Porcine deltacoronavirus nucleocapsid protein interacts with the Grb2 through its proline-rich motifs to induce activation of the Raf-MEK-ERK signal pathway and promote virus replication.

Porcine deltacoronavirus (PDCoV), an enteropathogenic coronavirus, causes severe watery diarrhoea, dehydration and high mortality in piglets, which has the potential for cross-species transmission in recent years. Growth factor receptor-bound protein 2 (Grb2) is a bridging protein that can couple cell surface receptors with intracellular signal transduction events. Here, we investigated the reciprocal regulation between Grb2 and PDCoV. It is found that Grb2 regulates PDCoV infection and promotes IFN-ß production through activating Raf/MEK/ERK/STAT3 pathway signalling in PDCoV-infected swine testis cells to suppress viral replication. PDCoV N is capable of interacting with Grb2. The proline-rich motifs in the N- or C-terminal region of PDCoV N were critical for the interaction between PDCoV-N and Grb2. Except for Deltacoronavirus PDCoV N, the Alphacoronavirus PEDV N protein could interact with Grb2 and affect the regulation of PEDV replication, while the N protein of Betacoronavirus PHEV and Gammacoronavirus AIBV could not interact with Grb2. PDCoV N promotes Grb2 degradation by K48- and K63-linked ubiquitin-proteasome pathways. Overexpression of PDCoV N impaired the Grb2-mediated activated effect on the Raf/MEK/ERK/STAT3 signal pathway. Thus, our study reveals a novel mechanism of how host protein Grb2 protein regulates viral replication and how PDCoV N escaped natural immunity by interacting with Grb2.

Cyclophilin A promotes porcine deltacoronavirus replication by regulating autophagy via the Ras/AKT/NF-κB pathway.

Porcine deltacoronavirus (PDCoV) is an important enteric coronavirus that has caused major worldwide economic losses in the pig industry. Previous studies have shown that cyclophilin A (CypA), a key player in aetiological agent infection, is involved in regulating viral infection. However, the role of CypA during PDCoV replication remains unknown. Therefore, in this study, the role of CypA in PDCoV replication was determined. The results demonstrated that PDCoV infection increased CypA expression in LLC-PK1 cells. CypA overexpression substantially promoted PDCoV replication. Proteomic analysis was subsequently used to assess changes in total protein expression levels after CypA overexpression. Gene Ontology (GO) functional analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were used to further determine the mechanisms by which CypA affects viral replication. Proteomic analysis revealed that CypA protein overexpression significantly upregulated 75 differentially expressed proteins and significantly downregulated 172 differentially expressed proteins. The differentially expressed proteins were involved mainly in autophagy and activation of the host innate immune pathway. Subsequent experimental results revealed that the CypA protein promoted viral replication by reducing the levels of natural immune cytokines and mitigated the inhibitory effect of chloroquine (CQ) on viral replication. Further investigation revealed that CypA could activate the Ras/AKT/NF-κB pathway, mediate autophagy signalling and promote PDCoV replication. In summary, the findings of this study may help elucidate the role of CypA in PDCoV replication.

Perfluoroalkyl compounds in groundwater alter the spatial pattern of health risk in an arsenic­cadmium contaminated region.

Integrated health risk assessment strategies for emerging organic pollutants and heavy metals that coexist in water/soil media are lacking. Contents of perfluoroalkyl compounds and potentially toxic elements in multiple media were determined by investigating a county where a landfill and a tungsten mine coexist. The spatial characteristics and sources of contaminants were predicted by Geostatistics-based and multivariate statistical analysis, and their comprehensive health risks were assessed. The average contents of perfluorooctane acid, perfluorooctanesulfonic acid, arsenic, and cadmium in groundwater were 3.21, 0.77, 1.69, and 0.14 µg L-1, respectively; the maximum content of cadmium in soils and rice highly reached 2.12 and 1.52 mg kg-1, respectively. In soils, the contribution of mine lag to cadmium was 99 %, and fertilizer and pesticide to arsenic was 59.4 %. While in groundwater, arsenic, cadmium and perfluoroalkyl compounds near the landfill mainly came from leachate leakage. Significant correlations were found between arsenic in groundwater and arsenic and cadmium in soils, as well as perfluoroalkyl compounds in groundwater and pH and sulfate. Based on these correlations, the geographically optimal similarity model predicted high-level arsenic in groundwater near the tungsten mine and cadmium/perfluoroalkyl compounds around the landfill. The combination of analytic network process, entropy weighting method and game theory-based trade-off method with risk assessment model can assess the comprehensive risks of multiple pollutants. Using this approach, a high health-risk zone located around the landfill, which was mainly attributed to the presence of arsenic, cadmium and perfluorooctanesulfonic acid, was found. Overall, perfluoroalkyl compounds in groundwater altered the spatial pattern of health risks in an arsenic­cadmium contaminated area.

Adenovirus-vectored PDCoV vaccines induce potent humoral and cellular immune responses in mice.

Porcine deltacoronavirus (PDCoV) is a novel swine enteropathogenic coronavirus that causes severe watery diarrhea, vomiting, dehydration and high mortality in piglets, resulting in significant economic losses by the global pig industry. Recently, PDCoV has also shown the potential for cross-species transmission. However, there are currently few vaccine studies and no commercially available vaccines for PDCoV. Hence, here, two novel human adenovirus 5 (Ad5)-vectored vaccines expressing codon-optimized forms of the PDCoV spike (S) glycoprotein (Ad-PD-tPA-Sopt) and S1 glycoprotein (Ad-PD-oriSIP-S1opt) were constructed, and their effects were evaluated via intramuscular (IM) injection in BALB/c mice with different doses and times. Both vaccines elicited robust humoral and cellular immune responses; moreover, Ad-PD-tPA-Sopt-vaccinated mice after two IM injections with 108 infectious units (IFU)/mouse had significantly higher anti-PDCoV-specific neutralizing antibody titers. In contrast, the mice immunized with Ad-PD-tPA-Sopt via oral gavage (OG) did not generate robust systemic and mucosal immunity. Thus, IM Ad-PD-tPA-Sopt administration is a promising strategy against PDCoV and provides useful information for future animal vaccine development.

Recombinant human adenovirus type 5 based vaccine candidates against GIIa- and GIIb-genotype porcine epidemic diarrhea virus induce robust humoral and cellular response in mice.

Porcine epidemic diarrhea virus (PEDV) is a porcine enteropathogenic coronavirus causing severe watery diarrhea, vomiting, dehydration, and death in piglets. However, most commercial vaccines are developed based on the GI genotype strains, and have poor immune protection against the currently dominant GII genotype strains. Therefore, four novel replication-deficient human adenovirus 5-vectored vaccines expressing codon-optimized forms of the GIIa and GIIb strain spike and S1 glycoproteins were constructed, and their immunogenicity was evaluated in mice by intramuscular (IM) injection. All the recombinant adenoviruses generated robust immune responses, and the immunogenicity of recombinant adenoviruses against the GIIa strain was stronger than that of recombinant adenoviruses against the GIIb strain. Moreover, Ad-XT-tPA-Sopt-vaccinated mice elicited optimal immune effects. In contrast, mice immunized with Ad-XT-tPA-Sopt by oral gavage did not induce strong immune responses. Overall, IM administration of Ad-XT-tPA-Sopt is a promising strategy against PEDV, and this study provides useful information for developing viral vector-based vaccines.

Identification of B-cell epitopes on structural proteins VP1 and VP2 of Senecavirus A and development of a multi-epitope recombinant protein vaccine.

Senecavirus A (SVA) is an important pathogenic cause of vesicular disease in pigs worldwide. In this study, we screened the B-cell epitopes of SVA using a bioinformatics approach combined with an overlapping synthetic polypeptide method. Four dominant B-cell epitopes (at amino acid (aa) positions: 7-26, 48-74, 92-109, and 129-144) from the VP1 protein and five dominant B-cell epitopes (aa: 38-57, 145-160, 154-172, 193-208, 249-284) from the VP2 protein were identified. Multi-epitope genes comprising the identified B-cell epitope domains were synthesized, prokaryotic expressed, and purified, and their immune protection efficacy was evaluated in piglets. Our results showed that the multi-epitope recombinant protein rP2 induced higher neutralizing antibodies and provided 80% protection against homologous SVA challenge. Thus, the B-cell epitope peptides identified in this study are potential candidates for SVA vaccine development, and rP2 may offer safety and efficacy in controlling infectious SVA.

Polydopamine-coated thalidomide nanocrystals promote DSS-induced murine colitis recovery through Macrophage M2 polarization together with the synergistic anti-inflammatory and anti-angiogenic effects.

High levels of proinflammatory cytokines, macrophage polarization status and immune-mediated angiogenesis play pivotal roles in the pathogenesis of inflammatory bowel disease (IBD). Thalidomide, an anti-inflammatory, immunomodulatory and antiangiogenic agent, is used off-label for treatment of IBD. The therapeutic potential of thalidomide is limited by its poor solubility and side effects associated with its systemic exposure. To address these issues and promote its therapeutic effects on IBD, thalidomide nanocrystals (Thali NCs) were prepared and coated with polydopamine (PDA), a potential macrophage polarization modulator, to form PDA coated Thali NCs (Thali@PDA). Thali@PDA possessed a high drug loading and displayed average particle size of 764.7 ± 50.30 nm. It showed a better anti-colitis effect than bare thalidomide nanocrystals at the same dose of thalidomide. Synergistic effects of polydopamine on anti-inflammatory and anti-angiogenic activities of thalidomide were observed. Furthermore, PDA coating could direct polarization of macrophages towards M2 phenotype, which boosted therapeutic effects of Thali@PDA on IBD. Upon repeated dosing of Thali@PDA for one week, symptoms of IBD in mice were significantly relieved, and histomorphology of the colitis colons were normalized. Key proinflammatory cytokine levels in the inflamed intestines were significantly decreased. Toxicity study also revealed that Thali@PDA is a safe formulation.

[Construction of recombinant adenovirus expressing capsid protein of serotype O foot-and-mouth disease virus and analysis of its immunogenicity].

In order to construct a recombinant replication deficient human type 5 adenovirus (Ad5) expressing a foot-and-mouth disease virus (FMDV) capsid protein, specific primers for P12A and 3B3C genes of FMDV-OZK93 were synthesized. The P12A and 3B3C genes were then amplified and connected by fusion PCR, and a recombinant shuttle plasmid pDC316-mCMV-EGFP-P12A3B3C expressing the FMDV-OZK93 capsid protein precursor P12A and 3B3C protease were obtained by inserting the P12A3B3C gene into the pDC316-mCMV-EGFP plasmid. The recombinant adenovirus rAdv-P12A3B3C-OZK93 was subsequently packaged, characterized and amplified using AdMaxTM adenovirus packaging system, and the expression was verified by infecting human embryonic kidney cell HEK-293. The humoral and cellular immunity levels of well-expressed and purified recombinant adenovirus immunized mice were evaluated. The results showed that rAdv-P12A3B3C-OZK93 could be stably passaged and the maximum virus titer reached 1×109.1 TCID50/mL. Western blotting and indirect immunofluorescence showed that rAdv-P12A3B3C-OZK93 expressed the FMDV-specific proteins P12A and VP1 in HEK-293 cells. In addition, the PK cell infection experiment confirmed that rAdv-P12A3B3C-OZK93 could infect porcine cells, which is essential for vaccination in pigs. Comparing with the inactivated vaccine group, the recombinant adenovirus could induce higher FMDV-specific IgG antibodies, γ-IFN and IL-10. This indicates that the recombinant adenovirus has good immunity for animal, which is very important for the subsequent development of foot-and-mouth disease vaccine.

Sexual hormones in a coastal river adjacent to the Bohai Sea: Characteristic pollutants and dominantly influencing factors.

Characteristic sexual hormones (SHs) and the factors that dominantly influence their occurrence in coastal ecosystems are less understood. This study verified the relationships between SHs and environmental factors and further inferred the possible controlling mechanisms of SH distribution. A characteristic pollutant of SHs was first proposed by determining the contamination level and ecological risks of SHs (seven species) in a coastal river adjacent to the Bohai Sea. The results showed that the 17ß-oestradiol (17ß-E2), estriol (E3), and 17α-ethynylestradiol (EE2) had high mean concentrations of 11.20 (±1.31), 10.17 (±4.91), and 16.71 (±0.88) ng L-1, respectively, in the river water. The concentration of estrone (E1) was positively related to microbial substances of DOMs (p < 0.05). The humification index (HIX) had a negative relationship with E3 (p < 0.05). In water, the distribution of total SHs was regulated by the HIX and fluorescence index (FI), which might be related to photodegradation reactions. The 17α-oestradiol (17α-E2) and EE2 were related to humified organic matter, while E3 and androstenedione (ADD) were influenced by sewage input. The 17ß-E2, E1, and 17α-E2 may be derived from animal sources, while E3, ADD, EE2, and progesterone were from human activities. Oestrogens, including E1, 17α-E2, 17ß-E2, and EE2, displayed higher ecological risks than androgens and progesterone, with medium to high risk in most sites. The 17ß-E2 was regarded as a characteristic pollutant of SHs throughout the river system, which displayed the highest risk. This paper may provide a reference for SH risk management and control.

Blockade of Platelets Using Tumor-Specific NO-Releasing Nanoparticles Prevents Tumor Metastasis and Reverses Tumor Immunosuppression.

The tumor microenvironment maintains a sufficient immunosuppressive state owing to the existence of the immunosuppressive factors. The most prominent such factor is transforming growth factor ß (TGF-ß), which is mainly provided by platelets. Moreover, platelets have been shown to be the main accomplice in assisting tumor metastasis. Therefore, blocking tumor-associated platelets is endowed with functions of enhancing immunity and reducing metastasis. Herein, we designed a tumor microenvironment-responsive nitric oxide (NO) release nanoparticle, Ptx@AlbSNO, which was able to specifically and safely co-deliver the antiplatelet agent NO and the chemotherapeutic agent paclitaxel (Ptx) into tumor tissues and inhibit platelet-tumor cell interactions. We discovered that Ptx@AlbSNO could successfully block tumor-specific platelet functions, thereby suppressing the process of tumor epithelial-mesenchymal transition (EMT), preventing platelet adhesion around circulating tumor cells (CTCs) and reducing distant metastasis. In vivo studies demonstrate that the co-delivery of NO and Ptx can suppress primary tumor growth. With the ability to effectively inhibit activated platelets and TGF-ß secretion in tumors, Ptx@AlbSNO can enhance intratumoral immune cell infiltration to reverse the immunosuppressive tumor microenvironment.

Evaluation of the Efficacy of a Recombinant Adenovirus Expressing the Spike Protein of Porcine Epidemic Diarrhea Virus in Pigs.

In recent years, many studies have shown that recombinant adenovirus live vector-based vaccines are a promising novel vaccine candidate against virus infection. Therefore, in this study, a new type of recombinant adenovirus expressing the spike (S) protein of porcine epidemic diarrhea virus (PEDV), rAd-PEDV-S, was generated, and its characteristics were determined. Then, its efficacy as a vaccine candidate was evaluated in 4-week-old pigs. The results showed that the S protein could be well expressed at a high level in rAd-PEDV-S-infected cells and that the viral titers could reach 1011 PFU/mL. Further animal experimental results showed that rAd-PEDV-S elicited a significant PEDV-specific humoral immune response after vaccination (P < 0.05). In addition, rAd-PEDV-S provided partial protection for pigs against the highly virulent PEDV challenge. The results presented in this study indicate that the adenovirus vector can be used as a vaccine delivery vector for the development of a PEDV vaccine and is a promising novel vaccine candidate for future prevention and control of porcine epidemic diarrhea (PED), but its efficacy still needs to be improved in the future.

A modified hydrophobic ion-pairing complex strategy for long-term peptide delivery with high drug encapsulation and reduced burst release from PLGA microspheres.

Poor encapsulation and high initial burst were two major obstacles for the water-soluble peptide drug loaded microspheres preparation using the industrial emulsification method. In the present study, we hypothesized that the hydrophobic ion-pairing (HIP) complex strategy with a further healing of the pores within the microspheres may improve drug encapsulation and initial burst release. DSS was chosen as the most suitable one among the three test ion-pairing agents (SDS, DSS and STC) due to its high binding efficiency with drug and reversible dissociation capacity in presence of counter ions. The formation of HIP complex between octreotide acetate and DSS successfully reversed the highly water-soluble nature of the drug. A specific S/O/W method was adopted to encapsulate such drug containing HIP complex. The encapsulation efficiency of the drug was greatly improved compared with the conventional W1/O/W2 method (from 44% to 90%). Under the optimal healing conditions (the healing time 6 h, temperature 40 °C and 4% DEP content), the pores within the microspheres were effectively healed. Initial burst amount of octreotide acetate in S/O/W microspheres decreased to 3.56%. The pore healing effect was further confirmed by the scanning electron microscopy and fluorescence microscopy results. In the process of testing the drug release performance of such new strategy in vitro and in vivo, a more satisfactory single phase release profile with sustained and steady drug release was observed. These results suggested that the modified HIP strategy could be a promising platform for water-soluble peptide encapsulation with high encapsulation efficiency, low initial burst and stable drug release mechanism.

Glutathione depletion and dual-model oxygen balance disruption for photodynamic therapy enhancement.

Photodynamic therapy (PDT) is a prospective approach to cure tumor diseases. However, tumor micro-environment is notably characterized with severe hypoxia and high expression of glutathione (GSH), which seriously limit its clinical application. Here, based on the characteristics of perfluorocarbon (PFC) to dissolve substantial oxygen (O2) and the sensitivity of reductive GSH to S-NO group, we designed GSH depletion and dual-model O2 supply strategies to promote PDT enhancement. The PFC nanoliposomes (FI@Lip) and biocompatible NO donor S-nitrosated human serum albumin (HSA-SNO) were combined to synergistically combat the obstacle of tumor micro-environment, reducing GSH concentration and increasing singlet oxygen (1O2) generation. In vitro, after irradiation with NIR laser, the PFC in FI@Lip dissolved more O2 to increase 1O2 generation. In addition, with co-delivery of HSA-SNO, it can effectively promote GSH depletion to recover 1O2 level and release NO concurrently to inhibit mitochondrial respiration. This combination strategy of FI@Lip and HSA-SNO obviously relieved intracellular hypoxia and decreased GSH to increase more toxic 1O2 generation for PDT enhancement. The present work will play as an enlightening role in PDT design and clinical application in the near future.

Proton Oriented-"Smart Depot" for Responsive Release of Ca2+ to Inhibit Peptide Acylation in PLGA Microspheres.

PURPOSE: The purpose of this study was to characterize and detail the mechanism of a smart Ca2+ release depot (Ca3(PO4)2) about its ability for sustainable inhibition on peptide acylation within PLGA microspheres. METHODS: The octreotide acetate release and acylation kinetics were analyzed by RP-HPLC. Changes of Ca2+ concentration and adsorption behavior were determined by a Calcium Colorimetric Assay Kit. The inner pH changes were delineated by a classic pH sensitive probe, Lysosensor yellow/ blue® dextran. Morphological changes of microspheres, adsorption between polymer and additive, transformation of Ca3(PO4)2 were characterized using SEM, FTIR and SSNMR separately. RESULTS: Before and after microspheres formulation, the property and effectiveness of Ca3(PO4)2 were investigated. Compared with a commonly used calcium salt (CaCl2), high encapsulation efficiency (96.56%) of Ca3(PO4)2 guarantees lasting effectiveness. In an increasingly acidic environment that simulated polymer degradation, the poorly water-soluble Ca3(PO4)2 could absorb protons and transform into the more and more soluble CaHPO4 and Ca(H2PO4)2 to produce sufficient Ca2+ according to severity of acylation. The corresponding Ca2+ produce capacity fully met the optimum inhibition requirement since the real-time adsorption sites (water-soluble carboxylic acids) inside the degrading microspheres were rare. A sustained retention of three switchable calcium salts and slow release of Ca2+ were observed during the microsphere incubation. FTIR results confirmed the long-term inhibition effect induced by Ca3(PO4)2 on the adsorption between drug and polymer. CONCLUSIONS: With the presence of the smart Ca2+ depot (Ca3(PO4)2) in the microspheres, a sustainable and long-term inhibition of peptide acylation was achieved.

Starvation-amplified CO generation for enhanced cancer therapy via an erythrocyte membrane-biomimetic gas nanofactory.

Carbon monoxide (CO)-based gas therapy has emerged as an attractive therapeutic strategy for cancer therapy. However, the main challenges are the in situ-triggered and efficient delivery of CO in tumors, which limit its further clinical application. Herein, we developed an erythrocyte membrane-biomimetic gas nanofactory (MGP@RBC) to amplify the in situ generation of CO for combined energy starvation of cancer cells and gas therapy. This nanofactory was constructed by encapsulating glucose oxidase (GOx) and Mn2(CO)10 (CO-donor) into the biocompatible polymer poly(lactic-co-glycolic acid), obtaining MGP nanoparticles, which are further covered by red blood cell (RBC) membrane. Because of the presence of proteins on RBC membranes, the nanoparticles could effectively avoid immune clearance in macrophages (Raw264.7) and significantly prolong their blood circulation time, thereby achieving higher accumulation at the tumor site. After that, the GOx in GMP@RBC could effectively catalyze the conversion of endogenous glucose to hydrogen peroxide (H2O2) in the presence of oxygen. The concomitant generation of H2O2 could efficiently trigger CO release to cause dysfunction of mitochondria and activate caspase, thereby resulting in apoptosis of the cancer cells. In addition, the depletion of intratumoral glucose could starve tumor cells by shutting down the energy supply. Altogether, the in vitro and in vivo studies of our synthesized biomimetic gas nanofactory exhibited an augmentative synergistic efficacy of CO gas therapy and energy starvation to inhibit tumor growth. It provides an attractive strategy to amplify CO generation for enhanced cancer therapy in an accurate and more efficient manner. STATEMENT OF SIGNIFICANCE: Carbon monoxide (CO) based gas therapy has emerged as an attractive therapeutic strategy for cancer therapy. In this study, we developed an erythrocyte membrane biomimetic gas nanofactory to amplify the in-situ generation of CO for combined cancer starvation and gas therapy. It is constructed by coating glucose oxidase (GOx) and CO donor-loaded nanoparticles with erythrocyte membrane. Due to the erythrocyte membrane, it can effectively prolong blood circulation time and achieve higher tumor accumulation. After accumulated in tumor, endogenous glucose can be effectively catalyzed to hydrogen peroxide, in-situ amplified CO release to induce the apoptosis of cancer cells. In addition, depleting glucose can also starve tumor cells by shutting down the energy supply. Overall, our biomimetic gas nanofactory exhibits an augmentative synergistic efficacy of CO gas therapy and starvation to increased tumor inhibition. It provide a novel strategy to deliver CO in an accurate and more efficient manner, promising for combined cancer therapy in future clinical application.

A switchable NO-releasing nanomedicine for enhanced cancer therapy and inhibition of metastasis.

Clinical chemotherapy for cancer is limited by the physiological barrier of tumors, resulting in low drug delivery to tumors, poor efficacy of drugs and inability to block tumor metastasis. Here we developed an intelligent switchable nitric oxide (NO)-releasing nanoparticle, IPH-NO, which loads a photosensitizer (IR780) and the chemotherapy drug paclitaxel (PTX) into NO donor-S-nitrosated human serum albumin (HSA-NO). NO exhibits two effects based on its concentration: enhancement of chemotherapy by increasing the enhanced permeability and retention (EPR) effect at low concentrations and direct killing of cancer cells at high concentrations. IPH-NO can slowly release NO in the presence of glutathione to boost tumor vascular permeability and improve drug accumulation. Near-infrared light irradiation was utilized to induce a quick release of NO that can directly kill cancer cells at high concentrations. This combination of phototherapy and NO gas therapy activated by NIR together with chemotherapy showed significant effects in tumor inhibition. Furthermore, IPH-NO blocked tumor metastasis by inhibiting epithelial mesenchymal transition. PH-NO provides a novel strategy to control NO release at tumor site for drug accumulation and combination therapies, consequently potentiating the anticancer efficacy and inhibiting tumor metastasis.

Proteus alimentorum sp. nov., isolated from pork and lobster in Ma'anshan city, China.

Two strains of Gram-stain-negative, facultatively anaerobic short-rod bacteria were recovered from two different food samples in Ma'anshan city, Anhui province, China in 2008. The bacteria were characterized in a polyphasic taxonomic study that included phenotypic, phylogenetic and genotypic methodologies. Phylogenetic analysis of the 16S rRNA gene demonstrated that the two strains belonged to the genus Proteus and were most similar to Proteus vulgaris ATCC 29905T with a score of 99.7 %. Phylogenetic analysis of the rpoB gene placed the two strains into a cluster with a distinctly interspecies phylogenetic branch that was clearly separated from six type strains of the genus Proteus, with the most closely related species being Proteus mirabilis ATCC 29906T. In silico genomic comparisons, including in silico DNA-DNA hybridization (isDDH) and average nucleotide identity (ANI) analysis showed that the representative strain, 08MAS0041T, and all six Proteus species share less than 70 % isDDH and have a 95 % ANI cutoff level, supporting the designation of the two strains as a novel species of the genus Proteus. The predominant cellular fatty acids of strain 08MAS0041T were C16 : 0 (24.8 %), C16 : 1ω7c/16 : 1ω6c (16.5 %), C18 : 1ω6c/C18 : 1ω7c (14.5 %), C17 : 0 cyclo (12.6 %) and C16 : 1iso I/C14 : 0 3-OH (10.6 %). The analysis of biochemical, phylogenetic and genomic data confirmed that the two strains were clearly different from all recognized species of the genus Proteus and represent a novel Proteus species, for which the name Proteus alimentorum sp. nov. is proposed. The type strain is 08MAS0041T (=DSM 104685T=CGMCC 1.15939T).

Tf-PEG-PLL-PLGA nanoparticles enhanced chemosensitivity for hypoxia-responsive tumor cells.

Hypoxia is an inseparable component of the solid tumor as well as the bone marrow microenvironment. In this study, we investigated the effect of the novel polyethylene glycol (PEG)-poly L-lysine (PLL)-poly lactic-co-glycolic acid (PLGA) based nanoparticles (NPs) modified by transferrin (Tf) loaded with daunorubicin (DNR) (DNR-Tf-PEG-PLL-PLGA-NPs, abbreviated as DNR-Tf-NPs) on leukemia cells (K562) under hypoxia. In vitro and in vivo tests to determine the effect of the enhanced chemosensitivity were evaluated using the immunofluorescence, flow cytometry, 3,-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-tetrazoliumbromide assay, Western blot analysis, histopathological examination, and immunohistochemistry analysis. Under hypoxia, K562 cells were hypoxia-responsive with the inhibitory concentration 50% (IC50) of DNR increased, resulting in chemotherapy insensitivity. By targeting the transferrin receptor (TfR) on the surface of K562 cells, DNR-Tf-NPs led to an increased intracellular DNR level, enhancing drug sensitivity of K562 cells to DNR with a decreased IC50, even under hypoxia. We further detected the protein levels of hypoxia-inducible factor-1α (HIF-1α), Bcl-2, Bax, and caspase-3 in K562 cells. The results indicated that DNR-Tf-NPs downregulated HIF-1α and induced apoptosis to overcome hypoxia. In the xenograft model, injection of DNR-Tf-NPs significantly suppressed tumor growth, and the immunosignals of Ki67 in DNR-Tf-NPs group was significantly lower than the other groups. It was therefore concluded that DNR-Tf-NPs could be a promising candidate for enhancing drug sensitivity under hypoxia in tumor treatment.

Dendritic cell targeted vaccines: Recent progresses and challenges.

Dendritic cells (DCs) are known to be a set of morphology, structure and function of heterogeneous professional antigen presenting cells (APCs), as well as the strongest functional antigen presenting cells, which can absorb, process and present antigens. As the key regulators of innate and adaptive immune responses, DCs are at the center of the immune system and capable of interacting with both B cells and T cells, thereby manipulating the humoral and cellular immune responses. DCs provide an essential link between the innate and adaptive immunity, and the strong immune activation function of DCs and their properties of natural adjuvants, make them a valuable target for antigen delivery. Targeting antigens to DC-specific endocytic receptors in combination with the relevant antibodies or ligands along with immunostimulatory adjuvants has been recently recognized as a promising strategy for designing an effective vaccine that elicits a strong and durable T cell response against intracellular pathogens and cancer. This opinion article provides a brief summary of the rationales, superiorities and challenges of existing DC-targeting approaches.