Sulforaphane-Enriched Extracts from Broccoli Exhibit Antimicrobial Activity against Plant Pathogens, Promising a Natural Antimicrobial Agent for Crop Protection.

Sulforaphane (SFN) is one of the hydrolysates of glucosinolates (GSLs), primarily derived from Brassica vegetables like broccoli. In clinical therapy, SFN has been proven to display antimicrobial, anticancer, antioxidant, and anti-inflammatory properties. However, the antimicrobial effects and mechanism of SFN against plant pathogens need to be further elucidated, which limits its application in agriculture. In this study, the genetic factors involved in SFN biosynthesis in 33 B. oleracea varieties were explored. The finding showed that besides the genetic background of different B. oleracea varieties, myrosinase and ESP genes play important roles in affecting SFN content. Subsequently, the molecular identification cards of these 33 B. oleracea varieties were constructed to rapidly assess their SFN biosynthetic ability. Furthermore, an optimized protocol for SFN extraction using low-cost broccoli curds was established, yielding SFN-enriched extracts (SFN-ee) containing up to 628.44 µg/g DW of SFN. The antimicrobial activity assay confirmed that SFN-ee obtained here remarkably inhibit the proliferation of nine tested microorganisms including four plant pathogens by destroying their membrane integrity. Additionally, the data demonstrated that exogenous application of SFN-ee could also induce ROS accumulation in broccoli leaves. These results indicated that SFN-ee should play a dual role in defense against plant pathogens by directly killing pathogenic cells and activating the ROS signaling pathway. These findings provide new evidence for the antimicrobial effect and mechanism of SFN against plant pathogens, and suggest that SFN-ee can be used as a natural plant antimicrobial agent for crop protection and food preservation.

A Structure-Guided Genetic Modification Strategy: Developing Seneca Valley Virus Therapy against Nonsensitive Nonsmall Cell Lung Carcinoma.

Numerous studies have illustrated that the Seneca Valley virus (SVV) shows sufficient oncolytic efficacy targeting small cell lung cancer (SCLC). However, the therapeutics of nonsmall cell lung carcinoma (NSCLC, accounts for 85% of lung cancer cases) using oncolytic virus have been resisting due to the filtration of neutralizing antibody and limited reproduction capacity. Here, we employed structural biology and reverse genetics to optimize novel oncolytic SVV mutants (viral receptor-associated mutant SVV-S177A and viral antigenic peptide-related variant SVV-S177A/P60S) with increased infectivity and lower immunogenicity. The results of the NSCLC-bearing athymic mouse model demonstrated that wild-type (wt) SVV-HB extended the median overall survival (mOS) from 11 days in the PBS group to 19 days. Notably, the newly discovered mutations significantly (P < 0.001) prolonged the mOS from 11 days in the control cohort to 23 days in the SVV-S177A cohort and the SVV-S177A/P60S cohort. Taken together, we present a structure-guided genetic modification strategy for oncolytic SVV optimization and provide a candidate for developing oncolytic viral therapy against nonsensitive NSCLC. IMPORTANCE Nonsmall cell lung cancer (NSCLC) accounts for approximately 85% of lung cancer cases (more than 1.85 million cases with 1.48 million deaths in 2020). In the present study, two novel oncolytic SVV mutants modified based on structural biology and reverse genetics (viral receptor-associated mutant SVV-S177A and viral antigenic peptide-related mutant SVV-S177A/P60S) with increased infectivity or lower immunogenicity significantly (P < 0.001) prolonged the mOS from 11 days in the control cohort to 23 days in the SVV-S177A cohort and the SVV-S177A/P60S cohort in the NSCLC-bearing athymic mouse model, which may provide the direction for modifying SVV to improve the effect of oncolysis.

Machine-Learned Molecular Surface and Its Application to Implicit Solvent Simulations.

Implicit solvent models, such as Poisson-Boltzmann models, play important roles in computational studies of biomolecules. A vital step in almost all implicit solvent models is to determine the solvent-solute interface, and the solvent excluded surface (SES) is the most widely used interface definition in these models. However, classical algorithms used for computing SES are geometry-based, so that they are neither suitable for parallel implementations nor convenient for obtaining surface derivatives. To address the limitations, we explored a machine learning strategy to obtain a level set formulation for the SES. The training process was conducted in three steps, eventually leading to a model with over 95% agreement with the classical SES. Visualization of tested molecular surfaces shows that the machine-learned SES overlaps with the classical SES in almost all situations. Further analyses show that the machine-learned SES is incredibly stable in terms of rotational variation of tested molecules. Our timing analysis shows that the machine-learned SES is roughly 2.5 times as efficient as the classical SES routine implemented in Amber/PBSA on a tested central processing unit (CPU) platform. We expect further performance gain on massively parallel platforms such as graphics processing units (GPUs) given the ease in converting the machine-learned SES to a parallel procedure. We also implemented the machine-learned SES into the Amber/PBSA program to study its performance on reaction field energy calculation. The analysis shows that the two sets of reaction field energies are highly consistent with a 1% deviation on average. Given its level set formulation, we expect the machine-learned SES to be applied in molecular simulations that require either surface derivatives or high efficiency on parallel computing platforms.

Cholesterol-25-Hydroxylase Suppresses Seneca Valley Virus Infection via Producing 25-Hydroxycholesterol to Block Adsorption Procedure.

Cholesterol-25-hydroxylase (CH25H) is a membrane protein associated with endoplasmic reticulum, and it is an interferon-stimulated factor regulated by interferon. CH25H catalyzes cholesterol to produce 25-hydroxycholesterol (25HC) by adding a second hydroxyl to the 25th carbon atom of cholesterol. Recent studies have shown that both CH25H and 25HC could inhibit the replication of many viruses. In this study, we found that ectopic expression of CH25H in HEK-293T and BHK-21 cell lines could inhibit the replication of Seneca Valley virus (SVV) and that there was no species difference. On the other hand, the knockdown of CH25H could enhance the replication of SVV in HEK-293T and BHK-21 cells, indicating the importance of CH25H. To some extent, the CH25H mutant without hydroxylase activity also lost its ability to inhibit SVV amplification. Further studies demonstrated that 25HC was involved in the entire life cycle of SVV, especially in repressing its adsorption process. This study reveals that CH25H exerts the advantage of innate immunity mainly by producing 25HC to block virion adsorption.

Selective autophagy receptor SQSTM1/ p62 inhibits Seneca Valley virus replication by targeting viral VP1 and VP3.

Macroautophagy/autophagy plays a critical role in antiviral immunity through targeting viruses and initiating host immune responses. The receptor protein, SQSTM1/p62 (sequestosome 1), plays a vital role in selective autophagy. It serves as a receptor targeting ubiquitinated proteins or pathogens to phagophores for degradation. In this study, we explored the reciprocal regulation between selective autophagy receptor SQSTM1 and Seneca Valley virus (SVV). SVV infection induced autophagy. Autophagy promoted SVV infection in pig cells but played opposite functions in human cells. Overexpression of SQSTM1 decreased viral protein production and reduced viral titers. Further study showed that SQSTM1 interacted with SVV VP1 and VP3 independent of its UBA domain. SQSTM1 targeted SVV VP1 and VP3 to phagophores for degradation to inhibit viral replication. To counteract this, SVV evolved strategies to circumvent the host autophagic machinery to promote viral replication. SVV 3Cpro targeted the receptor SQSTM1 for cleavage at glutamic acid 355, glutamine 392, and glutamine 395 and abolished its capacity to mediate selective autophagy. At the same time, the 3Cpro-mediated SQSTM1 cleavage products lost the ability to inhibit viral propagation. Collectively, our results provide evidence for selective autophagy in host against viruses and reveal potential viral strategies to evade autophagic machinery for successful pathogenesis.Abbreviations: Baf.A1: bafilomycin A1; Co-IP: co-immunoprecipitation; hpi: h post-infection; LIR: LC3-interacting region; MAP1LC3B/LC3B: microtubule-associated protein 1 light chain 3 beta; MOI: multiplicity of infection; PB1: N-terminal Phox/Bem1p; Rap.: rapamycin; Seneca Valley virus: SVV; SQSTM1/p62: sequestosome 1; SQSTM1-N355: residues 1 to 355 of SQSTM1; SQSTM1-C355: residues 355 to 478 of SQSTM1; SQSTM1-N392: residues 1 to 392 of SQSTM1; SQSTM1-C392: residues 392 to 478 of SQSTM1; SQSTM1-N388: residues 1 to 388 of SQSTM1; SQSTM1-N397: residues 1 to 397 of SQSTM1; UBA: ubiquitin association; Ubi: ubiquitin.

A Self-Assembling Ferritin Nanoplatform for Designing Classical Swine Fever Vaccine: Elicitation of Potent Neutralizing Antibody.

Protein-based self-assembling nanoplatforms exhibit superior immunogenicity compared with soluble antigens. Here, we present a comprehensive vaccine strategy for displaying classical swine fever virus (CSFV) E2 glycoprotein on the surface of ferritin (fe) nanocages. An E2-specific blocking antibody assay showed that the blocking rates in pE2-fe/Gel02 (84.3%) and a half-dose cohort of E2-fe/Gel02 (81.9%) were significantly higher (p < 0.05) than that in a ferritin-free cohort of pE2/Gel02 (62.7%) at 21 days post immunization (dpi) in vivo. Furthermore, quantitation of neutralizing potency revealed that a highly significant difference (p < 0.001) was observed between the pE2-fe/Gel02 cohort (1:32, equivalent to live-attenuated strain C at 1:32) and the pE2/Gel02 cohort (1:4) at 21 dpi. Moreover, the innate immune cytokines of IL-4 and IFN-γ activated by the half-dose (20 µg) cohort of E2-fe/Gel02 were equivalent to those elicited by the full dose (40 µg) of purified E2 in the pE2/Gel02 cohort at most time points. In conclusion, we successfully obtained an antigen-displaying E2-ferritin nanoplatform and confirmed high ferritin-assisted humoral and cellular immunities. Our results provided a novel paradigm of self-assembling nanovaccine development for the defense and elimination of potentially pandemic infectious viral pathogens.

Immunization with a recombinant fusion of porcine reproductive and respiratory syndrome virus modified GP5 and ferritin elicits enhanced protective immunity in pigs.

Porcine reproductive and respiratory syndrome (PRRS) has caused huge economic losses in the swine industry worldwide. Live and inactivated vaccines have only been partially successful in generating protective immune responses. The PRRS virus (PRRSV) glycoprotein 5 (GP5) is a major viral antigenic target and is thus suitable for development of genetically engineered PRRSV vaccines. Here, a modified GP5 and ferritin were fused and expressed using a baculovirus system to generate a GP5m-ferritin nanoparticle vaccine. We demonstrated that the GP5m-ferritin vaccine elicited higher serum antibody titers in pigs than inactivated PRRSV. Moreover, immunization with GP5m-Ft promoted a Th1-dominant cellular immune response and enhanced specific T-lymphocyte immune responses. GP5m-ferritin-vaccinated pigs had significantly lower mean rectal temperatures, respiratory scores, viremia, and macroscopic and microscopic lung lesion scores post-challenge compared with unvaccinated pigs. These results indicated that GP5m-ferritin subunit vaccines can elicit specific protective immune responses and represent promising vaccine candidates.

Efficient mucosal vaccination of a novel classical swine fever virus E2-Fc fusion protein mediated by neonatal Fc receptor.

Classical swine fever (CSF) remains one of the most important highly contagious and fatal viral disease of swine with high morbidity and mortality. CSF is caused by classical swine fever virus (CSFV), a small, enveloped RNA virus of the genus Pestivirus. The aim of this study was to construct the a novel CSFV Fc-fusion recombinant protein and evaluate the efficacy as a vaccine against CSFV. Here, we obtained a novel subunit vaccine expressing CSFV E2 recombinant fusion protein in CHO-S cells. Functional analysis revealed that CSFV Fc-fusion recombinant protein (CSFV-E2-Fc) could bind to FcγRI on antigen-presenting cells (APCs) and significantly increase IgA levels in serum and feces, inducing stronger mucosal immune response in swine. Additionally, CSFV-E2-Fc immunization enhanced CSFV-specific T cell immune response with a Th1-like pattern of cytokine secretion, remarkably stimulated the Th1-biased cellular immune response and humoral immune response. Further, the protective effects of CSFV-E2-Fc subunit vaccines were confirmed. The data suggest that CSFV E2-Fc recombinant fusion protein may be a promising candidate subunit vaccine to elicit immune response and protect against CSFV.

Palladium-Catalyzed Controllable Reductive/Oxidative Heck Coupling between Cyclic Enones and Thiophenes via C-H Activation.

Herein, we report a straightforward, environmentally friendly, and controllable palladium/ligand catalytic system to enable reductive/oxidative Heck reactions of cyclic enones with thiophene or furan derivatives via C-H activation. The key to this tunable reaction is the appropriate intercepting thienyl-Pd(II)-enolate during the enolization process. Such a controllable and economic protocol would not only provide efficient methods to construct various value-added ß-heteroarylated cyclic ketones/enones but also shed light on developing other conjugate addition reactions via C-H activation.

Enhanced protective immunity to CSFV E2 subunit vaccine by using IFN-γ as immunoadjuvant in weaning piglets.

The glycoprotein E2 of classical swine fever virus (CSFV) is a major immunogenic protein that induces neutralizing antibodies and protective immunity. Thus, E2 is a suitable target antigen for the development of genetically engineered CSFV vaccines. However, these vaccines cannot generate complete protective immunity in their hosts, thereby limiting the scope of applications under field conditions. IFN-γ is an immune adjuvant that has been shown to enhance antigen immune response in various experimental models. In this study, porcine IFN-γ was used to improve the immunogenicity of the CSFV E2 subunit vaccine in pigs. Pigs were immunized with E2 subunit vaccine alone or in combination with IFN-γ. Results demonstrated that porcine IFN-γ did not enhance the CSFV-specific antibody and neutralizing antibody titers compared with the E2 subunit vaccine alone. However, co-administration of the E2 and IFN-γ subunit vaccines significantly enhanced the CSFV-specific IFN-γ expression. These findings indicated that porcine IFN-γ can increase cellular immune responses to E2 protein in pigs. Furthermore, co-immunization with E2 + IFN-γ subunit vaccine and C-strain conferred complete protection against CSFV. In contrast, E2 subunit vaccines provided incomplete protection in pigs. These results indicated that using IFN-γ as an adjuvant with CSFV E2 subunit vaccines can enhance the specific protective immune response. Therefore, E2 + IFN-γ subunit vaccine is a promising marker vaccine candidate for the control and eradication of CSF.

Facile dynamic one-step modular assembly based on boronic acid-diol for construction of a micellar drug delivery system.

Nano-assembled amphiphilic micelles with characteristics including facile control, a simplified construction procedure, convenient and efficient drug loading, and a controlled release at pathological sites are in high demand. This study reports a facile and dynamic one-step modular assembly strategy based on boronic acid-diol for constructing focus-responsive micellar drug delivery systems. In this manner, a dopamine modified hydrophilic building block, phenylboronic acid modified hydrophobic building block and drug molecules (Dox) spontaneously one-step assembled into drug encapsulated distinct core/shell micelles (Dox/PBAE-M) in mild physiological media. After a simple adjustment of weight ratios between these three building blocks, Dox/PBAE-M, with the highest Dox-loading capacity (22.4%) and optimal physical dimensions, was generated. Furthermore, the desirable pH-dependent disassembly of Dox/PBAE-M was independently verified by morphological changes alongside in vitro release of Dox in different simulated environments. The experimental results here demonstrated that Dox/PBAE-M kept structural integrity in normal physiological environments, while accomplishing a selective nano-disassembly and Dox release within acid endo/lysosomes. As a result, Dox/PBAE-M exhibited the highest cytotoxicity and apoptosis induction among all of the tested groups on the 4T1 breast cancer xenograft model. This newly proposed assembly strategy gave new insight into easy fabrication and disassembly of multi-functional micellar drug delivery systems.

Neutrophil-mediated anticancer drug delivery for suppression of postoperative malignant glioma recurrence.

Cell-mediated drug-delivery systems have received considerable attention for their enhanced therapeutic specificity and efficacy in cancer treatment. Neutrophils (NEs), the most abundant type of immune cells, are known to penetrate inflamed brain tumours. Here we show that NEs carrying liposomes that contain paclitaxel (PTX) can penetrate the brain and suppress the recurrence of glioma in mice whose tumour has been resected surgically. Inflammatory factors released after tumour resection guide the movement of the NEs into the inflamed brain. The highly concentrated inflammatory signals in the brain trigger the release of liposomal PTX from the NEs, which allows delivery of PTX into the remaining invading tumour cells. We show that this NE-mediated delivery of drugs efficiently slows the recurrent growth of tumours, with significantly improved survival rates, but does not completely inhibit the regrowth of tumours.

Smart nanoparticles with a detachable outer shell for maximized synergistic antitumor efficacy of therapeutics with varying physicochemical properties.

Co-delivery systems capable of transporting hydrophobic chemotherapeutics and hydrophilic siRNA to the same cell population with simultaneous burst release of both drugs to maximize synergistic anticancer efficacy remains elusive. In this light, a multifunctional nanoparticle (HA-PSR) consisting of a redox-sensitive core and detachable crosslinked hyaluronic acid (HA) shell was developed. Octyl modified PEI containing disulfide linkages (PSR) were synthesized as the core materials for co-encapsulation of chemotherapeutics and siRNA, while a HAase-sensitive thiolated HA (HA-SH) was collaboratively assembled to the anionic shell for CD44-mediated active targeting along with enhanced and detachable protection for drug loaded inner cores. Resultantly, HA de-protected redox-sensitive inner cores achieved co-burst release of both cargoes when triggered by glutathione (GSH) rich environments in cytoplasm. Results of in-vivo and in-vitro testing indicated successful co-encapsulation of hydrophobic drugs and hydrophilic siRNA with adjustable ratios. Selective delivery to CD44 overexpressing tumors was achieved through passive and active targeting, followed by HAase-triggered HA de-shielding and GSH-triggered burst release of both cargos. Rapid intracellular trafficking maximized synergistic cytotoxicities of chemotherapeutics and siRNA for remarkable tumor inhibition in a xenograft animal tumor model. Consequently, the HA-PSR nanoparticle holds great potential for combined chemotherapeutics/siRNA treatment in cancer with maximized synergistic antitumor efficacy.

Genome Sequences of the Novel Porcine Parvovirus 3, Identified in Guangxi Province, China.

Porcine parvovirus 3 is a novel parvovirus that infects pigs. Here, we report two genome sequences of porcine parvovirus 3 strains GX1 and GX2, which are highly prevalent in Guangxi province. It will help in understanding the epidemiology and molecular characteristics of the porcine parvovirus 3.

Sequential intra-intercellular nanoparticle delivery system for deep tumor penetration.

To achieve deep tumor penetration of large-sized nanoparticles (NPs), we have developed a reversible swelling-shrinking nanogel in response to pH variation for a sequential intra-intercellular NP delivery. The nanogel had a crosslinked polyelectrolyte core, consisting of N-lysinal-N'-succinyl chitosan and poly(N-isopropylacrylamide), and a crosslinked bovine serum albumin shell, which was able to swell in an acidic environment and shrink back under neutral conditions. The swelling resulted in a rapid release of the encapsulated chemotherapeutics in the cancer cells for efficient cytotoxicity. After being liberated from the dead cells, the contractive nanogel could infect neighboring cancer cells closer to the center of the tumor tissue.

Novel nonsecosteroidal VDR agonists with phenyl-pyrrolyl pentane skeleton.

In order to find the vitamin D receptor (VDR) ligand whose VDR agonistic activity is separated from the calcemic activity sufficiently, novel nonsecosteroidal analogs with phenyl-pyrrolyl pentane skeleton were synthesized and evaluated for the VDR binding affinity, antiproliferative activity in vitro and serum calcium raising ability in vivo (tacalcitol used as control). Among them, several compounds showed varying degrees of VDR agonistic and growth inhibition activities of the tested cell lines. The most effective compound 2g (EC50: 1.06 nM) exhibited stronger VDR agonistic activity than tacalcitol (EC50: 7.05 nM), inhibited the proliferations of HaCaT and MCF-7 cells with IC50 of 2.06 µM and 0.307 µM (tacalcitol: 2.07 µM and 0.057 µM) and showed no significant effect on serum calcium.