A Facile Strategy for Catalyst Separation and Recycling Suitable for ATRP of Hydrophilic Monomers Using a Macroligand.

How to simply and efficiently separate and recycle catalyst has still been a constraint for the wide application of atom transfer radical polymerization (ATRP), especially for the polymerization systems with hydrophilic monomers because the polar functional groups may coordinate with transition metal salts, resulting in abundant catalyst residual in the resultant water-soluble polymers. In order to overcome this problem, a latent-biphasic system is developed, which can be successfully used for ATRP catalyst separation and recycling in situ for various kinds of hydrophilic monomers for the first time, such as poly(ethylene glycol) monomethyl ether methacrylate (PEGMA), 2-hydroxyethyl methacrylate (HEMA), 2-(dimethylamino)ethyl methacrylate (DMAEMA), N,N-dimethyl acrylamide (DMA), and N-isopropylacrylamide (NIPAM). Herein, random copolymer of octadecyl acrylate (OA), MA-Ln (2-(bis(pyridin-2-ylmethyl)amino)ethyl acrylate), and POA-ran-P(MA-Ln) is designed as the macroligand, and heptane/ethanol is selected as the biphasic solvent. Copper(II) bromide (CuBr2 ) is employed as the catalyst, PEG-bound 2-bromo-2-methylpropanoate (PEG350 -Br) as the water-soluble ATRP initiator and 2,2'-azobis(isobutyronitrile) (AIBN) as the azo-initiator to establish an ICAR (initiators for continuous activator regeneration) ATRP system. Importantly, well-defined water-soluble polymers are obtained even though the recyclable catalyst is used for sixth times.

Highly Efficient and Facile Photocatalytic Recycling System Suitable for ICAR ATRP of Hydrophilic Monomers.

Photoinduced initiators for continuous activator regeneration atom transfer radical polymerization (ATRP) of hydrophilic monomers in heptane/ethanol latent-biphasic system for copper catalyst separation and recycling have been realized for the first time at room temperature with different wavelengths of visible light LED (green, blue, purple, and white LED) as external stimulus, using 2-bromophenylacetate as the ATRP initiator and camphorquinone/triethylamine as the photoinitiator. In this system, hybrid catalyst complex (HCc) is synthesized as a novel nonpolar catalyst, which is preferentially dissolved in heptane. The hydrophilic polymers obtained catalyzed by HCc in heptane/ethanol mixture solvent show typical "living" features, for example, the values of Mn,GPC increase linearly with monomer conversion up to quantitative level (>96%) and the molecular weight distributions were kept narrow (Mw /Mn < 1.20) throughout the polymerization process. It should be noted that the excellent controllability of this novel polymerization system can be achieved even after 5 catalyst recycling experiments under LED irradiation.

Recent Progress on Transition Metal Catalyst Separation and Recycling in ATRP.

Atom transfer radical polymerization (ATRP) is a versatile and robust tool to synthesize a wide spectrum of monomers with various designable structures. However, it usually needs large amounts of transition metal as the catalyst to mediate the equilibrium between the dormant and propagating species. Unfortunately, the catalyst residue may contaminate or color the resultant polymers, which limits its application, especially in biomedical and electronic materials. How to efficiently and economically remove or reduce the catalyst residue from its products is a challenging and encouraging task. Herein, recent advances in catalyst separation and recycling are highlighted with a focus on (1) highly active ppm level transition metal or metal free catalyzed ATRP; (2) post-purification method; (3) various soluble, insoluble, immobilized/soluble, and reversible supported catalyst systems; and (4) liquid-liquid biphasic catalyzed systems, especially thermo-regulated catalysis systems.

Diffusion-regulated phase-transfer catalysis for atom transfer radical polymerization of methyl methacrylate in an aqueous/organic biphasic system.

A concept based on diffusion-regulated phase-transfer catalysis (DRPTC) in an aqueous-organic biphasic system with copper-mediated initiators for continuous activator regeneration is successfully developed for atom transfer radical polymerization (ICAR ATRP) (termed DRPTC-based ICAR ATRP here), using methyl methacrylate (MMA) as a model monomer, ethyl α-bromophenylacetate (EBrPA) as an initiator, and tris(2-pyridylmethyl)amine (TPMA) as a ligand. In this system, the monomer and initiating species in toluene (organic phase) and the catalyst complexes in water (aqueous phase) are simply mixed under stirring at room temperature. The trace catalyst complexes transfer into the organic phase via diffusion to trigger ICAR ATRP of MMA with ppm level catalyst content once the system is heated to the polymerization temperature (75 °C). It is found that well-defined PMMA with controlled molecular weights and narrow molecular weight distributions can be obtained easily. Furthermore, the polymerization can be conducted in the presence of limited amounts of air without using tedious degassed procedures. After cooling to room temperature, the upper organic phase is decanted and the lower aqueous phase is reused for another 10 recycling turnovers with ultra low loss of catalyst and ligand loading. At the same time, all the recycled catalyst complexes retain nearly perfect catalytic activity and controllability, indicating a facile and economical strategy for catalyst removal and recycling.

Bifunctional nanoparticles with magnetism and NIR fluorescence: controlled synthesis from combination of AGET ATRP and 'click' reaction.

In this work, bifunctional nanoparticles (NPs) capable of emitting near infrared (NIR) fluorescence and generating superparamagnetism under an external magnetic field were prepared by combination of 'click' reaction and surface-initiated activators generated by electron transfer for atom transfer radical polymerization (AGET ATRP) of water-soluble poly(ethylene glycol) monomethyl ether methacrylate (PEGMA) and glycidyl methacrylate (GMA) using biocompatible iron as the catalyst on the surface of silica-coated iron oxide (Fe3O4@SiO2) NPs. The nanosized Fe3O4@SiO2@PPEGMA-co-PGMA@N3 was prepared through AGET ATRP and alkynyl bearing NIR dye was also prepared; afterwards they were integrated together by 'click' reaction. The different stages of surface modification were approved by employing different characterization techniques such as TEM, XRD, XPS, VSM and FT-IR, and the properties of the final NPs were thoroughly studied. Their suitability as dual model imaging agents for magnetic resonance (MR) and fluorescence imaging was investigated, indicating them to be a competitive candidate for imaging contrast agents.

Highly active ppm level organic copper catalyzed photo-induced ICAR ATRP of methyl methacrylate.

A novel photo-induced homogeneous atom transfer radical polymerization (ATRP) system is constructed using an organic copper salt (Cu(SC(S)N(C2 H5 )2 )2 ) as a photo-induced catalyst at 30 °C. Herein, N,N,N',N'',N''-pentamethyldiethylenetriamine (PMDETA) is used as a ligand, ethyl 2-bromophenylacetate (EBPA) as an ATRP initiator, and (2,4,6-trimethylbenzoyl) diphenylphosphine oxide (TPO) as a photo-induced radical initiator to establish an ICAR (initiators for continuous activator regeneration) ATRP using methyl methacrylate (MMA) as a modal monomer. The effect of the concentration of the organic copper on the polymerization is investigated in detail. It is found that well-controlled polymerization can be obtained even with the amount of (Cu(SC(S)N(C2 H5 )2 )2 decreasing to a 1.56 ppm level, with the molecular weight of the resultant polymers increasing linearly with monomer conversion while maintaining a narrow molecular weight distribution (M¯w/M¯n < 1.3).

Facile "living" radical polymerization of methyl methacrylate in the presence of iniferter agents: homogeneous and highly efficient catalysis from copper(II) acetate.

A facile homogeneous polymerization system involving the iniferter agent 1-cyano-1-methylethyl diethyldithiocarbamate (MANDC) and copper(II) acetate (Cu(OAc)2 ) is successfully developed in bulk using methyl methacylate (MMA) as a model monomer. The detailed polymerization kinetics with different molar ratios (e.g., [MMA]0 /[MANDC]0 /[Cu(OAc)2 ]0 = 500/1/x (x = 0.1, 0.2, 0.5, 1.0)) demonstrate that this system has the typical "living"/controlled features of "living" radical polymerization, even with ppm level catalyst Cu(OAc)2 , first order polymerization kinetics, a linear increase in molecular weight with monomer conversion and narrow molecular weight distributions for the resultant PMMA. (1) H NMR spectra and chain-extension experiments further confirm the "living" characteristics of this process. A plausible mechanism is discussed.

Cu(II)-mediated atom transfer radical polymerization of methyl methacrylate via a strategy of thermo-regulated phase-separable catalysis in a liquid/liquid biphasic system: homogeneous catalysis, facile heterogeneous separation, and recycling.

A strategy of thermo-regulated phase-separable catalysis (TPSC) is applied to the Cu(II)-mediated atom transfer radical polymerization (ATRP) of methyl methacrylate (MMA) in a p-xylene/PEG-200 biphasic system. Initiators for continuous activator regeneration ATRP (ICAR ATRP) are used to establish the TPSC-based ICAR ATRP system using water-soluble TPMA as a ligand, EBPA as an initiator, CuBr2 as a catalyst, and AIBN as a reducing agent. By heating to 70 °C, unlimited miscibility of both solvents is achieved and the polymerization can be carried out under homogeneous conditions; then on cooling to 25 °C, the mixture separates into two phases again. As a result, the catalyst complex remains in the PEG-200 phase while the obtained polymers stay in the p-xylene phase. The catalyst can therefore be removed from the resultant polymers by easily separating the two different layers and can be reused again. It is important that well-defined PMMA with a controlled molecular weight and narrow molecular weight distribution could be obtained using this TPSC-based ICAR ATRP system.

Facile iron-mediated dispersant-free suspension polymerization of methyl methacrylate via reverse ATRP in water.

An iron-mediated reverse ATRP of methyl methacrylate (MMA) is successfully carried out in water in the absence of any dispersants, using a water-soluble 2,2'-azobis(2-methylpropionamidine) dihydrochloride (V-50) as the initiator and the stabilizer, and using an oil-soluble N,N-butyldithiocarbamate ferrum (Fe(S2 CN(C4 H9 )2 )3 ) as the catalyst without adding any additional ligands. Micron-sized PMMA particles with UV light-sensitive -S2 CN(C4 H9 )2 end group are obtained, and monomer droplet nucleation and suspension polymerization mechanism are proposed. Polymerization results demonstrated typical "living"/controlled characteristics of ATRP: first-order polymerization kinetics, linear increase of molecular weights with monomer conversion and narrow molecular weight distributions for the resultant PMMA particles. NMR spectroscopy and chain-extension experiments under UV light irradiation confirm the attachment and livingness of UV light-sensitive -S2 CN(C4 H9 )2 group in the chain end.

Flanking N- and C-terminal domains of PrsA in Streptococcus suis type 2 are crucial for inducing cell death independent of TLR2 recognition.

Streptococcus suis type 2 (SS2), a major emerging/re-emerging zoonotic pathogen found in humans and pigs, can cause severe clinical infections, and pose public health issues. Our previous studies recognized peptidyl-prolyl isomerase (PrsA) as a critical virulence factor promoting SS2 pathogenicity. PrsA contributed to cell death and operated as a pro-inflammatory effector. However, the molecular pathways through which PrsA contributes to cell death are poorly understood. Here in this study, we prepared the recombinant PrsA protein and found that pyroptosis and necroptosis were involved in cell death stimulated by PrsA. Specific pyroptosis and necroptosis signalling inhibitors could significantly alleviate the fatal effect. Cleaved caspase-1 and IL-1ß in pyroptosis with phosphorylated MLKL proteins in necroptosis pathways, respectively, were activated after PrsA stimulation. Truncated protein fragments of enzymatic PPIase domain (PPI), N-terminal (NP), and C-terminal (PC) domains fused with PPIase, were expressed and purified. PrsA flanking N- or C-terminal but not enzymatic PPIase domain was found to be critical for PrsA function in inducing cell death and inflammation. Additionally, PrsA protein could be anchored on the cell surface to interact with host cells. However, Toll-like receptor 2 (TLR2) was not implicated in cell death and recognition of PrsA. PAMPs of PrsA could not promote TLR2 activation, and no rescued phenotypes of death were shown in cells blocking of TLR2 receptor or signal-transducing adaptor of MyD88. Overall, these data, for the first time, advanced our perspective on PrsA function and elucidated that PrsA-induced cell death requires its flanking N- or C-terminal domain but is dispensable for recognizing TLR2. Further efforts are still needed to explore the precise molecular mechanisms of PrsA-inducing cell death and, therefore, contribution to SS2 pathogenicity.

Deletion of lacD gene affected stress tolerance and virulence of Streptococcus suis serotype 2.

Streptococcus suis type 2 (S. suis type 2, SS2), an infectious pathogen which is zoonotic and can induce severely public health concern. Our previous research identified a newly differential secreted effector of tagatose-bisphosphate aldolase (LacD) mediated by VirD4 factor within the putative type IV secretion system of SS2, whereas the functional basis and roles in virulence of LacD remain elusive. Here in this study, the LacD was found enzymatic and can be activated to express under oxidative stress. Gene mutant and its complemental strain (ΔlacD and cΔlacD) were constructed to analyze the phenotypes, virulence and transcriptomic profiles as compared with the parental strain. The lacD gene deletion showed no effect on growth capability and cells morphology of SS2. However, reduced tolerance to oxidative and heat stress conditions, increased antimicrobial susceptibility to ciprofloxacin and kanamycin were found in ΔlacD strain. Further, the LacD deficiency led to weakened invasion and attenuated virulence since an easier phagocytosed and more prone to be cleared of SS2 in macrophages were shown in ΔlacD mutant. Distinctive transcriptional profiling in ΔlacD strain and typical down-regulated genes with significant mRNA changes including alcohol dehydrogenase, GTPase, integrative and conjugative elements, and iron ABC transporters which were mainly involved in cell division, stress response, antimicrobial susceptibility and virulence regulation, were examined and confirmed by RNA sequencing and real time qPCR. In summary, the results demonstrated for the first time that LacD was a pluripotent protein mediated the metabolic, stress and virulent effect of SS2.

Development of a recombinase polymerase amplification assay for rapid detection of Streptococcus suis type 2 in nasopharyngeal swab samples.

Streptococcus suis serotype 2 (SS2), an emerging zoonotic pathogen, may induce severe infections and symptoms manifested as septicemia, meningitis and even death both in human and pigs. The aim of this article was to develop a new methodology as real-time recombinase polymerase amplification (RT-RPA) assay targeting cps2J gene for the detection of SS2 (or SS1/2). The sensitivity and reproducibility of RT-RPA results were evaluated and compared with a real-time quantitative PCR (RT-qPCR). The established RT-RPA reaction could be completed in 20 minutes with distinguishable specificity against the predominant S. suis infection serotypes of 3, 4, 5, 7, 9, 14, and 31. Lower detection limit for RT-RPA was 102 genomic DNA copies per reaction. The specimen performance of RT-RPA was tested in nasopharyngeal swab samples with the sensitivity and specificity as 97.5% and 100%, respectively. Thus, this RT-RPA method is a rapid and potential molecular diagnostic tool for SS2 detection.

Generated SecPen_NY-ESO-1_ubiquitin-pulsed dendritic cell cancer vaccine elicits stronger and specific T cell immune responses.

Dendritic cell-based cancer vaccines (DC vaccines) have been proved efficient and safe in immunotherapy of various cancers, including melanoma, ovarian and prostate cancer. However, the clinical responses were not always satisfied. Here we proposed a novel strategy to prepare DC vaccines. In the present study, a fusion protein SNU containing a secretin-penetratin (SecPen) peptide, NY-ESO-1 and ubiquitin was designed and expressed. To establish the DC vaccine (DC-SNU), the mouse bone marrow-derived DCs (BMDCs) were isolated, pulsed with SNU and maturated with cytokine cocktail. Then peripheral blood mononuclear cells (PBMCs) from C57BL/6 mice inoculated intraperitoneally with DC-SNU were separated and cocultured with MC38/MC38 NY-ESO-1 tumor cells or DC vaccines. The results show that SNU was successfully expressed. This strategy made NY-ESO-1 entering cytoplasm of BMDCs more efficiently and degraded mainly by proteasome. As we expected, mature BMDCs expressed higher CD40, CD80 and CD86 than immature BMDCs. Thus, the PBMCs released more IFN-γ and TNF-α when stimulated with DC-SNU in vitro again. What's more, the PBMCs induced stronger and specific cytotoxicity towards MC38 NY-ESO-1 tumor cells. Given the above, it demonstrated that DC-SNU loaded with SecPen and ubiquitin-fused NY-ESO-1 could elicit stronger and specific T cell immune responses. This strategy can be used as a platform for DC vaccine preparation and applied to various cancers treatment.

5-(pyridinon-1-yl)indazoles and 5-(furopyridinon-5-yl)indazoles as MCH-1 antagonists.

A new series of 5-(pyridinon-1-yl)indazoles with MCH-1 antagonist activity were synthesized. Potential cardiovascular risk for these compounds was assessed based upon their interaction with the hERG potassium channel in a mini-patch clamp assay. Selected compounds were studied in a 5-day diet-induced obese mouse model to evaluate their potential use as weight loss agents. Structural modification of the 5-(pyridinon-1-yl)indazoles to give 5-(furopyridinon-5-yl)indazoles provided compounds with enhanced pharmacokinetic properties and improved efficacy.

Tetrahydrocarboline analogs as MCH-1 antagonists.

A new series of tetrahydrocarbolines with potent MCH-1 antagonist activity were synthesized, using a conformationally constrained design approach towards optimizing pharmacokinetic properties. Two compounds from this series were progressed to a 5-day diet-induced obesity mouse screening model to evaluate their potential as weight loss agents. Both compounds produced a highly significant reduction in weight, which was attributed to their improved pharmacokinetic profile.

PrsA contributes to Streptococcus suis serotype 2 pathogenicity by modulating secretion of selected virulence factors.

Streptococcus suis serotype 2 (S. suis 2) is a major zoonotic pathogen. Parvulin-type peptidyl-prolyl isomerase (PrsA) in S. suis 2 is found surface-associated, pro-inflammatory and cytotoxic. To further explore the roles of PrsA in S. suis 2 infection, we constructed a prsA deletion mutant (ΔprsA) and a complemented strain (CΔprsA). The ΔprsA mutant showed increased length of bacterial chains and decreased growth. Deletion of prsA increased bacterial adhesion to host epithelial cells but with weakened invasion. The ΔprsA mutant had reduced survival in RAW264.7 macrophages and pig whole blood, and significantly attenuated in virulence to mice. All these phenotypes of the mutant could be reversed largely to the levels of its parental strain by gene complementation. Western blotting revealed that suilysin was markedly reduced both in surface-associated (SAP) and secreted fractions (SecP) of ΔprsA, which might be responsible for reduced hemolytic activity. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and enolase were significantly increased in both SAP and SecP fractions as a result of prsA deletion. Increased adhesion of the ΔprsA mutant to bEND.3 cells was prevented using polyclonal antibodies against GAPDH and enolase. Overall, we propose that S. suis 2 deploys PrsA to control translocation of important virulence factors, thereby favoring its survival in the host with enhanced pathogenicity by compromising its interactions with the host cells. Further investigation is required to find out how PrsA modulates protein translocation to benefit S. suis infection and if there are other S. suis 2 substrates of potential virulence regulated by PrsA.

Peptidyl isomerase PrsA is surface-associated on Streptococcus suis and offers cross-protection against serotype 9 strain.

PrsA, a peptidyl isomerase encoded by the prsA gene, plays pleiotropic roles in bacterial physiology and pathogenicity. This study was attempted to characterize the distribution of prsA in different serotypes of Streptococcus suis isolates and on the bacterial cells and to evaluate its immunogenicity in a murine model. PrsA is present in different S. suis types and surface-associated as tested in a S. suis type 2 strain. The prsA gene from the serotype 2 strain was cloned for its expression in Escherichia coli. Recombinant PrsA (SsPrsA) showed good reactivity with anti-sera to S. suis serotype 2 and 9 strains. Immunization of mice with SsPrsA elicited a significant antibody response and conferred partial protection against lethal challenge with a S. suis serotype 2 strain (50% protection) or a serotype 9 strain (66% protection). The anti-SsPrsA sera showed good reactivity to the surface-associated proteins of both serotype 2 and serotype 9 strains. Higher abundance of surface-associated PrsA in the serotype 9 strain (than the serotype 2 strain) might account, in part, for higher protection against its challenge infection. These results suggest that SsPrsA may serve as a novel subunit vaccine candidate with cross-protective potential.

A Facial Strategy for Catalyst and Reducing Agent Synchronous Separation for AGET ATRP Using Thiol-Grafted Cellulose Paper as Reducing Agent.

Atom Transfer Radical Polymerization (ATRP) has been a powerful tool to synthesize well-defined functional polymers, which are widely used in biology, drug/gene delivery and antibacterial materials, etc. However, the potential toxic residues in polymer reduced its service life and limited its applications. In order to overcome the problem, in this work, a novel polymerization system of activators generated by electron transfer for atom transfer radical polymerization (AGET ATRP) for synchronous separation of the metal catalyst and byproduct of reducing agent was developed, using thiol-grafted cellulose paper (Cell-SH) as a solid reducing agent. The polymerization kinetics were investigated in detail, and the "living" features of the novel polymerization system were confirmed by chain-end analysis and chain extension experiment for the resultant polymethyl methacrylate (PMMA). It is noted that the copper residual in obtained PMMA was less than 20 ppm, just by filtering the sheet-like byproduct of the reducing agent.

Iron-Mediated Homogeneous ICAR ATRP of Methyl Methacrylate under ppm Level Organometallic Catalyst Iron(III) Acetylacetonate.

Atom Transfer Radical Polymerization (ATRP) is an important polymerization process in polymer synthesis. However, a typical ATRP system has some drawbacks. For example, it needs a large amount of transition metal catalyst, and it is difficult or expensive to remove the metal catalyst residue in products. In order to reduce the amount of catalyst and considering good biocompatibility and low toxicity of the iron catalyst, in this work, we developed a homogeneous polymerization system of initiators for continuous activator regeneration ATRP (ICAR ATRP) with just a ppm level of iron catalyst. Herein, we used oil-soluble iron (III) acetylacetonate (Fe(acac)3) as the organometallic catalyst, 1,1'-azobis (cyclohexanecarbonitrile) (ACHN) with longer half-life period as the thermal initiator, ethyl 2-bromophenylacetate (EBPA) as the initiator, triphenylphosphine (PPh3) as the ligand, toluene as the solvent and methyl methacrylate (MMA) as the model monomer. The factors related with the polymerization system, such as concentration of Fe(acac)3 and ACHN and polymerization kinetics, were investigated in detail at 90 °C. It was found that a polymer with an acceptable molecular weight distribution (Mw/Mn = 1.43 at 45.9% of monomer conversion) could be obtained even with 1 ppm of Fe(acac)3, making it needless to remove the residual metal in the resultant polymers, which makes such an ICAR ATRP process much more industrially attractive. The "living" features of this polymerization system were further confirmed by chain-extension experiment.

Porcine Circovirus Type 2 Activates CaMMKß to Initiate Autophagy in PK-15 Cells by Increasing Cytosolic Calcium.

Porcine circovirus type 2 (PCV2) induces autophagy via the 5' adenosine monophosphate-activated protein kinase (AMPK)/extracellular signal-regulated kinase (ERK)/tuberous sclerosis complex 2 (TSC2)/mammalian target of rapamycin (mTOR) pathway in pig kidney PK-15 cells. However, the underlying mechanisms of AMPK activation in autophagy induction remain unknown. With specific inhibitors and RNA interference (RNAi), we show that PCV2 infection upregulated calcium/calmodulin-dependent protein kinase kinase-beta (CaMKKß) by increasing cytosolic Ca(2+) via inositol 1,4,5-trisphosphate receptor (IP3R). Elevation of cytosolic calcium ion (Ca(2+)) did not seem to involve inositol 1,4,5-trisphosphate (IP3) release from phosphatidylinositol 4,5-bisphosphate (PIP2) by phosphoinositide phospholipase C-gamma (PLC-γ). CaMKKß then activated both AMPK and calcium/calmodulin-dependent protein kinase I (CaMKI). PCV2 employed CaMKI and Trp-Asp (WD) repeat domain phosphoinositide-interacting protein 1 (WIPI1) as another pathway additional to AMPK signaling in autophagy initiation. Our findings could help better understanding of the signaling pathways of autophagy induction as part of PCV2 pathogenesis. Further research is warranted to study if PCV2 interacts directly with IP3R or indirectly with the molecules that antagonize IP3R activity responsible for increased cytosolic Ca(2+) both in PK-15 cells and PCV2-targeted primary cells from pigs.