Structural basis of water-mediated cis Watson-Crick/Hoogsteen base-pair formation in non-CpG methylation.

Non-CpG methylation is associated with several cellular processes, especially neuronal development and cancer, while its effect on DNA structure remains unclear. We have determined the crystal structures of DNA duplexes containing -CGCCG- regions as CCG repeat motifs that comprise a non-CpG site with or without cytosine methylation. Crystal structure analyses have revealed that the mC:G base-pair can simultaneously form two alternative conformations arising from non-CpG methylation, including a unique water-mediated cis Watson-Crick/Hoogsteen, (w)cWH, and Watson-Crick (WC) geometries, with partial occupancies of 0.1 and 0.9, respectively. NMR studies showed that an alternative conformation of methylated mC:G base-pair at non-CpG step exhibits characteristics of cWH with a syn-guanosine conformation in solution. DNA duplexes complexed with the DNA binding drug echinomycin result in increased occupancy of the (w)cWH geometry in the methylated base-pair (from 0.1 to 0.3). Our structural results demonstrated that cytosine methylation at a non-CpG step leads to an anti→syntransition of its complementary guanosine residue toward the (w)cWH geometry as a partial population of WC, in both drug-bound and naked mC:G base pairs. This particular geometry is specific to non-CpG methylated dinucleotide sites in B-form DNA. Overall, the current study provides new insights into DNA conformation during epigenetic regulation.

Amphiphilic Block Copolymers Containing Benzenesulfonyl Azide Groups as Visible Light-Responsive Drug Carriers for Image-Guided Delivery.

Nanoparticles (NPs) containing light-responsive polymers and imaging agents show great promise for controlled drug delivery. However, most light-responsive NPs rely on short-wavelength excitation, resulting in poor tissue penetration and potential cytotoxicity. Moreover, excessively sensitive NPs may prematurely release drugs during storage and circulation, diminishing their efficacy and causing off-target toxicity. Herein, we report visible-light-responsive NPs composed of an amphiphilic block copolymer containing responsive 4-acrylamide benzenesulfonyl azide (ABSA) and hydrophilic N,N'-dimethylacrylamide (DMA) units. The polymer pDMA-ABSA was loaded with the chemotherapy drug dasatinib and zinc tetraphenylporphyrin (ZnTPP). ZnTPP acted as an imaging reagent and a photosensitizer to reduce ABSA upon visible light irradiation, converting hydrophobic units to hydrophilic units and disrupting NPs to trigger drug release. These NPs enabled real-time fluorescence imaging in cells and exhibited synergistic chemophotodynamic therapy against multiple cancer cell lines. Our light-responsive NP platform holds great promise for controlled drug delivery and cancer theranostics, circumventing the limitations of traditional photosensitive nanosystems.

SpRab11a-Regulated Exosomes Inhibit Bacterial Infection through the Activation of Antilipopolysaccharide Factors in Crustaceans.

Exosomes, secreted by most cells, are critical antimicrobial immune factors in animals. Recent studies of certain key regulators of vesicular transport, the Rab GTPases, have linked Rab dysfunction to regulation of innate immune signaling. However, the relationship between exosomes and Rab GTPases, resulting in antimicrobial activity in vertebrates and invertebrates during pathogenic infection, has not been addressed. In this study, SpRab11a was reported to have a protective effect on the survival rate of mud crabs Scylla paramamosain after Vibrio parahaemolyticus challenge through the stimulation of exosome secretion and modulation of anti-LPS factor (ALF) expression. Furthermore, Sp14-3-3 was confirmed to be densely packaged in exosomes after V. parahaemolyticus infection, which could recruit the MyD88 and TLR by binding the Toll/IL-1R domain to the plasma membrane, promoting the translocation of Dorsal from the cytoplasm into the nucleus, and thereby regulating ALFs expression in the hemocytes of mud crab in response to the bacterial infection. The findings therefore provide, to our knowledge, a novel mechanism that underlies the cross-talk between SpRab11a-regulated exosome formation and ALFs expression in innate immune response in invertebrates, with a crustacean species, mud crab S. paramamosain, as a model study.

Exosome-mediated apoptosis pathway during WSSV infection in crustacean mud crab.

MicroRNAs are regulatory molecules that can be packaged into exosomes to modulate cellular response of recipients. While the role of exosomes during viral infection is beginning to be appreciated, the involvement of exosomal miRNAs in immunoregulation in invertebrates has not been addressed. Here, we observed that exosomes released from WSSV-injected mud crabs could suppress viral replication by inducing apoptosis of hemocytes. Besides, miR-137 and miR-7847 were found to be less packaged in mud crab exosomes during viral infection, with both miR-137 and miR-7847 shown to negatively regulate apoptosis by targeting the apoptosis-inducing factor (AIF). Our data also revealed that AIF translocated to the nucleus to induce DNA fragmentation, and could competitively bind to HSP70 to disintegrate the HSP70-Bax (Bcl-2-associated X protein) complex, thereby activating the mitochondria apoptosis pathway by freeing Bax. The present finding therefore provides a novel mechanism that underlies the crosstalk between exosomal miRNAs and apoptosis pathway in innate immune response in invertebrates.

Supramolecular Self-Assembly in Living Cells.

Supramolecular interactions rely on non-covalent forces, such as hydrophobic effects, hydrogen-bonding, and electrostatic interactions, which govern many intracellular biological pathways. In cellulo supramolecular self-assembly is mainly based on host-guest interactions, changes in pH, enzymes, and polymerization-induced self-assembly to accurately induce various unnatural reactions without disturbing natural biological processes. This process can produce synthetic biocompatible macromolecules to control cell properties and regulate biological functions, such as cell proliferation and differentiation. This Minireview focuses on the latest reports in the field of in cellulo supramolecular self-assembly and anticipates future advances regarding its activation in response to internal and external stimuli, such as pH changes, reactive oxygen species, and enzymes, as well as external light illumination.

C=C Bond Oxidative Cleavage of BODIPY Photocages by Visible Light.

Photocages for protection and the controlled release of bioactive compounds have been widely investigated. However, the vast majority of these photocages employ the cleavage of single bonds and high-energy ultraviolet light. The construction of a photoactivation system that uses visible light to cleave unsaturated bonds still remains a challenge. Herein, we report a regioselective oxidative cleavage of C=C bonds from a boron-dipyrrolemethene (BODIPY)-based photocage by illumination at 630 nm, resulting in a free aldehyde and a thiol fluorescent probe. This strategy was demonstrated in live HeLa cells, and the generated α-formyl-BODIPY allowed real-time monitoring of aldehyde release in the cells. In particular, it is shown that a mannose-functionalized photocage can target HepG2 cells.

Transcriptome and metabolome integration analysis of mud crab Scylla paramamosain challenged to Vibrio parahaemolyticus infection.

Vibrio parahaemolyticus (V. parahaemolyticus) is a common pathogen for marine crustacean, which causes severe illnesses in aquatic animals. Therefore, it is meaningful to explore the mechanism during V. parahaemolyticus infection. In this study, to investigate the immune responses of mud crab Scylla paramamosain (S. paramamosain) to V. parahaemolyticus, we established the metabolic and transcriptional profiles of mud crab hemocytes challenged with V. parahaemolyticus. The results indicated that V. parahaemolyticus infection could induce a series of metabolism alterations at both metabolome and transcriptome levels, including biosynthesis of amino acids and Aminoacyl-tRNA, Purine and pyrimidine metabolism, TCA cycle and glutamine metabolism. In this context, through the integration of metabolomics and transcriptomics, our study provided a more comprehensive understanding of the biological process in mud crab against pathogen infection.

The miRNAs profiling revealed by high-throughput sequencing upon WSSV infection in mud crab Scylla paramamosain.

microRNAs (miRNAs) are known to regulate various immune functions by silencing the target genes in both vertebrates and invertebrates. However, in mud crab Scylla paramamosain, the role of miRNAs during the response to virus invasion remains unclear. To investigate the roles of miRNAs in S. paramamosain during virus infection, the mud crab was challenged with white spot syndrome virus (WSSV) and then subjected to the transcriptional analysis at different conditions. The results of high-throughput sequencing revealed that 940,379 and 1,306,023 high-quality mappable reads were detected in the hemocyte of normal and WSSV-infected mud crabs, respectively. Besides, the total number of 261 unique miRNAs were identified. Among them, 131 miRNAs were specifically expressed in the hemocytes of normal mud crabs, 46 miRNAs were specifically transcribed in those of WSSV-infected individuals, the other 84 miRNAs were expressed in both normal and WSSV-infected individuals. Furthermore, a number of 152 (89 down-regulated and 63 up-regulated) miRNAs were found to be differentially expressed in the WSSV-infected hemocytes, normalized to the controls. The identified miRNAs were subjected to GO analysis and target gene prediction and the results suggested that the differentially regulated miRNAs were mainly correlated with the changes of the immune responses of the hemocytes, including phagocytosis, melanism, and apoptosis as well. Taken together, the results demonstrated that the expressed miRNAs during the virus infection were mainly involved in the regulation of immunological pathways in mud crabs. Our findings not only enrich the understanding of the functions of miRNAs in the innate immune system but also provide some novel potential targets for the prevention of WSSV infection in crustaceans.

SpBAG1 promotes the WSSV infection by inhibiting apoptosis in mud crab (Scylla paramamosain).

Bcl-2 associated athanogene-1 (BAG1) is involved in various signalling pathways including apoptosis, cell proliferation, gene transcriptional regulation and signal transduction in animals. However the functions of BAG1 during the antiviral response of mud crab Scylla paramamosain is still unclear. In this study, the mud crab BAG1 (SpBAG1) was characterized to consist of 1761 nucleotides, containing an opening frame of 630bp encoding 209 amino acids with an ubiquitin domain and a BAG1 domain. SpBAG1 was found to be significantly up-regulated at 6 h-24 h, but down-regulated from 48 h-72 h in the hemocytes of mud crab after challenge with white spot syndrome virus (WSSV). RNAi knock-down of SpBAG1 significantly reduced the copies of WSSV and increased the apoptotic rate in mud crabs. The finding from this study suggested that SpBAG1 could promote the WSSV infection by inhibiting apoptosis in mud crab. Therefore, to the best of our knowledge, this is the first study demonstrating the role of SpBAG1 as a novel apoptosis inhibitor to promote virus infection in mud crab.

Antiviral effect of the viroporin inhibitors against Taiwan isolates of infectious bronchitis virus (IBV).

Coronaviruses (CoVs) are significant animal and human pathogens, characterized by being enveloped RNA viruses with positive-sense single-stranded RNA. The Coronaviridae family encompasses four genera, among which gammacoronaviruses pose a major threat to the poultry industry, which infectious bronchitis virus (IBV) being the most prominent of these threats. Particularly, IBV adversely affects broiler growth and egg production, causing substantial losses. The IBV strains currently circulating in Taiwan include the IBV Taiwan-I (TW-I) serotype, IBV Taiwan-II (TW-II) serotype, and vaccine strains. Therefore, ongoing efforts have focused on developing novel vaccines and discovering antiviral agents. The envelope (E) proteins of CoVs accumulate in the endoplasmic reticulum-Golgi intermediate compartment prior to virus budding. These E proteins assemble into viroporins, exhibiting ion channel activity that leads to cell membrane disruption, making them attractive targets for antiviral therapy. In this study, we investigated the E proteins of IBV H-120, as well as IBV serotypes TW-I and TW-II. E protein expression resulted in inhibited bacteria growth, increased permeability of bacteria to ß-galactosidase substrates, and blocked protein synthesis of bacteria by hygromycin B (HygB). Furthermore, in the presence of E proteins, HygB also impeded protein translation in DF-1 cells and damaged their membrane integrity. Collectively, these findings confirm the viroporin activity of the E proteins from IBV H-120, IBV serotype TW-I, and IBV serotype TW-II. Next, the viroporin inhibitors, 5-(N,N-hexamethylene) amiloride (HMA) and 4,4'-diisothiocyano stilbene-2,2'-disulphonic acid (DIDS) were used to inhibit the viroporin activities of the E proteins of IBV H-120, IBV serotype TW-I, and IBV serotype TW-II. In chicken embryos and chickens infected with IBV serotypes TW-I and IBV TW-II, no survivors were observed at 6 and 11 days post-infection (dpi), respectively. However, treatments with both DIDS and HMA increased the survival rates in infected chicken embryos and chickens and mitigated histopathological lesions in the trachea and kidney. Additionally, a 3D pentameric structure of the IBV E protein was constructed via homology modeling. As expected, both inhibitors were found to bind to the lipid-facing surface within the transmembrane domain of the E protein, inhibiting ion conduction. Taken together, our findings provide comprehensive evidence supporting the use of viroporin inhibitors as promising antiviral agents against IBV Taiwan isolates.

Ultrasound-responsive glycopolymer micelles for targeted dual drug delivery in cancer therapy.

Controlled drug release of nanoparticles was achieved by irreversibly disrupting polymer micelles through high-intensity focused ultrasound (HIFU) induction. An ultrasound-responsive block copolymer was synthesized, comprising an end-functional Eosin Y fluorophore, 2-tetrahydropyranyl acrylate (THPA), and acrylate mannose (MAN). The block copolymer was then self-assembled to produce micelles. The chemotherapy drug dasatinib (DAS) and the sonodynamic therapy agent methylene blue (MB) were encapsulated by the self-assembly of the block copolymer. This targeted nanoparticle enables sonodynamic therapy through high-intensity focused ultrasound while triggering nanoparticle disassembly for controlled drug release. The ultrasound-mediated, non-invasive strategy provides external spatiotemporal control for targeted tumour treatment.

SpBAG3 assisted WSSV infection in mud crab (Scylla paramamosain) by inhibiting apoptosis.

The function of B-cell lymphoma-2 (Bcl-2) family proteins can be divided into two categories: anti-apoptotic and pro-apoptotic. As an anti-apoptotic protein, Bcl2-associated athanogene 3 (BAG3) plays a key role in regulating apoptosis, development, cell movement, and autophagy, and mediating the adaptability of cells to stimulation. However, SpBAG3 has not been reported in mud crab (Scylla paramamosain), and the regulatory effect of SpBAG3 on apoptosis in mud crab and its function in antiviral immunity is still unknown. In this study, SpBAG3 was found, and characterized, which encoded a total of 175 amino acid (molecular mass 19.3 kDa), including a specific conserved domain of the BAG family. SpBAG3 was significantly down-regulated at 0-48 h post-infection with WSSV in vivo. The antiviral effect of SpBAG3 was investigated using RNA interference. The results indicated that SpBAG3 might be involved in assisting the replication of WSSV in the host. SpBAG3 could change the mitochondrial membrane potential (△ψm), and affect cell apoptosis through mitochondrial apoptotic pathways. Therefore, the results of this study suggested that SpBAG3 could assist WSSV infection by inhibiting the apoptosis of the hemocytes in mud crab.

Tumor-Targeting Polymer-Drug Conjugate for Liver Cancer Treatment In Vitro.

Bufalin (buf) has poor solubility in aqueous solution, poor tumor targeting, and many non-specific toxic and side effects. The advantages of high-molecular-weight polymer conjugates are that they can improve the water solubility of buf, prolong plasma half-life, and reduce non-specific toxicity. A novel water-soluble polymer-drug conjugate with buf and fluorescein pendants was prepared by the combination of reversible addition-fragmentation transfer (RAFT) polymerization and click chemistry. Its anticancer performance and cellular uptake behavior against liver cancer were investigated in vitro. The polymer-buf conjugates exhibit controlled release and tumor-targeting capabilities, showing promise for clinical applications.

A nanomedicine enables synergistic chemo/photodynamic therapy for pancreatic cancer treatment.

Pancreatic cancer is one of the leading causes of cancer-related deaths worldwide. Gemcitabine (Gem) has been a key chemotherapy agent for pancreatic cancer treatment by suppressing cell proliferation and inducing apoptosis. However, the overexpression of inhibitors of apoptosis (IAP) family of proteins during the carcinogenesis of pancreatic cancer can develop resistance to chemotherapy treatment and result in poor efficacy. To achieve the synergistic combinations of multiple strategies for this dismal disease, we developed a robust nanomedicine system, consisting of a photodynamic therapeutic agent (chlorine e6, Ce6) and a pro-apoptotic peptide-Gem conjugate. To have spatiotemporally controlled drug release, the pro-apoptotic peptide-Gem conjugate was designed to have a vinyldithioether linker that was sensitive to reactive oxygen species (ROS). The nanomedicine was fabricated by the direct self-assembly of the pro-apoptotic peptide-Gem conjugate with Ce6. After being delivered into tumors, the nanomedicine disassembled and rapidly released Gem, Ce6, and the pro-apoptotic peptide upon light illumination (660 nm). Both in vitro and in vivo studies in pancreatic cancer models confirmed the tumor inhibition efficacy with low systemic toxicity to animals.

Targeting the N-Terminus Domain of the Coronavirus Nucleocapsid Protein Induces Abnormal Oligomerization via Allosteric Modulation.

Epidemics caused by coronaviruses (CoVs), namely the severe acute respiratory syndrome (SARS) (2003), Middle East respiratory syndrome (MERS) (2012), and coronavirus disease 2019 (COVID-19) (2019), have triggered a global public health emergency. Drug development against CoVs is inherently arduous. The nucleocapsid (N) protein forms an oligomer and facilitates binding with the viral RNA genome, which is critical in the life cycle of the virus. In the current study, we found a potential allosteric site (Site 1) using PARS, an online allosteric site predictor, in the CoV N-N-terminal RNA-binding domain (NTD) to modulate the N protein conformation. We identified 5-hydroxyindole as the lead via molecular docking to target Site 1. We designed and synthesized four 5-hydroxyindole derivatives, named P4-1 to P4-4, based on the pose of 5-hydroxyindole in the docking model complex. Small-angle X-ray scattering (SAXS) data indicate that two 5-hydroxyindole compounds with higher hydrophobic R-groups mediate the binding between N-NTD and N-C-terminal dimerization domain (CTD) and elicit high-order oligomerization of the whole N protein. Furthermore, the crystal structures suggested that these two compounds act on this novel cavity and create a flat surface with higher hydrophobicity, which may mediate the interaction between N-NTD and N-CTD. Taken together, we discovered an allosteric binding pocket targeting small molecules that induces abnormal aggregation of the CoV N protein. These novel concepts will facilitate protein-protein interaction (PPI)-based drug design against various CoVs.

Corrigendum: Targeting the N-terminus domain of the coronavirus nucleocapsid protein induces abnormal oligomerization via allosteric modulation.

[This corrects the article DOI: 10.3389/fmolb.2022.871499.].

Targeting protein-protein interaction interfaces in COVID-19 drug discovery.

To date, the COVID-19 pandemic has claimed over 1 million human lives, infected another 50 million individuals and wreaked havoc on the global economy. The crisis has spurred the ongoing development of drugs targeting its etiological agent, the SARS-CoV-2. Targeting relevant protein-protein interaction interfaces (PPIIs) is a viable paradigm for the design of antiviral drugs and enriches the targetable chemical space by providing alternative targets for drug discovery. In this review, we will provide a comprehensive overview of the theory, methods and applications of PPII-targeted drug development towards COVID-19 based on recent literature. We will also highlight novel developments, such as the successful use of non-native protein-protein interactions as targets for antiviral drug screening. We hope that this review may serve as an entry point for those interested in applying PPIIs towards COVID-19 drug discovery and speed up drug development against the pandemic.

Elucidation of metabolic responses in mud crab Scylla paramamosain challenged to WSSV infection by integration of metabolomics and transcriptomics.

White spot syndrome virus (WSSV) is a severe pathogen of mud crab Scylla paramamosain (S. paramamosain). Hemolymph, containing three types of hemocytes, is the key immunoregulatory tool of mud crab in response to pathogens. Herein, the metabonomics and transcriptomics analysis of hemocytes were adopted to investigate the immune response of S. paramamosain challenged to WSSV. We established the metabolic and transcriptional profiles of mud crab hemocytes with different treatments, including the control group (WT), WSSV early infected group (WSSV-6 h) and WSSV later infected group (WSSV-72 h). The results showed that 68 metabolites were dysregulated both in WSSV-infected mud crab of early stage and later stage, while 4452 genes were up-regulated and 9746 genes were down-regulated in WSSV-6 h, and 2016 genes were up-regulated and 6229 genes were down-regulated compared in WSSV-72 h. We found that several pathways were dysregulated at both metabolic and transcriptional levels, including ABC transporters, purine metabolism, taurine and hypotaurine metabolism in the WSSV early infected group, cysteine metabolism, methionine metabolism and biosynthesis of unsaturated fatty acids in the WSSV later infected group. In this context, through the integration of metabolomics and transcriptomics, our study provided a more comprehensive understanding of the biological process in mud crab against viral invasion.

Elucidation of Gut Microbiota in Mud Crab Scylla paramamosain Challenged to WSSV and Aeromonas hydrophila.

Mud crab Scylla paramamosain (S. paramamosain) is an economically important marine crab species around the world. White spot syndrome virus (WSSV) and Aeromonas hydrophila (AH) are pathogens during mud crab mariculture. It has been reported that gut microbiota possessed a great impact on the host development, nutrition, immunity, and disease resistance. However, little information was known about the impacts of WSSV or AH infection on the structure, composition, and function of the gut microbiotain of mud crabs. In this study, the gut microbiota of mud crabs infected with A. hydrophila and WSSV were characterized. The results showed that the composition and bacteria correlation of the gut microbiota were significantly decreased. During A. hydrophila infection, the pathogens played a major regulatory role in host. While in the mud crabs infected with WSSV, many beneficial strains had a great impact on the host expect for the pathogens. Therefore, our study revealed the effect of pathogens infection on gut microbiota of mud crabs and clarified the difference between viral infection and bacterial infection.

miR-9875 functions in antiviral immunity by targeting PDCD6 in mud crab (Scylla paramamosain).

Programmed cell death 6 (PDCD6) is a well-known apoptosis regulator that is involved in the immunity of mammals. However, the effects of miRNA-mediated regulation of PDCD6 expression on apoptosis and virus infection in organisms, especially in marine invertebrates, have not been extensively explored. In this study, PDCD6 of mud crab (Scylla paramamosain) (Sp-PDCD6) was characterized. The results showed that Sp-PDCD6 contains five EF-hands domains and could suppress virus infection via apoptosis promotion. It also presented that Sp-PDCD6 was directly targeted by miR-9875 in vitro and in vivo, miR-9875 served as a positive regulator during the virus invasion. The findings indicated that the miR-9875-PDCD6 pathway possessed fundamental effects on the immune response to virus infection in mud crab. Therefore, our research provided a novel insight into the roles of both miR-9875 and PDCD6 in the regulation of apoptosis and virus defense in mud crab.