Polymer-Protein Nanovaccine Synthesized via Reactive Self-Assembly with Potential Application in Cancer Immunotherapy: Physicochemical and Biological Characterization In Vitro and In Vivo.

Nanovaccines composed of polymeric nanocarriers and protein-based antigens have attracted much attention in recent years because of their enormous potential in the prevention and treatment of diseases such as viral infections and cancer. While surface-conjugated protein antigens are known to be more immunoactive than encapsulated antigens, current surface conjugation methods often result in low and insufficient protein loading. Herein, reactive self-assembly is used to prepare nanovaccine from poly(ε-caprolactone) (PCL) and ovalbumin (OVA)-a model antigen. A rapid thiol-disulfide exchange reaction between PCL with pendant pyridyl disulfide groups and thiolated OVA results in the formation of nanoparticles with narrow size distribution. High OVA loading (≈70-80 wt%) is achieved, and the native secondary structure of OVA is preserved. Compared to free OVA, the nanovaccine is much superior in enhancing antigen uptake by bone marrow-derived dendritic cells (BMDCs), promoting BMDC maturation and antigen presentation via the MHC I pathway, persisting at the injection site and draining lymph nodes, activating both Th1 and Th2 T cell immunity, and ultimately, resisting tumor challenge in mice. This is the first demonstration of reactive self-assembly for the construction of a polymer-protein nanovaccine with clear potential in advancing cancer immunotherapy.

Prevalence and affective correlates of subjective cognitive decline in patients with de novo Parkinson's disease.

OBJECTIVES: The novel concept of subjective cognitive decline (SCD) in Parkinson's disease (PD) refers to subjective cognitive impairment without concurrent objective cognitive deficits. This study aimed to determine the prevalence and affective correlates of SCD in de novo PD patients. MATERIALS AND METHODS: A total of 139 de novo PD patients underwent comprehensive neuropsychological evaluation. PD patients with SCD (PD-SCD) did not meet the diagnostic criteria for mild cognitive impairment in PD (PD-MCI) based on the Movement Disorder Society Level II Criteria and were defined by a Domain-5 Score ≥1 on the Non-Motor Symptoms Questionnaire. Affective symptoms were measured using the Hamilton Depression Scale (HAMD) and Hamilton Anxiety Scale (HAMA). RESULTS: In de novo PD cohort, the prevalence of SCD was 28.1%. PD-SCD patients performed significantly better than PD-MCI patients on tests of five cognitive domains. The more commonly affected domains in PD-SCD patients were memory (28.2%) and attention/working memory (25.6%). Multivariable linear regression analysis revealed that PD-SCD was significantly associated with both HAMD (ß = 4.518, 95% CI = 0.754-8.281, p = .019) and HAMA scores (ß = 4.259, 95% CI = 1.054-7.464, p = .010). Furthermore, binary logistic regression analysis revealed that higher HAMD (OR = 1.128, 95% CI = 1.019-1.249, p = .020) and HAMA scores (OR = 1.176, 95% CI = 1.030-1.343, p = .017) increased the risk of PD-SCD. CONCLUSIONS: Our findings suggest that SCD is highly prevalent in de novo PD patients. The presence of PD-SCD is suggestive of an underlying affective disorder.

Fraxinol attenuates LPS-induced acute lung injury by equilibrating ACE-Ang II-AT1R and ACE2-Ang (1-7)-Mas and inhibiting NLRP3.

CONTEXT: Acute lung injury (ALI) is a serious heterogenous pulmonary disorder. Fraxinol was selected for this study since it is a simple coumarin compound, not previously investigated in ALI. OBJECTIVES: This study investigates the ALI therapeutic effect and mechanisms of fraxinol. MATERIALS AND METHODS: Male BALB/c mice were treated with fraxinol (20, 40, and 80 mg/kg) following intranasal injection of lipopolysaccharide (LPS; 10 µg in 50 µL). The mice in control group were intratracheally injected with 50 µL phosphate buffered saline (PBS). Raw264.7 cells were treated with fraxinol by 100 ng/mL LPS for 6 h, then treated by different concentrations of fraxinol (5, 10, and 25 µM) for 48 h. Cells in control group were treated with PBS. RESULTS: Fraxinol with doses of 20, 40, and 80 mg/kg significantly attenuated LPS-induced lung injury in mice (lung injury score, 10.4, 31.2, 50.3%). Fraxinol attenuated the apoptosis and nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing-3 (NLRP3) activation induced by LPS (apoptosis, 18.3, 30.2, 55.6%; NLRP3, 30.0, 47.7, 63.6%). The anti-apoptosis and anti-inflammation effects of fraxinol were also confirmed in Raw264.7 cells (apoptosis, 38.8, 55.3, 68.9%; NLRP3, 20.6, 55.7, 73.9%). DISCUSSION AND CONCLUSION: The anti-ALI effects of fraxinol maybe by equilibrating ACE-Ang II-AT1R and ACE2-Ang (1-7)-Mas axis and inhibiting NLRP3 inflammasome. Our research provides a candidate drug in the treatment of ALI.

A numerical simulation method of natural fragment formation and injury to human thorax.

OBJECTIVE: Fragment injury is a type of blast injury that is becoming more and more common in military campaigns and terrorist attacks. Numerical simulation methods investigating the formation of natural fragments and injuries to biological targets are expected to be developed. METHODS: A cylindrical warhead model was established and the formation process of natural fragments was simulated using the approach of tied nodes with failure through the explicit finite element (FE) software of LS-DYNA. The interaction between the detonation product and the warhead shell was simulated using the fluid-structure interaction algorithm. A method to simulate the injury of natural fragments to a biological target was presented by transforming Lagrange elements into smooth particle hydrodynamics (SPH) particles after the natural fragments were successfully formed. A computational model of the human thorax was established to simulate the injury induced by natural fragments by the node-to-surface contact algorithm with erosion. RESULTS: The discontinuous velocities of the warhead shell at different locations resulted in the formation of natural fragments with different sizes. The velocities of natural fragments increased rapidly at the initial stage and slowly after the warhead shell fractured. The initial velocities of natural fragments at the central part of the warhead shell were the largest, whereas those at both ends of the warhead shell were the smallest. The natural fragments resulted in bullet holes that were of the same shape as that of the fragments but slightly larger in size than the fragments in the human thorax after they penetrated through. Stress waves propagated in the ribs and enhanced the injury to soft tissues; additionally, ballistic pressure waves ahead of the natural fragments were also an injury factor to the soft tissues. CONCLUSION: The proposed method is effective in simulating the formation of natural fragments and their injury to biological targets. Moreover, this method will be beneficial for simulating the combined injuries of natural fragments and shock waves to biological targets.

E3 Ubiquitin Ligase c-cbl Inhibits Microglia Activation After Chronic Constriction Injury.

E3 ubiquitin ligase c-Caritas B cell lymphoma (c-cbl) is associated with negative regulation of receptor tyrosine kinases, signal transduction of antigens and cytokine receptors, and immune response. However, the expression and function of c-cbl in the regulation of neuropathic pain after chronic constriction injury (CCI) are unknown. In rat CCI model, c-cbl inhibited the activation of spinal cord microglia and the release of pro-inflammatory factors including tumor necrosis factor alpha (TNF-α), interleukin 1 beta (IL-1ß) and interleukin 6 (IL-6), which alleviated mechanical and heat pain through down-regulating extracellular signal-regulated kinase (ERK) pathway. Additionally, exogenous TNF-α inhibited c-cbl protein level vice versa. In the primary microglia transfected with c-cbl siRNA, when treated with TNF-α or TNF-α inhibitor, the corresponding secretion of IL-1ß and IL-6 did not change. In summary, CCI down-regulated c-cbl expression and induced the activation of microglia, then activated microglia released inflammatory factors via ERK signaling to cause pain. Our data might supply a novel molecular target for the therapy of CCI-induced neuropathic pain.

Bioassemblies Fabricated by Coassembly of Protein Molecules and Monotethered Single-Chain Polymeric Nanoparticles.

Molecular nanoparticles have been used as building blocks in the synthesis of functional materials. The grand challenges in the synthesis of the functional materials are precise control of the structures and functionalities of the materials by using nanoparticles with different architectures and properties. Monotethered single-chain polymeric nanoparticles (SCPN) are a type of nanosized asymmetric particles formed by intramolecular cross-linking of linear diblock copolymer chains. Monotethered SCPNs can be used as elemental building blocks for the fabrication of well-defined advanced structures. In this research, synthesis of biohybrid materials based on coassembly of bovine serum albumin (BSA) molecules and monotethered SCPNs is investigated. Due to the asymmetric structure of the SCPNs, positively charged SCPNs and negatively charged protein molecules coassemble into biohybrid vesicles with SCPNs on the layers and protein molecules in the walls. The self-assembled structures were analyzed by using dynamic light scattering, transmission electron microscopy, cryo-transmission electron microscopy, and atomic force microscopy. The average size of the biohybrid vesicles can be controlled by the molar ratio of SCPNs to BSA. The protein molecules in the biohybrid vesicles maintain most of the activities. This research paves a new way for the synthesis of functional biohybrid structures, and the materials can be used as protein carriers.

Fabrication of Polymer-Protein Hybrids.

Rapid developments in organic chemistry and polymer chemistry promote the synthesis of polymer-protein hybrids with different structures and biofunctionalities. In this feature article, recent progress achieved in the synthesis of polymer-protein conjugates, protein-nanoparticle core-shell structures, and polymer-protein nanogels/hydrogels is briefly reviewed. The polymer-protein conjugates can be synthesized by the "grafting-to" or the "grafting-from" approach. In this article, different coupling reactions and polymerization methods used in the synthesis of bioconjugates are reviewed. Protein molecules can be immobilized on the surfaces of nanoparticles by covalent or noncovalent linkages. The specific interactions and chemical reactions employed in the synthesis of core-shell structures are discussed. Finally, a general introduction to the synthesis of environmentally responsive polymer-protein nanogels/hydrogels by chemical cross-linking reactions or molecular recognition is provided.

Covalently Connected Polymer-Protein Nanostructures Fabricated by a Reactive Self-Assembly Approach.

The synthesis of polymer-protein nanostructures opens up a new avenue for the development of new biomaterials. In this research, covalently connected polymer-protein nanostructures were fabricated through a reactive self-assembly approach. Poly(tert-butyl methacrylate-co-pyridyl disulfide methacrylamide) (PtBMA-co-PPDSMA) was synthesized by reversible addition fragmentation chain transfer (RAFT) polymerization. Covalently connected nanostructures (CCNs) with hydrophobic polymer cores and hydrophilic protein coronae were prepared by adding solutions of PtBMA-co-PPDSMA/DMF to aqueous solutions of bovine serum albumin (BSA). The thiol-disulfide exchange reaction between pyridyl disulfide groups on the polymer chains and thiol groups on the protein molecules plays a key role in the fabrication of CCNs. The self-assembly process was investigated by dynamic light scattering (DLS) and stopped-flow techniques. DLS results indicated that the sizes of the CCNs were determined by the initial polymer concentration, the BSA concentration, and the average number of thiol groups on BSA molecules. TEM and sodium dodecyl sulfate polyacrylamide gel electrophoresis were used to analyze the nanostructures. Far-UV circular dichroism results demonstrated that the original folded conformations of BSA molecules were basically maintained in the reactive self-assembly process. Compared with native BSA, the secondary structure and conformation change of coronal BSA induced by urea or thermal treatment were remarkably suppressed. The cytotoxicity assays demonstrated that the CCNs were essentially nontoxic to Hela and COS-7 cells.

Antiviral Activity of Ailanthone from Ailanthus altissima on the Rice Stripe Virus.

Rice stripe disease caused by the rice stripe virus (RSV), which infects many Poaceae species in nature, is one of the most devastating plant viruses in rice that causes enormous losses in production. Ailanthone is one of the typical C20 quassinoids synthesized by the secondary metabolism of Ailanthus altissima, which has been proven to be a biologically active natural product with promising prospects and great potential for use as a lead structure for pesticide development. Based on the achievement of the systemic infection and replication of RSV in Nicotiana benthamiana plants and rice protoplasts, the antiviral properties of Ailanthone were investigated by determining its effects on viral-coding RNA gene expression using reverse transcription polymerase chain reaction, and Western blot analysis. Ailanthone exhibited a dose-dependent inhibitory effect on RSV NSvc3 expression in the assay in both virus-infected tobacco plants and rice protoplasts. Further efforts revealed a potent inhibitory effect of Ailanthone on the expression of seven RSV protein-encoding genes, among which NS3, NSvc3, NS4, and NSvc4 are the most affected genes. These facts promoted an extended and greater depth of understanding of the antiviral nature of Ailanthone against plant viruses, in addition to the limited knowledge of its anti-tobacco mosaic virus properties. Moreover, the leaf disc method introduced and developed in the study for the detection of the antiviral activity of Ailanthone facilitates an available and convenient screening method for anti-RSV natural products or synthetic chemicals.

Colonic expression of glutathione S-transferase alpha 4 and 4-hydroxynonenal adducts is correlated with the pathology of murine colitis-associated cancer.

Chronic inflammation-induced oxidative stress is an important driving force for developing colitis-associated cancer (CAC). 4-hydroxynonenal (4-HNE) is a highly reactive aldehyde derived from lipid peroxidation of ω-6 polyunsaturated fatty acids that contributes to colorectal carcinogenesis. Glutathione S-transferase alpha 4 (Gsta4) specifically conjugates glutathione to 4-HNE and thereby detoxifies 4-HNE. The correlation of these oxidative biomarkers with the pathological changes in CAC is, however, unclear. In this study, we investigated the expression of Gsta4 and 4-HNE adducts in azoxymethane/dextran sulfate sodium (AOM/DSS)-induced murine CAC, and analyzed the correlations of 4-HNE and Gsta4 with inflammatory cytokines and the pathological scores in the colon biopsies. Real-time quantitative PCR showed that expression of IL6, TNFα, and Gsta4 sequentially increased in colon tissues for mice treated with DSS for 1, 2, and 3 cycles, respectively. Moreover, immunohistochemical staining showed remarkably increased expression of 4-HNE adducts, Gsta4, TNFα, and IL6 in the colon biopsies after 3 cycles of DSS treatment. Correlation analysis demonstrated that 4-HNE adducts in the colon biopsies were positively correlated with Gsta4 expression. Additionally, the expression of Gsta4 and 4-HNE adducts were strongly correlated with the pathological changes of colon, as well as the expression of TNFα and IL6 in colon tissues. These results provide evidence for the association of oxidative biomarkers Gsta4 and 4-HNE with the pathological changes of CAC and may help developing novel histopathological biomarkers and prevention targets for CAC.

Fabrication of PεCL-AuNP-BSA core-shell-corona nanoparticles for flexible spatiotemporal drug delivery and SERS detection.

Nanoparticles with protein coronae can be used as promising multifunctional platforms for nanomedicine due to the possibility of performing surface functionalization on protein molecules and the achievement of biomedical properties. In this research, nanoparticles (NPs) with poly(ε-caprolactone) (PεCL) cores, gold NP (AuNP) shells and BSA coronae were fabricated by a self-assembly approach. The hydrophobic PεCL cores were used to encapsulate curcumin (CUR), the AuNP shells were decorated with a Raman probe, and the protein molecules in the coronae were functionalized with folic acid (FA). The self-assembly behaviors, drug delivery and the surface-enhanced Raman scattering (SERS) effect of the hybrid NPs were investigated in this research. The sizes of the core-shell-corona NPs (CSCNPs) are dependent on the initial concentrations of PεCL and AuNPs. The CUR in CSCNPs show enzyme-triggered release properties. The added lipase or trypsin can facilitate the CUR release from the hybrid NPs. The functionalization of CSCNPs with FA can significantly improve the internalization of NPs into 4T1 tumor cells due to the overexpressed folate receptors on the cells. In addition, the SERS effect of CSCNPs can be achieved when the AuNPs are decorated with 2-naphthalenethiol. The hybrid CSCNPs can be used as a promising platform for spatiotemporal drug delivery, cell imaging, and theranostics. Based on the same CSCNP platform, flexible functions can be adjusted according to the application needs.

CXCL10 and CXCR3 in the Trigeminal Ganglion Contribute to Trigeminal Neuropathic Pain in Mice.

PURPOSE: Trigeminal neuropathic pain is very common clinically, but effective treatments are lacking. Chemokines and their receptors have been implicated in the pathogenesis of chronic pain. This study explored the role of the chemokine CXCL10 and its receptor, CXCR3, in trigeminal neuropathic pain in mice. MATERIALS AND METHODS: Trigeminal neuropathic pain was established by partial infraorbital nerve ligation (pIONL) in wild-type and Cxcr3 -/- mice. Facial mechanical allodynia was evaluated by behavioral testing. A lentivirus containing Cxcr3 shRNA (LV-Cxcr3 shRNA) was microinjected into the trigeminal ganglion (TG) to knock down Cxcr3 expression. Quantitative polymerase chain reaction assays and immunofluorescence staining were used to examine Cxcl10/Cxcr3 mRNA expression and protein distribution. Western blotting was performed to examine activation of extracellular signal-regulated kinase (ERK) and AKT in the TG. Intra-TG injection of an AKT inhibitor was performed to examine the role of AKT in trigeminal neuropathic pain. RESULTS: pIONL induced persistent trigeminal neuropathic pain, which was alleviated in Cxcr3 -/- mice. Intra-TG injection of LV-Cxcr3 shRNA attenuated pIONL-induced mechanical allodynia. Furthermore, pIONL increased the expression of CXCR3 and its major ligand, CXCL10, in TG neurons. Intra-TG injection of CXCL10 induced pain hypersensitivity in wild-type mice but not in Cxcr3 -/- mice. CXCL10 also induced activation of ERK and AKT in the TG of wild-type mice. Finally, pIONL-induced activation of ERK and AKT was reduced in Cxcr3 -/- mice. Intra-TG injection of the AKT inhibitor alleviated pIONL-induced mechanical allodynia in WT mice but not in Cxcr3 -/- mice. CONCLUSION: CXCL10 acts on CXCR3 to induce ERK and AKT activation in TG neurons and contributes to the maintenance of trigeminal neuropathic pain.

Fruquintinib inhibits VEGF/VEGFR2 axis of choroidal endothelial cells and M1-type macrophages to protect against mouse laser-induced choroidal neovascularization.

Wet age-related macular degeneration, which is characterized by choroidal neovascularization (CNV) and induces obvious vision loss. Vascular endothelial growth factor (VEGF) family member VEGF-A (also named as VEGF) and its receptor VEGFR2 contribute to the pathogenesis of CNV. Choroidal endothelial cells (CECs) secret C-C motif chemokine ligand 2 (CCL2), which attracts macrophages to CNV lesion and promotes macrophage M1 polarization. Accordingly, infiltrating macrophages secret inflammatory cytokines to promote CNV. In vivo, intravitreal injection of fruquintinib (HMPL-013), an antitumor neovascularization drug, alleviated mouse CNV formation without obvious ocular toxicity. Meanwhile, HMPL-013 inhibited VEGF/VEGFR2 binding in CECs and macrophages, as well as macrophage M1 polarization. In vitro, noncontact coculture of human choroidal vascular endothelial cells (HCVECs) and macrophages under hypoxia conditions was established. HMPL-013 downregulated VEGF/VEGFR2/phosphoinositide-3-kinase/protein kinase B (AKT)/nuclear factor kappa B pathway and CCL2 secretion in HCVECs, as well as VEGF/VEGFR2-induced macrophage M1 polarization under hypoxia condition. In addition, HMPL-013 inhibited HCEVC derived CCL2-induced macrophage migration and M1 polarization, along with macrophage M1 polarization-induced HCVECs proliferation, migration, and tube formation. Altogether, HMPL-013 alleviated CNV formation might via breaking detrimental cross talk between CECs and macrophages.

Autophagy dysfunction in neuropathic pain.

Autophagy is a lysosomal degradation pathway that maintains tissue homeostasis by recycling damaged and aged cellular components, which plays important roles in development of the nervous system, as well as in neuronal function and survival. In addition, autophagy dysfunction underlies neuropathic pain. Thus, the modulation of autophagy can alleviate neuropathic pain. Here, we describe the definition, mechanisms of autophagy and neuropathic pain. On this basis, we further discuss the role of autophagy dysfunction in neuropathic pain. This review updates our knowledge on autophagy mechanisms which propose potential therapeutic targets for the treatment of neuropathic pain.

ß4GalT1 Mediates PPARγ N-Glycosylation to Attenuate Microglia Inflammatory Activation.

The inflammatory activation of microglia has double-edged effects in central nervous system (CNS) diseases. The ligand-activated transcriptional factor peroxisome proliferator-activated receptor γ (PPARγ) inhibits the inflammatory response. ß-1,4-Galactosyltransferase Ι (ß1, 4GalT1) mediates N-glycosylation. In this study, the N-glycosylation of PPARγ, as well as two N-linked glycosylation sites in its DNA binding domain (DBD), was identified. Disruption of both sites by site-directed mutagenesis completely abrogated the N-glycosylation of PPARγ. PPAR wild-type (WT) transfection inhibited the inflammatory activation of microglia, while the anti-inflammatory function of unglycosylated PPARγ was down-regulated. In addition, ß1, 4GalT1 was shown to interact with PPARγ and to mediate PPARγ glycosylation. ß1, 4GalT1 promoted PPARγ's anti-transcription and anti-inflammatory functions. Collectively, our findings define that ß-1, 4GalT1 mediated PPARγ glycosylation to attenuate the inflammatory activation of microglia, which has implications for potential therapies for CNS inflammatory diseases.

[Oxidation buffer capacity of sewage sludge barrier for immobilization of heavy metals].

Benefit from the microbial activities especially the anaerobic sulfate reduction processes, sewage sludge could be used as a barrier to immobilize the heavy metals leached from tailings. With respect to the redox reaction between sewage sludge and acid mine drainage (AMD), oxidation titration test was carried out to study the effect of oxidation buffer capacity (OBC) of sewage sludge on the immobilization of heavy metals. Test results showed that OBC of sludge suspensions was decreased slightly with the solid-liquid ratio of the suspensions, but increased with the anaerobic incubation time, and that more than 50% of OBC was contributed by the sludge existed in strongly-reduction conditions (Eh < or = - 150 mV). During oxidation titration test, Zn was released obviously when Eh > or = - 150 mV, while Cu and Pb released obviously when Eh > or = 150 mV. According to the test results, a mathematical model was established to predict the OBC consumption of the sludge barrier under AMD penetrating conditions. The simulation results showed that a sludge barrier with 2m thickness, even undergone 38 787-years oxidation by AMD under 10m water head, keep in a strongly-reduced condition and, therefore, promote an immobilization of heavy metals from AMD in the barrier.

[Acid buffer capacity of sewage sludge barrier for immobilization of heavy metals].

Employing the anaerobic activities of microorganisms, sewage sludge can be used as a barrier to immobilize the heavy metals leached from tailings. Due to the interactions between sewage sludge barrier and acid mine drainage (AMD), it is possible that the heavy metals that have been immobilized previously might be released out. The acid buffering capacity (ABC) of sewage sludge suspensions with various anaerobic incubation time and the effect of ABC on the mobility of heavy metals were investigated by acid titration tests. Test results showed that ABC of sewage sludge suspensions was increased with the solid-liquid ratio of the suspensions and the anaerobic incubation time, and that carbonate and organics play an important role in acid buffer of sewage sludge suspensions. During the acid titration test, Zn, Pb and Cu were released out obviously following the order of Zn > Cu > Pb as pH was decreased less than 6.2. A mathematical model was established to predict the ABC consumption of the sewage sludge barrier under AMD penetration condition. The simulation results showed that a sewage sludge barrier with 2.0 m thickness, even undergoing 666-years acidification by AMD under 10.0 m water head, can maintain a condition of pH > or = 6.2 and, therefore, keep immobilize the heavy metals of AMD in the barrier.

[Feasibility of sewage sludge used as filling material in permeable reactive barrier].

Permeable reactive barriers (PRB) have been used widely as an alternative technique to treatment of acid mine drainage (AMD). Selection of the appropriate filling materials is the most important procedure to application of this treatment. Batch adsorption tests and bacteria culture batch tests were conducted to assess the possibility of sewage sludge served as filling material for PRB. Results from batch adsorption tests showed that the maximum adsorption capacities of the sewage sludge were 13.62 mg x g(-1) (Zn2+) and 15.60 mg x g(-1) (Cd2+). Bacteria culture batch tests indicated that SO4(2-) concentrations in reactors decreased from initial concentrations of 700 mg x L(-1) to below 300 mg x L(-1). Sulfate removal efficiency ranged from 60% to 70%. Fe and heavy metals, including Zn and Cd, were removed completely in two reactors. This study suggested that sewage sludge is a suitable filling material for PRB.