTX3L possesses single-stranded nucleic acids binding ability and given the fact that nucleic acids have recently emerged as substrates for ADP-ribosylation, we asked whether DELTEX E3s might catalyse ubiquitylation of an ADPr moiety linked to nucleic acids. Indeed, we show that DTX3L and DTX2 are capable of ubiquitylating ADP-ribosylated DNA and RNA synthesized by PARPs, including PARP14. Furthermore, we demonstrate that the Ub-ADPr-nucleic acids conjugate can be reversed by two groups of hydrolases, which remove either the whole adduct (e.g. SARS-CoV-2 Mac1 or PARP14 macrodomain 1) or just the Ub (e.g. SARS-CoV-2 PLpro). Overall, this study reveals ADPr ubiquitylation as a general function of the DELTEX family E3s and presents the evidence of reversible ubiquitylation of ADP-ribosylated nucleic acids.

Okadaic acid enhances NfKB, TLR-4, caspase 3, ERK ½, c-FOS, and 8-OHdG signaling pathways activation in brain tissues of zebrafish larvae.

This study was designed to investigate the potential neuronal damage mechanism of the okadaic acid (OA) in the brain tissues of zebrafish embryos by evaluating in terms of immunofluorescence of Nf KB, TLR-4, caspase 3, ERK ½, c-FOS and 8-OHdG signaling pathways. We also evaluated body malformations. For this purpose, zebrafish embryos were exposed to 0.5 µg/ml, 1 µg/ml and 2.5 µg/ml of OA for 5 days. After application, FITC/GFP labeled protein-specific antibodies were used in immunofluorescence assay for NfKB, TLR-4, caspase 3, ERK ½, c-FOS and 8-OHdG respectively. The results indicated that OA caused immunofluorescence positivity of NfKB, TLR-4, caspase 3, ERK ½, c-FOS and 8-OHdG in a dose-dependent manner in the brain tissues of zebrafish embryos. Pericardial edema (PE), nutrient sac edema (YSE) and body malformations, tail malformation, short tail and head malformation (BM) were detected in zebrafish embryos. These results suggest that OA induces neuronal damage by affecting the modulation of DNA damage, apoptotic, and inflammatory activities in the brain tissues of zebrafish embryos. The increase in signaling pathways shows that OA can cause damage in the structure and function of brain nerve cells. Our results provide a new basis for the comprehensive assessment of the neural damage of OA and will offer enable us to better understand molecular the mechanisms underlying the pathophysiology of OA toxicity.

Purification, structural characterization, and neuroprotective effect of 3,6-diisobutyl-2,5-piperazinedione from Halomonas pacifica CARE-V15 against okadaic acid-induced neurotoxicity in zebrafish model.

Halomonas pacifica CARE-V15 was isolated from the southeastern coast of India to determine its genome sequence. Secondary metabolite gene clusters were identified using an anti-SMASH server. The concentrated crude ethyl acetate extract was evaluated by GC-MS. The bioactive compound from the crude ethyl acetate extract was fractionated by gel column chromatography. HPLC was used to purify the 3,6-diisobutyl-2,5-piperazinedione (DIP), and the structure was determined using FTIR and NMR spectroscopy. Purified DIP was used in an in silico molecular docking analysis. Purified DIP exhibits a stronger affinity for antioxidant genes like glutathione peroxidase (GPx), glutathione-S-transferase (GST), and glutathione reductase (GSR). Using in silco molecular docking analysis, the protein-ligand binding affinities of GSR (-4.70 kcal/mol), GST (-5.27 kcal/mol), and GPx (-5.37 kcal/mol) were measured. The expression of antioxidant genes were investigated by qRT-PCR. The in vivo reactive oxygen species production, lipid peroxidation, and cell death levels were significantly (p ≤ 0.05) increased in OA-induced group, but all these levels were significantly (p ≤ 0.05) decreased in the purified DIP pretreated group. Purified DIP from halophilic bacteria could thus be a useful treatment for neurological disorders associated with oxidative stress.

Effects of marine biotoxins on drug-metabolizing cytochrome P450 enzymes and their regulation in mammalian cells.

Marine biotoxins are a heterogenous group of natural toxins, which are able to trigger different types of toxicological responses in animals and humans. Health effects arising from exposure to marine biotoxins are ranging, for example, from gastrointestinal symptoms to neurological effects, depending on the individual toxin(s) ingested. Recent research has shown that the marine biotoxin okadaic acid (OA) can strongly diminish the expression of drug-metabolizing cytochrome P450 (CYP) enzymes in human liver cells by a mechanism involving proinflammatory signaling. By doing so, OA may interfere with the metabolic barrier function of liver and intestine, and thus alter the toxico- or pharmacokinetic properties of other compounds. Such effects of marine biotoxins on drug and xenobiotic metabolism have, however, not been much in the focus of research yet. In this review, we present the current knowledge on the effects of marine biotoxins on CYP enzymes in mammalian cells. In addition, the role of CYP-regulating nuclear receptors as well as inflammatory signaling in the regulation of CYPs by marine biotoxins is discussed. Strong evidence is available for effects of OA on CYP enzymes, along with information about possible molecular mechanisms. For other marine biotoxins, knowledge on effects on drug metabolism, however, is scarce.

Transcriptomics Analysis of the Immune Effects of Okadaic Acid on Caco-2 Cells.

Okadaic Acid, a type of diarrhetic shellfish poison, is widely distributed and harmful, causing symptoms such as diarrhea, vomiting, and more in humans. Recent studies have demonstrated that OA can lead to various toxicities such as cytotoxicity, neurotoxicity, embryotoxicity, and hepatotoxicity. In order to investigate the immunotoxicity of OA on intestinal cells, a transcriptome analysis was conducted to compare the differences in the Caco-2 cell transcriptional group before and after administration. The CCK-8 experiment demonstrated that OA had a detrimental effect on the activity of Caco-2 cells, with an IC50 value of 33.98 nM. Transcriptome data revealed changes in immune-related genes between the experimental and control groups, including inflammatory factors, heat shock proteins, and zinc finger proteins. The analysis of the results suggests that OA can induce the production of inflammatory factors and apoptosis in cells, and may also affect cell ferroptosis. These findings indicate that OA has a significant impact on intestinal immunity, providing valuable insights for the study of immune toxicity associated with OA.

Assessment of the health risks associated with the consumption of bivalve mollusks potentially contaminated with phycotoxins from the coastal ecosystem of the Ebrié lagoon, Côte d'Ivoire.

This work focused on assessing of the risk associated with the consumption of bivalve mollusks, potentially contaminated with phycotoxins. The studied phycotoxins are saxitoxin (STX), okadaic acid (OA), dinophysistoxins (DTXs), yessotoxins (YTXs), pectenotoxins (PTX), azaspiracids (AZAs), and domoic acid (DA). These toxins were investigated in three species of bivalve mollusks (Anadara senilis, Crassostrea gasar, and Perna perna), originating from the Ebrié lagoon. Chemical analyses were carried out by LC-MS/MS, HPLC-FLD, and HPLC-UV. The level of OA and DTXs, STX, and DA was 10.92 µg OA eq./kg, 9.6 µg STX eq./kg, and 0.17 mg DA eq./kg, respectively. The level of PTXs and AZAs was 3.3 µg PTX-2 eq./kg and 13.86 µg AZA-1 eq./kg; that of YTXs was 0.01 mg YTX eq./kg. The daily exposure dose (DED) was 0.019 µg OA eq./kg bw for OA and DTXs; 0.285 µg DA eq./kg bw for DA; 0.006 µg PTX-2 eq./kg bw for PTXs; 0.016 µg STX eq./kg bw for STX; 0.01 µg YTX eq./kg bw for YTXs; and 0.024 µg AZA-1 eq./kg bw for AZAs for the oyster Crassostrea gasar. These estimated values are lower than the acute reference dose (ARfD) of each phycotoxin recommended by the European Food Safety Agency (EFSA). The risk of harmful effects is acceptable. The absence of risk is valid only for the study period (11 months) and concerns coastal populations living near the sampling points.

Functional Selection of Tau Oligomerization-Inhibiting Aptamers.

Pathological hyperphosphorylation and aggregation of microtubule-associated Tau protein contribute to Alzheimer's Disease (AD) and other related tauopathies. Currently, no cure exists for Alzheimer's Disease. Aptamers offer significant potential as next-generation therapeutics in biotechnology and the treatment of neurological disorders. Traditional aptamer selection methods for Tau protein focus on binding affinity rather than interference with pathological Tau. In this study, we developed a new selection strategy to enrich DNA aptamers that bind to surviving monomeric Tau protein under conditions that would typically promote Tau aggregation. Employing this approach, we identified a set of aptamer candidates. Notably, BW1c demonstrates a high binding affinity (Kd=6.6 nM) to Tau protein and effectively inhibits arachidonic acid (AA)-induced Tau protein oligomerization and aggregation. Additionally, it inhibits GSK3ß-mediated Tau hyperphosphorylation in cell-free systems and okadaic acid-mediated Tau hyperphosphorylation in cellular milieu. Lastly, retro-orbital injection of BW1c tau aptamer shows the ability to cross the blood brain barrier and gain access to neuronal cell body. Through further refinement and development, these Tau aptamers may pave the way for a first-in-class neurotherapeutic to mitigate tauopathy-associated neurodegenerative disorders.

The early molecular events leading to COFILIN phosphorylation during mouse sperm capacitation are essential for acrosomal exocytosis.

The actin cytoskeleton reorganization during sperm capacitation is essential for the occurrence of acrosomal exocytosis (AR) in several mammalian species. Here, we demonstrate that in mouse sperm, within the first minutes of exposure upon capacitating conditions, the activity of RHOA/C and RAC1 is essential for LIMK1 and COFILIN phosphorylation. However, we observed that the signaling pathway involving RAC1 and PAK4 is the main player in controlling actin polymerization in the sperm head necessary for the occurrence of AR. Moreover, we show that the transient phosphorylation of COFILIN is also influenced by the Slingshot family of protein phosphatases (SSH1). The activity of SSH1 is regulated by the dual action of two pathways. On one hand, RHOA/C and RAC1 activity promotes SSH1 phosphorylation (inactivation). On the other hand, the activating dephosphorylation is driven by okadaic acid-sensitive phosphatases. This regulatory mechanism is independent of the commonly observed activating mechanisms involving PP2B and emerges as a new finely tuned modulation that is, so far, exclusively observed in mouse sperm. However, persistent phosphorylation of COFILIN by SSH1 inhibition or okadaic acid did not altered actin polymerization and the AR. Altogether, our results highlight the role of small GTPases in modulating actin dynamics required for AR.

Inhibition of protein phosphatase 2A by okadaic acid induces translocation of nucleocytoplasmic O-GlcNAc transferase.

Post-translational modification (PTM) is crucial for many biological events, such as the modulation of bone metabolism. Phosphorylation and O-GlcNAcylation are two examples of PTMs that can occur at the same site in the protein: serine and threonine residues. This phenomenon may cause crosstalk and possible interactions between the molecules involved. Protein phosphatase 2 A (PP2A) is widely expressed throughout the body and plays a major role in dephosphorylation. At the same location where PP2A acts, O-GlcNAc transferase (OGT) can introduce uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) molecules and mediates O-GlcNAc modifications. To examine the effects of PP2A inhibition on OGT localization and expression, osteoblastic MC3T3-E1 cells were treated with Okadaic Acid (OA), a potent PP2A inhibitor. In the control cells, OGT was strictly localized in the nucleus. However, OGT was observed diffusely in the cytoplasm of the OA-treated cells. This change in localization from the nucleus to the cytoplasm resulted from an increase in mitochondrial OGT expression and translocation of the nucleocytoplasmic isoform. Furthermore, knockdown of PP2A catalytic subunit α isoform (PP2A Cα) significantly affected OGT expression (p < 0.05), and there was a correlation between PP2A Cα and OGT expression (r = 0.93). These results suggested a possible interaction between PP2A and OGT, which strengthens the notion of an interaction between phosphorylation and O-GlcNAcylation.

Sterols of morphologically distinct, okadaic acid-producing Prorocentrum texanum var. texanum and var. cuspidatum isolated from the Gulf of Mexico.

Prorocentrum texanum var. texanum and its morphologically distinct yet genetically identical (as based on the sequences of five genes) variety P. texanum var. cuspidatum represent a species of Prorocentrum recently isolated from the Gulf of Mexico. Together, these two varieties represent a sister species to Prorocentrum micans. P. micans has had its sterols, which are ringed lipids common to eukaryotic cell membranes, shown in some studies to be comprised of cholesterol (cholest-5-en-3ß-ol), 23,24-dimethyl-cholesta-5,22-dien-3ß-ol, 23,24-dimethyl-5α-cholest-22E-en-3ß-ol, dinosterol, and 4α,23,24-trimethyl-5α-cholestan-3ß-ol (dinostanol) as major sterols, thus placing it within a previously identified cluster of dinoflagellates characterized by the predominance of cholesterol and dinosterol. In this study we have determined the sterol compositions of these two varieties of P. texanum to be abundant in cholesterol, 23,24-dimethyl-cholesta-5,22-dien-3ß-ol, 23,24-dimethyl-5α-cholest-22E-en-3ß-ol, dinosterol, and dinostanol such that the varieties are virtually indistinguishable from each other, making them both in general agreement with the sterols of P. micans, its closest species relative. This expands our knowledge of the sterols of this environmentally important dinoflagellate genus.

Sample pooling and incurred samples improve analytical throughput and quality control of lipophilic phycotoxins screening in bivalve mollusks.

Lipophilic marine biotoxins (LMBs) are one of the main risks associated with the consumption of mussels and oysters. Sanitary and analytical control programs are developed to detect the occurrence of these toxins in seafood before they reach toxic levels. To ensure quick results, methods must be easy and fast to perform. In this work, we demonstrated that incurred samples were a viable alternative to validation and internal quality control studies for the analysis of LMBs in bivalve mollusks. These samples were used to optimize, validate, and monitor a simple and fast ultrasound-assisted extraction (UAE) procedure. An internal quality control material containing okadaic acid (227 ± 46 µg kg-1) was produced and characterized. This material had its homogeneity and stability verified and was included as a quality control in all batches of analytical routine. Besides, a sample pooling protocol for extracts analysis was developed, based on tests for COVID-19. Up to 10 samples could be analyzed simultaneously, reducing the instrumental time of analysis by up to 80%. The UAE and sample pooling approaches were then applied to more than 450 samples, of which at least 100 were positive for the okadaic acid group of toxins.

A survey of Dinophysis spp. and their potential to cause diarrhetic shellfish poisoning in coastal waters of the United States.

Multiple species of the genus Dinophysis produce diarrhetic shellfish toxins (okadaic acid and Dinophysis toxins, OA/DTXs analogs) and/or pectenotoxins (PTXs). Only since 2008 have DSP events (illnesses and/or shellfish harvesting closures) become recognized as a threat to human health in the United States. This study characterized 20 strains representing five species of Dinophysis spp. isolated from three US coastal regions that have experienced DSP events: the Northeast/Mid-Atlantic, the Gulf of Mexico, and the Pacific Northwest. Using a combination of morphometric and DNA-based evidence, seven Northeast/Mid-Atlantic isolates and four Pacific Northwest isolates were classified as D. acuminata, a total of four isolates from two coasts were classified as D. norvegica, two isolates from the Pacific Northwest coast were identified as D. fortii, and three isolates from the Gulf of Mexico were identified as D. ovum and D. caudata. Toxin profiles of D. acuminata and D. norvegica varied by their geographical origin within the United States. Cross-regional comparison of toxin profiles was not possible with the other three species; however, within each region, distinct species-conserved profiles for isolates of D. fortii, D. ovum, and D. caudata were observed. Historical and recent data from various State and Tribal monitoring programs were compiled and compared, including maximum recorded cell abundances of Dinophysis spp., maximum concentrations of OA/DTXs recorded in commercial shellfish species, and durations of harvesting closures, to provide perspective regarding potential for DSP impacts to regional public health and shellfish industry.

Dinophysis acuminata or Dinophysis acuta: What Makes the Difference in Highly Stratified Fjords?

Dinophysis acuminata and D. acuta, which follows it seasonally, are the main producers of lipophilic toxins in temperate coastal waters, including Southern Chile. Strains of the two species differ in their toxin profiles and impacts on shellfish resources. D. acuta is considered the major cause of diarrhetic shellfish poisoning (DSP) outbreaks in Southern Chile, but there is uncertainty about the toxicity of D. acuminata, and little information on microscale oceanographic conditions promoting their blooms. During the austral summer of 2020, intensive sampling was carried out in two northern Patagonian fjords, Puyuhuapi (PUY) and Pitipalena (PIT), sharing D. acuminata dominance and D. acuta near detection levels. Dinophysistoxin 1 (DTX 1) and pectenotoxin 2 (PTX 2) were present in all net tow samples but OA was not detected. Although differing in hydrodynamics and sampling dates, D. acuminata shared behavioural traits in the two fjords: cell maxima (>103 cells L-1) in the interface (S ~ 21) between the estuarine freshwater (EFW)) and saline water (ESW) layers; and phased-cell division (µ = 0.3-0.4 d-1) peaking after dawn, and abundance of ciliate prey. Niche analysis (Outlying Mean Index, OMI) of D. acuta with a high marginality and much lower tolerance than D. acuminata indicated an unfavourable physical environment for D. acuta (bloom failure). Comparison of toxin profiles and Dinophysis niches in three contrasting years in PUY-2020 (D. acuminata bloom), 2018 (exceptional bloom of D. acuta), and 2019 (bloom co-occurrence of the two species)-shed light on the vertical gradients which promote each species. The presence of FW (S < 11) and thermal inversion may be used to provide short-term forecasts of no risk of D. acuta blooms and OA occurrence, but D. acuminata associated with DTX 1 pose a risk of DSP events in North Patagonian fjords.

Discovering a New Okadaic Acid Derivative, a Potent HIV Latency Reversing Agent from Prorocentrum lima PL11: Isolation, Structural Modification, and Mechanistic Study.

Marine toxins (MTs) are a group of structurally complex natural products with unique toxicological and pharmacological activities. In the present study, two common shellfish toxins, okadaic acid (OA) (1) and OA methyl ester (2), were isolated from the cultured microalgae strain Prorocentrum lima PL11. OA can significantly activate the latent HIV but has severe toxicity. To obtain more tolerable and potent latency reversing agents (LRAs), we conducted the structural modification of OA by esterification, yielding one known compound (3) and four new derivatives (4-7). Flow cytometry-based HIV latency reversal activity screening showed that compound 7 possessed a stronger activity (EC50 = 46 ± 13.5 nM) but was less cytotoxic than OA. The preliminary structure-activity relationships (SARs) indicated that the carboxyl group in OA was essential for activity, while the esterification of carboxyl or free hydroxyls were beneficial for reducing cytotoxicity. A mechanistic study revealed that compound 7 promotes the dissociation of P-TEFb from the 7SK snRNP complex to reactivate latent HIV-1. Our study provides significant clues for OA-based HIV LRA discovery.

Cytotoxicity and intestinal permeability of phycotoxins assessed by the human Caco-2 cell model.

Phycotoxins are a class of multiple natural metabolites produced by microalgae in marine and freshwater ecosystems that bioaccumulate in food webs, particularly in shellfish, having a great impact on human health. Phycotoxins are mainly leached and absorbed in the small intestine when human consumers accidentally ingest toxic aquatic products contaminated by them. To assess the intestinal uptake and damage of phycotoxins, a typical in vitro model was developed and widely applied using the human colorectal adenocarcinoma Caco-2 cell line. In this review, the application cases were summarized for multiple phycotoxins, including microcystins (MCs), cylindrospermopsins (CYNs), domoic acids (DAs), saxitoxins (STXs), palytoxins (PLTXs), okadaic acids (OAs), pectenotoxins (PTXs) and azaspiracids (AZAs). The results of the previous studies showed that each group of phycotoxins presented different cytotoxicity and mechanisms to Caco-2 cells, and significant discrepancies in the transport of phycotoxin across the Caco-2 cell monolayers. Therefore, this review describes the evaluation assays of the Caco-2 cell monolayer model, illustrates the principles of several primary cytotoxicity evaluation assays, and summarizes the cytotoxicity of each group of phycotoxins to Caco-2 cells line and their cellular transport, and finally proposes the development of multicellular intestinal models for future comprehensive studies on the toxicity and absorption of phycotoxins in the intestine. It will improve the understanding of Caco-2 cell monolayer models in the toxicology studies on phycotoxins and the potentially detrimental effects of microalgal toxins on the human intestine.

Polystyrene nanoplastics promote the apoptosis in Caco-2 cells induced by okadaic acid more than microplastics.

Microplastics (MPs) are widespread in the environment and can be ingested through food, water, and air, posing a threat to human health. In addition, MPs can have a potential combined effect with other toxic compounds. Polystyrene (PS) has been shown to enhance the cytotoxicity of okadaic acid (OA). However, it remains unclear whether this enhancement effect is related to the size of PS particles. In this study, we investigated the mechanism of the combined effect of PS microplastics (PS-MPs) or PS nanoplastics (PS-NPs) and OA on Caco-2 cells. The results indicated that PS-NPs enhanced the cytotoxicity of OA and induced endoplasmic reticulum (ER) stress-mediated apoptosis in Caco-2 cells, compared to PS-MPs. Specifically, PS-NPs and OA cause more severe oxidative stress, lactate dehydrogenase (LDH) release, and mitochondrial membrane depolarization. Furthermore, it induced intracellular calcium overload through store-operated channels (SOCs) and activated the PERK/ATF-4/CHOP pathway to cause ER stress. ER stress promoted mitochondrial damage and finally activated the caspase family to induce apoptosis. This study provided an indirect basis for the assessment of the combined toxicity of MPs or NPs with OA.

Exposure to okadaic acid could disrupt the colonic microenvironment in rats.

Okadaic acid (OA) is one of the most prevalent marine phycotoxin with complex toxicity, which can lead to toxic symptoms such as diarrhea, vomiting, nausea, abdominal pain, and gastrointestinal discomfort. Studies have shown that the main affected tissue of OA is digestive tract. However, its toxic mechanism is not yet fully understood. In this study, we investigated the changes that occurred in the epithelial microenvironment following OA exposure, including the epithelial barrier and gut bacteria. We found that impaired epithelial cell junctions, mucus layer destruction, cytoskeletal remodeling, and increased bacterial invasion occurred in colon of rats after OA exposure. At the same time, the gut bacteria decreased in the abundance of beneficial bacteria and increased in the abundance of pathogenic bacteria, and there was a significant negative correlation between the abundance of pathogenic bacteria represented by Escherichia/Shigella and animal body weight. Metagenomic analysis inferred that Escherichia coli and Shigella spp. in Escherichia/Shigella may be involved in the process of cytoskeletal remodeling and mucosal layer damage caused by OA. Although more evidence is needed, our results suggest that opportunistic pathogens may be involved in the complex toxicity of OA during OA-induced epithelial barrier damage.

Upregulation of Peridinin-Chlorophyll A-Binding Protein in a Toxic Strain of Prorocentrum hoffmannianum under Normal and Phosphate-Depleted Conditions.

Some strains of the dinoflagellate species Prorocentrum hoffmannianum show contrasting ability to produce diarrhetic shellfish poisoning (DSP) toxins. We previously compared the okadaic acid (OA) production level between a highly toxic strain (CCMP2804) and a non-toxic strain (CCMP683) of P. hoffmannianum and revealed that the cellular concentration of OA in CCMP2804 would increase significantly under the depletion of phosphate. To understand the molecular mechanisms, here, we compared and analyzed the proteome changes of both strains growing under normal condition and at phosphate depletion using two-dimensional gel electrophoresis (2-DE). There were 41 and 33 differential protein spots observed under normal condition and phosphate depletion, respectively, of which most were upregulated in CCMP2804 and 22 were common to both conditions. Due to the lack of matched peptide mass fingerprints in the database, de novo peptide sequencing was applied to identify the differentially expressed proteins. Of those upregulated spots in CCMP2804, nearly 60% were identified as peridinin-chlorophyll a-binding protein (PCP), an important light-harvesting protein for photosynthesis in dinoflagellates. We postulated that the high expression of PCP encourages the production of DSP toxins by enhancing the yields of raw materials such as acetate, glycolate and glycine. Other possible mechanisms of toxicity related to PCP might be through triggering the transcription of non-ribosomal peptide synthetase/polyketide synthase genes and the transportation of dinophysistoxin-4 from chloroplast to vacuoles.

Identification of Uncaria rhynchophylla in the Potential Treatment of Alzheimer's Disease by Integrating Virtual Screening and In Vitro Validation.

Uncaria rhynchophylla (Gouteng in Chinese, GT) is the main medicine in many traditional recipes in China. It is commonly used to alleviate central nervous system (CNS) disorders, although its mechanism in Alzheimer's disease is still unknown. This study was designed to predict and validate the underlying mechanism in AD treatment, thus illustrating the biological mechanisms of GT in treating AD. In this study, a PPI network was constructed, KEGG analysis and GO analysis were performed, and an "active ingredient-target-pathway" network for the treatment of Alzheimer's disease was constructed. The active ingredients of GT were screened out, and the key targets were performed by molecular docking. UHPLC-Q-Exactive Orbitrap MS was used to screen the main active ingredients and was compared with the network pharmacology results, which verified that GT did contain the above ingredients. A total of targets were found to be significantly bound up with tau, Aß, or Aß and tau through the network pharmacology study. Three SH-SY5Y cell models induced by okadaic acid (OA), Na2S2O4, and H2O2 were established for in vitro validation. We first found that GT can reverse the increase in the hyperphosphorylation of tau induced by OA to some extent, protecting against ROS damage. Moreover, the results also indicated that GT has significant neuroprotective effects. This study provides a basis for studying the potential mechanisms of GT in the treatment of AD.

Engineering Robust Aptamers with High Affinity by Key Fragment Evolution and Terminal Fixation.

Researchers have been looking for ways to fix the structural stability of aptamers so as to achieve the high affinity of aptamers and thus the high sensitivity of analytical methods. Herein, we report a post-selection strategy to facilitate the formation of aptameric structures and enhance their affinity. Key fragments containing crucial bases of parent aptamers were identified and evolved by iterative embedding to form chimeras. The termini of the optimized chimera were then fixed by hybridization to limit their flexibility. Robust aptamers with more stable structures and higher affinity were thus engineered. An anti-okadaic acid aptamer, anti-dinophysistoxin aptamer, and anti-phosphatidylserine (PS) aptamer were engineered in this way, with the affinity enhanced by 160.5-fold, 50.36-fold, and 39.28-fold over that of the parent aptamers, respectively. Furthermore, the practicability of the anti-PS aptamer was validated with a polyA-nanotetrahedron-assisted electrochemical aptasensor. The aptasensor achieved high sensitivity, with the limit of detection as low as 1.741 nM, good accuracy, and good selectivity when monitoring PS in real biosynthesis samples. This study offers a facile and efficient approach to generate robust aptamers and aptasensors for real-world applications.