Inhibitors of dermatan sulfate epimerase 1 decreased accumulation of glycosaminoglycans in mucopolysaccharidosis type I fibroblasts.

Genetic deficiency of alpha-L-iduronidase causes mucopolysaccharidosis type I (MPS-I) disease, due to accumulation of glycosaminoglycans (GAGs) including chondroitin/dermatan sulfate (CS/DS) and heparan sulfate (HS) in cells. Currently, patients are treated by infusion of recombinant iduronidase or by hematopoietic stem cell transplantation. An alternative approach is to reduce the L-iduronidase substrate, through limiting the biosynthesis of iduronic acid. Our earlier study demonstrated that ebselen attenuated GAGs accumulation in MPS-I cells, through inhibiting iduronic acid producing enzymes. However, ebselen has multiple pharmacological effects, which prevents its application for MPS-I. Thus, we continued the study by looking for novel inhibitors of dermatan sulfate epimerase 1 (DS-epi1), the main responsible enzyme for production of iduronic acid in CS/DS chains. Based on virtual screening of chemicals towards chondroitinase AC, we constructed a library with 1,064 compounds that were tested for DS-epi1 inhibition. Seventeen compounds were identified to be able to inhibit 27%-86% of DS-epi1 activity at 10 µM. Two compounds were selected for further investigation based on the structure properties. The results show that both inhibitors had a comparable level in inhibition of DS-epi1while they had negligible effect on HS epimerase. The two inhibitors were able to reduce iduronic acid biosynthesis in CS/DS and GAG accumulation in WT and MPS-I fibroblasts. Docking of the inhibitors into DS-epi1 structure shows high affinity binding of both compounds to the active site. The collected data indicate that these hit compounds may be further elaborated to a potential lead drug used for attenuation of GAGs accumulation in MPS-I patients.

Glymphatic System Impairment Contributes to the Formation of Brain Edema After Ischemic Stroke.

BACKGROUND: Blood-brain barrier damage has traditionally been considered to determine the occurrence and development of poststroke brain edema, a devastating and life-threatening complication. However, no treatment strategy targeting blood-brain barrier damage has been proven clinically effective in ameliorating brain edema. METHODS: In mice with stroke models induced by transient middle cerebral artery occlusion (MCAO), the changes in glymphatic system (GS) function impairment were detected by ex vivo fluorescence imaging, 2-photon in vivo imaging, and magnetic resonance imaging within 1 week after MCAO, and the effects of GS impairment and recovery on the formation and resolution of brain edema were evaluated. In addition, in patients with ischemic stroke within 1 week after onset, changes in GS function and brain edema were also observed by magnetic resonance imaging. RESULTS: We found that the extravasation of protein-rich fluids into the brain was not temporally correlated with edema formation after MCAO in mice, as brain edema reabsorption preceded blood-brain barrier closure. Strikingly, the time course of edema progression matched well with the GS dysfunction after MCAO. Pharmacological enhancement of the GS function significantly alleviated brain edema developed on day 2 after MCAO, accompanied by less deposition of Aß (amyloid-ß) and better cognitive function. Conversely, functional suppression of the GS delayed the absorption of brain edema on day 7 after MCAO. Moreover, patients with ischemic stroke revealed a consistent trend of GS dysfunction after reperfusion as MCAO mice, which was correlated with the severity of brain edema and functional outcomes. CONCLUSIONS: GS is a key contributor to the formation of brain edema after ischemic stroke, and targeting the GS may be a promising strategy for treating brain edema in ischemic stroke. REGISTRATION: URL: https://www.chictr.org.cn/showproj.html?proj=162857; Unique identifier: NFEC-2019-189.

Metagenomic and metabolomic profiling of dried shrimp (Litopenaeus vannamei) prepared by a procedure traditional to the south China coastal area.

Sun-drying is a traditional process for preparing dried shrimp in coastal area of South China, but its impacts on nutrition and the formation of flavor-contributory substances in dried shrimp remain largely unknown. This study aimed to examine the effects of the production process on the microbiota and metabolites in dried shrimp. 16S rDNA amplicon sequencing was employed to identify 170 operational taxonomic units (OTUs), with Vibrio, Photobacterium, and Shewanella emerging as the primary pathogenic bacteria in shrimp samples. Lactococcus lactis was identified as the principal potential beneficial microorganism to accrue during the dried shrimp production process and found to contribute significantly to the development of desirable shrimp flavors. LC-MS-based analyses of dried shrimp sample metabolomes revealed a notable increase in compounds associated with unsaturated fatty acid biosynthesis, arachidonic acid metabolism, amino acid biosynthesis, and flavonoid and flavanol biosynthesis throughout the drying process. Subsequent exploration of the relationship between metabolites and bacterial communities highlighted the predominant coexistence of Bifidobacterium, Clostridium, and Photobacterium contributing heterocyclic compounds and metabolites of organic acids and their derivatives. Conversely, Arthrobacter and Staphylococcus were found to inhibit each other, primarily in the presence of heterocyclic compounds. This comprehensive investigation provides valuable insights into the dynamic changes in the microbiota and metabolites of dried shrimps spanning different drying periods, which we expect to contribute to enhancing production techniques and safety measures for dried shrimp processing.

Mechanism Analysis of Antimicrobial Peptide NoPv1 Related to Potato Late Blight through a Computer-Aided Study.

Phytophthora infestans (Mont.) de Bary, the oomycotic pathogen responsible for potato late blight, is the most devastating disease of potato production. The primary pesticides used to control oomycosis are phenyl amide fungicides, which cause environmental pollution and toxic residues harmful to both human and animal health. To address this, an antimicrobial peptide, NoPv1, has been screened to target Plasmopara viticola cellulose synthase 2 (PvCesA2) to inhibit the growth of Phytophthora infestans (P. infestans). In this study, we employed AlphaFold2 to predict the three-dimensional structure of PvCesA2 along with NoPv peptides. Subsequently, utilizing computational methods, we dissected the interaction mechanism between PvCesA2 and these peptides. Based on this analysis, we performed a saturation mutation of NoPv1 and successfully obtained the double mutants DP1 and DP2 with a higher affinity for PvCesA2. Meanwhile, dynamics simulations revealed that both DP1 and DP2 utilize a mechanism akin to the barrel-stave model for penetrating the cell membrane. Furthermore, the predicted results showed that the antimicrobial activity of DP1 was superior to that of NoPv1 without being toxic to human cells. These findings may offer insights for advancing the development of eco-friendly pesticides targeting various oomycete diseases, including late blight.

Rationally Designed Novel Antimicrobial Peptides Targeting Chitin Synthase for Combating Soybean Phytophthora Blight.

Soybean phytophthora blight is a severe menace to global agriculture, causing annual losses surpassing USD 1 billion. Present crop loss mitigation strategies primarily rely on chemical pesticides and disease-resistant breeding, frequently surpassed by the pathogens' quick adaptive evolution. In this urgent scenario, our research delves into innovative antimicrobial peptides characterized by low drug resistance and environmental friendliness. Inhibiting chitin synthase gene activity in Phytophthora sojae impairs vital functions such as growth and sporulation, presenting an effective method to reduce its pathogenic impact. In our study, we screened 16 previously tested peptides to evaluate their antimicrobial effects against Phytophthora using structure-guided drug design, which involves molecular docking, saturation mutagenesis, molecular dynamics, and toxicity prediction. The in silico analysis identified AMP_04 with potential inhibitory activity against Phytophthora sojae's chitin synthase. Through three rounds of saturation mutagenesis, we pin-pointed the most effective triple mutant, TP (D10K, G11I, S14L). Molecular dynamic simulations revealed TP's stability in the chitin synthase-TP complex and its transmembrane mechanism, employing an all-atom force field. Our findings demonstrate the efficacy of TP in occupying the substrate-binding pocket and translocation catalytic channel. Effective inhibition of the chitin synthase enzyme can be achieved. Specifically, the triple mutant demonstrates enhanced antimicrobial potency and decreased toxicity relative to the wild-type AMP_04, utilizing a mechanism akin to the barrel-stave model during membrane translocation. Collectively, our study provides a new strategy that could be used as a potent antimicrobial agent in combatting soybean blight, contributing to sustainable agricultural practices.

De Novo Potent Peptide Nucleic Acid Antisense Oligomer Inhibitors Targeting SARS-CoV-2 RNA-Dependent RNA Polymerase via Structure-Guided Drug Design.

Global reports of novel SARS-CoV-2 variants and recurrence cases continue despite substantial vaccination campaigns, raising severe concerns about COVID-19. While repurposed drugs offer some treatment options for COVID-19, notably, nucleoside inhibitors like Remdesivir stand out as curative therapies for COVID-19 that are approved by the US Food and Drug Administration (FDA). The emergence of highly contagious SARS-CoV-2 variants underscores the imperative for antiviral drugs adaptable to evolving viral mutations. RNA-dependent RNA polymerase (RdRp) plays a key role in viral genome replication. Currently, inhibiting viral RdRp function remains a pivotal strategy to tackle the notorious virus. Peptide nucleic acid (PNA) therapy shows promise by effectively targeting specific genome regions, reducing viral replication, and inhibiting infection. In our study, we designed PNA antisense oligomers conjugated with cell-penetrating peptides (CPP) aiming to evaluate their antiviral effects against RdRp target using structure-guided drug design, which involves molecular docking simulations, drug likeliness and pharmacokinetic evaluations, molecular dynamics simulations, and computing binding free energy. The in silico analysis predicts that chemically modified PNAs might act as antisense molecules in order to disrupt ribosome assembly at RdRp's translation start site, and their chemically stable and neutral backbone might enhance sequence-specific RNA binding interaction. Notably, our findings demonstrate that PNA-peptide conjugates might be the most promising inhibitors of SARS-CoV-2 RdRp, with superior binding free energy compared to Remdesivir in the current COVID-19 medication. Specifically, PNA-CPP-1 could bind simultaneously to the active site residues of RdRp protein and sequence-specific RdRp-RNA target in order to control viral replication.

Identification of a Pentasaccharide Lead Compound with High Affinity to the SARS-CoV-2 Spike Protein via In Silico Screening.

The spike (S) protein on the surface of the SARS-CoV-2 virus is critical to mediate fusion with the host cell membrane through interaction with angiotensin-converting enzyme 2 (ACE2). Additionally, heparan sulfate (HS) on the host cell surface acts as an attachment factor to facilitate the binding of the S receptor binding domain (RBD) to the ACE2 receptor. Aiming at interfering with the HS-RBD interaction to protect against SARS-CoV-2 infection, we have established a pentasaccharide library composed of 14,112 compounds covering the possible sulfate substitutions on the three sugar units (GlcA, IdoA, and GlcN) of HS. The library was used for virtual screening against RBD domains of SARS-CoV-2. Molecular modeling was carried out to evaluate the potential antiviral properties of the top-hit pentasaccharide focusing on the interactive regions around the interface of RBD-HS-ACE2. The lead pentasaccharide with the highest affinity for RBD was analyzed via drug-likeness calculations, showing better predicted druggable profiles than those currently reported for RBD-binding HS mimetics. The results provide significant information for the development of HS-mimetics as anti-SARS-CoV-2 agents.

Design, Synthesis and Biological Evaluation of Conjugates of 3-O-Descladinose-azithromycin and Nucleobases against rRNA A2058G- or A2059G-Mutated Strains.

Structurally unrelated antibiotics MLSB (macrolide-lincosamide-streptogramin B) compromised with clinically resistant pathogens because of the cross-resistance resulting from the structural modification of rRNA A2058. The structure-activity relationships of a novel 3-O-descladinose azithromycin chemotype conjugating with nucleobases were fully explored with the aid of engineered E. coli SQ110DTC and SQ110LPTD. The conjugates of macrolides with nucleobases, especially adenine, displayed antibacterial superiority over telithromycin, azithromycin and clindamycin against rRNA A2058/2059-mutated engineered E. coli strains at the cost of lowering permeability and increasing vulnerability to efflux proteins against clinical isolates.

Antiviral Disaccharide Lead Compounds against SARS-CoV-2 through Computer-Aided High-Throughput Screen.

SARS-CoV-2 infects human epithelial cells through specific interaction with angiotensin-converting enzyme 2 (ACE2). In addition, heparan sulfate proteoglycans act as the attachment factor to promote the binding of viral spike protein receptor binding domain (RBD) to ACE2 on host cells. Though the rapid development of vaccines has contributed significantly to preventing severe disease, mutated SARS-CoV-2 strains, especially the SARS-CoV-2 Omicron variant, show increased affinity of RBD binding to ACE2, leading to immune escape. Thus, there is still an unmet need for new antiviral drugs. In this study, we constructed pharmacophore models based on the spike RBD of SARS-CoV-2 and SARS-CoV-2 Omicron variant and performed virtual screen for best-hit compounds from our disaccharide library. Screening of 96 disaccharide structures identified two disaccharides that displayed higher binding affinity to RBD in comparison to reported small molecule antiviral drugs. Further, screening PharmMapper demonstrated interactions of the disaccharides with a number of inflammatory cytokines, suggesting a potential for disaccharides with multiple-protein targets.

Virtual Evolution of HVEM Segment for Checkpoint Inhibitor Discovery.

Immune therapy has emerged as an effective treatment against cancers. Inspired by the PD-1/PD-L1 antibodies, which have achieved great success in clinical, other immune checkpoint proteins have drawn increasing attention in cancer research. B and T lymphocyte attenuator (BTLA) and herpes virus entry mediator (HVEM) are potential targets for drug development. The co-crystal structure of BTLA/HVEM have revealed that HVEM (26-38) fragment is the core sequence which directly involved on the interface. Herein, we conducted virtual evolution with this sequence by using saturation mutagenesis in silico and mutants with lower binding energy were selected. Wet-lab experiments confirmed that several of them possessed higher affinity with BTLA. Based on the best mutant of the core sequence, extended peptides with better efficacy were obtained. Furthermore, the mechanism of the effects of mutations was revealed by computational analysis. The mutated peptide discovered here can be a potent inhibitor to block BTLA/HVEM interaction and its mechanism may extend people's view on inhibitor discovery for the checkpoint pair.

Persistence of immunity in children immunised with 13-valent pneumococcal conjugate vaccine and impact on nasopharyngeal carriage: a cross-sectional study.

In 500 children aged ≤10 years after 13-valent pneumococcal conjugate vaccine (PCV)13 immunisation in different schedules, serotypes 19A-specific and 19F-specific immunoglobulin G (IgG) were predicted to persist above 0.35 µg/mL for ≥10 years in all groups, likely due to PCV13-induced memory with natural boosting from residual diseases and colonisation. Generally, serotype-specific IgG could persist above 0.35 µg/mL longer (≥5 years) in the catch-up group than in the 2+1 and 3+1 immunisation groups. 14.5% of the carriage isolates belonged to PCV13 serotypes; statistical analysis revealed that a high serum IgG level (>10.96 µg/mL) will be required to eliminate the point-prevalence nasopharyngeal carriage of serotype 19A.

Mechanism and Inhibitor Exploration with Binuclear Mg Ketol-Acid Reductoisomerase: Targeting the Biosynthetic Pathway of Branched-Chain Amino Acids.

Binuclear Mg ketol-acid reductoisomerase (KARI), which converts (S)-2-acetolactate into (R)-2,3-dihydroxyisovalerate, is responsible for the second step of the biosynthesis of branched-chain amino acids in plants and microorganisms and thus serves as a key inhibition target potentially without effects on mammals. Here, through the use of density functional calculations and a chemical model, the KARI-catalyzed reaction has been demonstrated to include the initial deprotonation of the substrate C2 hydroxy group, bridged by the two Mg ions, alkyl migration from the C2-alkoxide carbon atom to the C3-carbonyl carbon atom, and hydride transfer from a nicotinamide adenine dinucleotide phosphate [NAD(P)H] cofactor to C2. A dead-end mechanism with a hydride transferred to the C3 carbonyl group has been ruled out. The nucleophilicity (migratory aptitude) of the migrating carbon atom and the provision of additional negative charge to the di-Mg coordination sphere have significant effects on the steps of alkyl migration and hydride transfer, respectively. Other important mechanistic characteristics are also revealed. Inspired by the mechanism, an inhibitor (2-carboxylate-lactic acid) was designed and predicted by barrier analysis to be effective in inactivating KARI, hence probably enriching the antifungal and antibacterial library. Two types of slow substrate analogues (2-trihalomethyl acetolactic acids and 2-glutaryl lactic acid) were also found.

Protein labeling approach to improve lysosomal targeting and efficacy of antibody-drug conjugates.

An anti-EGFR nanobody was labeled at the C-terminus with a lysosome-sorting NPGY (Asn-Pro-Gly-Tyr) motif via sortase-mediated ligation to enhance the engagement of the clathrin-mediated endocytosis. The synergistic effects of NPGY motif and nona-arginine peptide were found to induce robust internalization and lysosomal trafficking, which in turn improved anti-tumor activity of an antibody-drug conjugate.

Comparative transcriptomic analysis of surf clams (Paphia undulate) infected with two strains of Vibrio spp. reveals the identity of key immune genes involved in host defense.

BACKGROUND: Vibrio spp. is the major infection-producing marine bacteria in commercially important bivalve Paphia undulata. The host resistance is the major determining factor for the development of pathogenesis. To explore defense mechanisms, researchers have focused primarily on the study of differential expression of individual or specific groups of host immune genes during pathogen-challenge. RESULTS: We compared the expression profile in the surf clams infected with avirulent V. alginolyticus and virulent V. parahaemolyticus to mark the possible molecular mechanisms of pathogenesis. Comparison of the differentially expressed genes between the two groups of Vibrio-infected clams revealed that the number of down-regulate genes in V. parahaemolyticus injected clams (1433) were significantly higher than the other group (169). Based on Gene Ontology classification, a large proportion of these down-regulate genes were found to be associated with cellular and molecular mechanisms for pathogen recognition, and immunity development thereby explaining the low survival rate for the V. parahaemolyticus-treated clams and suggesting a higher virulence of this bacterium towards the surf clams. Quantitative real-time PCR of 24 candidate genes related to immunity involving the JAK-STAT signaling pathway, complementary cascade, cytokine signaling pathway, oxidative stress, phagocytosis and apoptosis down regulated under V. parahaemolyticus infection, indicating compromised host defense. Furthermore, we could demonstrate a central role of JAK-STAT pathway in bacterial clearance. dsRNA mediated depletion of a clam STAT homolog gene results in dramatic increase in the infection by V. alginolyticus, a mildly pathogenic strain under control conditions. CONCLUSIONS: The difference in gene expression profiles in surf clams treated with two Vibrio species with a differential pathogenicity to P. undulate and downstream molecular analysis could enlighten on the probable molecular mechanisms of the Vibrio pathogenesis and the virulence of V. parahaemolyticus in surf clams, which also benefits to develop new strategies for disease control in surf calm aquaculture.

Genomic analysis of NF-κB signaling pathway reveals its complexity in Crassostrea gigas.

NF-κB signaling pathway is an evolutionarily conserved pathway that plays highly important roles in several developmental, cellular and immune response processes. With the recent release of the draft Pacific oyster (Crassostra gigas) genome sequence, we have sought to identify the various components of the NF-κB signaling pathway in these mollusks and investigate their gene structure. We further constructed phylogenetic trees to establish the evolutionary relationship of the oyster proteins with their homologues in vertebrates and invertebrates using BLASTX and neighbor-joining method. We report the presence of two classic NF-κB/Rel homologues in the pacific oyster namely Cgp100 and CgRel, which possess characteristic RHD domain and a consensus nuclear localization signal, similar to mammalian homologues and an additional CgRel-like protein, unique to C. gigas. Further, in addition to two classical IκB homologues, CgIκB1 and CgIκB2, we have identified three atypical IκB family members namely CgIκB3, CgIκB4 and CgBCL3 which lack the IκB degradation motif and consist of only one exon that might have arisen by retrotransposition from CgIκB1. Finally, we report the presence of three IKKs and one NEMO genes in oyster genome, named CgIKK1, CgIKK2, CgIKK3 and CgNEMO, respectively. While CgIKK1 and CgIKK3 domain structure is similar to their mammalian homologues, CgIKK2 was found to lack the HLH and NBD domains. Overall, the high conservation of the NF-κB/Rel, IκB and IKK family components in the pacific oyster and their structural similarity to the vertebrate and invertebrate homologues underline the functional importance of this pathway in regulation of critical cellular processes across species.

LRFN (leucine-rich repeat and fibronectin type-III domain-containing protein) recognizes bacteria and promotes hemocytic phagocytosis in the Pacific oyster Crassostrea gigas.

In bivalve mollusks, circulating hemocyte mediated phagocytosis is one of the primary ways to eliminate invading microbes. Here, we have identified one CgLRFN (leucine-rich repeat and fibronectin type-III domain-containing protein) in the Crassostrea gigas as a novel transmembrane LRR (Leucine-rich repeat) domain containing protein in C. gigas, homologous to the jawless fish VLR protein, that plays an important role in recognizing bacteria and promoting hemocytic phagocytosis. Tissue distribution analysis of CgLRFN in Pacific oyster showed that it is widely expressed in various tissues like the gills, adductor muscles, digestive glands, gonads, heart and in the hemocytes. Furthermore, infection of Pacific oysters with two marine Vibrio strains V. alginolyticus and V. parahaemolyticus was found to significantly increase CgLRFN expression in the hemocytes. Analysis of subcellular localization showed that CgLRFN is primarily localized in the cell membrane. Additionally, CgLRFN was found to be able to bind both the bacterial strains, indicating its possible role as a cell surface receptor. Flow cytometry analysis revealed that CgLRFN coated bacteria was phagocytosed by oyster hemocytes at a significantly higher rate compared to the uncoated bacteria. Finally, RNAi mediated knockdown of CgLRFN in vivo resulted in reduced clearance of both the bacterial strains from the oyster hemolymph. Overall, our study demonstrates that CgLRFN acts as a pattern recognition receptor for Vibrio spp. and promotes hemocytic phagocytosis in the Pacific oyster, which is critical for understanding the mechanism of bacterial infection in lower invertebrates, and also contributes to disease management of this economically and ecologically important marine mollusk.

Identification and functional characterization of NEMO in Crassostrea gigas reveals its crucial role in the NF-κB activation.

NEMO (NF-κB essential modulator) is one of the important regulatory subunits of the IκB kinase (IκK) complex that controls the activation of the NF-κB signaling pathway. Here, we have identified the homolog of NEMO from the pacific oyster Crassostrea gigas. CgNEMO harbors the conserved the IκK binding region, NEMO ubiquitin binding domain and Zinc finger domain. In terms of tissue distribution, CgNEMO is expressed in various tissues with an observed highest expression in the hemocytes. Furthermore, infection by two related Vibrio strains significantly increased CgNEMO expression in the hemocytes. Cell culture based luciferase reporter assays showed that CgNEMO activates the NF-κB reporter in a dose-pendent manner. Moreover, CgNEMO was also found to counter the IkB-dependent inhibitory effect on NF-κB activation, providing a plausible mechanism of NF-κB activation by CgNEMO. Meanwhile, site-directed mutagenesis demonstrated that the putative ubiquitination site K535 is required for the activation of NF-κB, implying that ubiquitination of NEMO may be involved in regulating its activity. Finally, RNAi mediated knockdown of CgNEMO in vivo significantly compromised the bacterial induction of key cytokines TNF-α and IL-17, strongly suggesting a role for CgNEMO in acute immune defense in oyster. In conclusion, this study provides new insights into our understanding about the evolution of NEMO mediated NF-κB activation and the induction of cytokine. Our findings may provide valuable information about diseases control and management in oyster aquaculture.

From NAD+ to Nickel Pincer Complex: A Significant Cofactor Evolution Presented by Lactate Racemase.

Lactate racemase (LarA), a new nickel enzyme discovered recently, catalyzes the racemization between d- and l-lactates with a novel nickel pincer cofactor (Ni-PTTMN) derived from nicotinic acid. In this study, by using DFT and a 200-atom active-site model, LarA is revealed to employ a modified proton-coupled hydride-transfer mechanism in which a hydride is transferred to a cofactor pyridine carbon from the substrate α-carbon along with proton transfer from the substrate hydroxy group to a histidine, and then moved back from the opposite side. Tyr294 and Lys298 provide significant acceleration effects by orientating substrates and stabilizing the negative charge developing at the substrate hydroxy oxygen. The barrier was determined to be 12.0 kcal mol-1 , which reveals enhanced racemase activity relative to the LarA reaction using NAD+ -like cofactors. Compared with NAD+ , Ni-PTTMN has a stronger hydride-addition reactivity in moderate and high environmental polarity and may fit perfectly the moderately polar active site of LarA.

The first mollusk spätzle homolog gene in the clam, Paphia undulate.

Spätzle, is the only identified endogenous Toll receptor ligand, plays a critical role in initiatinge innate immune responses and controlling dorsal-ventral axis formation in Drosophila. Here we identified the first spätzle gene homolog, Pu-Spz, in the marine mollusk Paphia undulate. The full-length of Pu-Spz cDNA is 1248 bp, including an open reading frame (ORF) of 702 bp, a 5'-untranslated region (UTR) of 26 bp and a 3'-UTR of 203 bp. The ORF encodes a 233-amino-acid protein with conserved domains; it includes a putative signal peptide and a C-terminal cystine-knot. Sequence alignment revealed that the cystine-knot domain of Pu-Spz contains six highly conserved Cys residues, which maintain a molecular conformation suitable for Toll receptor binding. Unlike Spätzle, Pu-Spz lacks a seventh Cys residue, which is essential for forming intermolecular disulfide bridge. Phylogenetic analysis revealed that Pu-Spz is closer to the homologs found in crustaceans than to those in the insect branch. Transcript abundance of Pu-Spz was increased after challenging P. undulate with either heat-killed Listeria monocytogenes or heat-killed Vibrio alginolyticus, suggesting Spätzle is involved in P. undulate host defense. Our results demonstrate convergent evolution of the spätzle-Toll system between the mollusk and arthropod lineages.

Unraveling the Mechanisms of Cannabidiol's Pharmacological Actions: A Comprehensive Research Overview.

Cannabis sativa has long been used for neurological and psychological healing. Recently, cannabidiol (CBD) extracted from cannabis sativa has gained prominence in the medical field due to its non-psychotropic therapeutic effects on the central and peripheral nervous systems. CBD, also acting as a potent antioxidant, displays diverse clinical properties such as anticancer, antiinflammatory, antidepressant, antioxidant, antiemetic, anxiolytic, antiepileptic, and antipsychotic effects. In this review, we summarized the structural activity relationship of CBD with different receptors by both experimental and computational techniques and investigated the mechanism of interaction between related receptors and CBD. The discovery of structural activity relationship between CBD and target receptors would provide a direction to optimize the scaffold of CBD and its derivatives, which would give potential medical applications on CBD-based therapies in various illnesses.