Influenza virus evolves constantly in an unpredictable fashion, making it necessary to vaccinate people annually for effective prevention and control of influenza. In general, however, during the first wave of an influenza outbreak caused by a newly emerging virus strain, influenza morbidity and mortality have been observed to rise sharply due to the lack of a matching vaccine. This necessitates the exploration of novel intervention approaches, particularly those prophylactic or therapeutic agents that have a broad range of antiviral activities and are also proven to be non-toxic. Here, we reported that stimulation of the innate immune system by intranasal administration of chitosan as a single agent was sufficient to completely protect BALB/c mice from lethal infection by H7N9 virus, a newly emerged viral strain that is highly pathogenic to humans. Remarkably, animals could still be protected against lethal challenge by H7N9 (10×LD50), even ten days after the intranasal chitosan administration. The significantly enhanced infiltration of leukocytes in the bronchoalveolar lavage and elevated levels of proinflammatory cytokines in the bronchia/lung tissues revealed the potent activation of mucosal immune responses by intranasally delivered chitosan. We also observed that chitosan can protect mice from three other virus strains. The marked breadth and magnitude of protection against diverse viral strains makes chitosan an attractive candidate as a universal anti-influenza agent.

Although cancer is a genetic disease, epigenetic alterations are involved in its initiation and progression. Previous studies have shown that reprogramming of colon cancer cells using Oct3/4, Sox2, Klf4, and cMyc reduces cancer malignancy. Therefore, cancer reprogramming may be a useful treatment for chemo- or radiotherapy-resistant cancer cells. It was also reported that the introduction of endogenous small-sized, non-coding ribonucleotides such as microRNA (miR) 302s and miR-369-3p or -5p resulted in the induction of cellular reprogramming. miRs are smaller than the genes of transcription factors, making them possibly suitable for use in clinical strategies. Therefore, we reprogrammed colon cancer cells using miR-302s and miR-369-3p or -5p. This resulted in inhibition of cell proliferation and invasion and the stimulation of the mesenchymal-to-epithelial transition phenotype in colon cancer cells. Importantly, the introduction of the ribonucleotides resulted in epigenetic reprogramming of DNA demethylation and histone modification events. Furthermore, in vivo administration of the ribonucleotides in mice elicited the induction of cancer cell apoptosis, which involves the mitochondrial Bcl2 protein family. The present study shows that the introduction of miR-302s and miR-369s could induce cellular reprogramming and modulate malignant phenotypes of human colorectal cancer, suggesting that the appropriate delivery of functional small-sized ribonucleotides may open a new avenue for therapy against human malignant tumors.

Host cells infected by Theileria annulata schizonts show the character of permanent proliferation in vitro, also named transformation. To explore the molecular mechanism a T. annulata Cyp1 (TaCyp1) protein potentially involved in regulating cell transformation was used as bait to screen for its interacting proteins by yeast-two-hybrid assay. Additional GST-pull down experiments confirmed that only MED21 specifically interacted with TaCyp1. Moreover, the distribution of TaCyp1 around T. annulata schizonts facilitated interaction with host cell MED21. As a component of mediator complex, MED21 is normally involved in regulating the transcription of nearly all RNA polymerase II-dependent genes. Therefore, to explore its influence on NF-κB signaling MED21 RNA interference and parasite killing with BW720c treatment were performed. Knock down of MED21 resulted in a significant decrease in NF-κB1/2 mRNA expressions, but no significant change in P105, P52 levels, nor detectable alteration in levels of phosphorylated IκBα/β. By contrast, BW720c treatment induced an obvious decrease in the phosphorylation status of P52 and IκBα/β, but no obvious change in that of P105. This suggests that BW720c-induced parasite death had a significant negative influence on NF-κB signaling, whereas knock down of MED21 had no obvious effect on NF-κB signaling. Characterization of TaCyp1 provides information on the function of parasite cyclophilins and leads to a better understanding of the interactions between T. annulata and its host leukocytes.

Since the first case of human avian influenza A (H7N9) virus infection in 2013, five H7N9 epidemics have occurred in China, all of which caused severe diseases, including pneumonia and acute respiratory distress syndrome, and the fatality rates of these epidemics were as high as 30-40%. To control the prevalence of H7N9 influenza, an effective vaccine is urgently needed. In the present study, we used chitosan and recombinant human interleukin-2 as an adjuvant (JY) to promote the systemic and mucosal immune responses induced by the H7N9 vaccine. Mice were immunized intranasally with the inactivated split influenza A (H7N9) virus (A/Shanghai/02/2013) vaccine with or without JY. The hemagglutination inhibition (HI) titers of mice immunized with the JY-adjuvanted vaccine were significantly higher than those of mice immunized with the vaccine without adjuvant (21.11 ± 9.58 vs. 5.04 ± 3, P < 0.05). The JY-adjuvanted H7N9 nasal spray vaccine induced higher HI titers (8 ± 0.82 vs. 6.7 ± 0.67, P = 0.0035) than those did the poly (I:C)-adjuvanted H7N9 vaccine or the LTB-adjuvanted H7N9 vaccine (8 ± 0.82 vs. 6.9 ± 0.88, P = 0.0186). The optimal immunization regimen for the nasal spray H7N9 vaccine was determined to be a 21-day interval between the primary immunization and booster, with a dose of 4.5 μg hemagglutinin per mouse. The immunogenicities of the nasal spray H7N9 vaccine and intramuscular vaccine (containing only the inactivated split virus) were compared in mice. Two doses of the nasal spray H7N9 vaccine induced higher titers of HI (6.7 ± 0.67 vs. 5.3 ± 1.16, P = 0.004) and anti-HA IgG in sera (19.26 ± 0.67 vs. 13.97 ± 0.82, P < 0.0001) and of anti-HA sIgA (7.13 ± 2.54 vs. 0, P = 0.0000) in bronchoalveolar lavage fluid (BALF) than one dose of intramuscular H7N9 vaccine 3 weeks after the last immunization. However, when we immunized the mice with two doses of both vaccines separately, the nasal spray H7N9 vaccine induced higher titers of anti-HA IgG (19.26 ± 0.67 vs. 17.56 ± 0.57, P < 0.0001) and anti-HA sIgA (7.13 ± 2.54 vs. 4.02 ± 0.33, P = 0.0026) than did the intramuscular H7N9 vaccine, and there was no difference in HI titer between the two groups (P = 0.3745). This finding indicates that the JY-adjuvanted nasal spray H7N9 vaccine induced not only the systemic immune response but also a local mucosal response, which may improve the efficacy of H7N9 influenza prevention through respiratory tract transmission.

Nanodevices for magnetic resonance imaging of cancer were self-assembled to core-shell micellar structures by metal complex formation of K(2)PtCl(6) with diethylenetriaminepentaacetic acid gadolinium (III) dihydrogen (Gd-DTPA), a T(1)-contrast agent, and poly(ethylene glycol)-b-poly{N-[N'-(2-aminoethyl)-2-aminoethyl]aspartamide} (PEG-b-PAsp(DET)) copolymer in aqueous solution. Gd-DTPA-loaded polymeric micelles (Gd-DTPA/m) showed a hydrodynamic diameter of 45 nm and a core size of 22 nm. Confining Gd-DTPA inside the core of the micelles increased the relaxivity of Gd-DTPA more than 13 times (48 mM(-1) s(-1)). In physiological conditions Gd-DTPA/m sustainedly released Gd-DTPA, while the Pt(IV) complexes remain bound to the polymer. Gd-DTPA/m extended the circulation time in plasma and augmented the tumor accumulation of Gd-DTPA leading to successful contrast enhancement of solid tumors. μ-Synchrotron radiation-X-ray fluorescence results confirmed that Gd-DTPA was delivered to the tumor site by the micelles. Our study provides a facile strategy for incorporating contrast agents, dyes and bioactive molecules into nanodevices for developing safe and efficient drug carriers for clinical application.

Human cytomegalovirus (HCMV) causes serious HCMV-related diseases in immunocompromised people. Vaccination is the most effective measure to control infection with the pathogen, yet no vaccine has been licensed till now. We performed a head-to-head comparison of the protective abilities of multiple DNA vaccines in murine model of murine cytomegalovirus (MCMV) infection.

The conventional chemotherapeutics could not be traced in vivo and provide timely feedback on the clinical effectiveness of drugs. In this study, poly(L-γ-glutamyl-glutamine)-paclitaxel (PGG-PTX), as a model polymer, was chemically conjugated with Gd-DTPA (Gd-diethylenetriaminepentaacetic acid), a T1-contrast agent of MRI, to prepare a Gd-DTPA-conjugated PGG-PTX (PGG-PTX-DTPA-Gd) delivery system used for tumor theranostics. PGG-PTX-DTPA-Gd can be self-assembled to NPs in water with a z-average hydrodynamic diameter about 35.9 nm. The 3 T MRI results confirmed that the relaxivity of PGG-PTX-DTPA-Gd NPs (r1 = 18.98 mM-1S-1) was increased nearly 4.9 times compared with that of free Gd-DTPA (r1 = 3.87 mM-1S-1). The in vivo fluorescence imaging results showed that PGG-PTX-DTPA-Gd NPs could be accumulated in the tumor tissue of NCI-H460 lung cancer animal model by EPR effect, which was similar to PGG-PTX NPs. The MRI results showed that compared with free Gd-DTPA, PGG-PTX-DTPA-Gd NPs showed significantly enhanced and prolonged signal intensity in tumor tissue, which should be attributed to the increased relaxivity and tumor accumulation. PGG-PTX-DTPA-Gd NPs also showed effective antitumor effect in vivo. These results indicated that PGG-PTX-DTPA-Gd NPs are an effective delivery system for tumor theranostics, and should have a potential value in personalized treatment of tumor.

Babesiosis, the hemolytic disease caused by Babesia, which is a tick-transmitted obligate intraerythrocytic protozoan parasite. This disease is responsible for significant mortality and morbidity rates and enormous economic losses to the livestock industry in tropical and subtropical regions worldwide. In this study, blood samples were collected from 141 pet dogs from Gansu, China, and analyzed for Babesia or Theileria spp. infection using specific PCR and sequencing based on 18S rRNA gene fragments. The results indicated that 18S rRNA gene sequences from 11 samples were similar to the 18S rRNA gene sequences in Babesia canis vogeli (2) and Theileria sinensis (9). The total infected rates of B. canis vogeli and T. sinensis were 1.4% (2/141) and 6.4% (9/141), respectively. This represents the first molecular report of T. sinensis in dogs worldwide and of B. canis vogeli in dogs from Gansu province of China. Furthermore, the finding of T. sinensis in dogs may represent the common infection of this parasite occurring in Gansu.

Wild birds, especially those in wetlands and aquatic environments, are considered to be natural reservoirs of avian influenza viruses. It is accepted that water is an important component in the transmission cycle of avian influenza virus. Monitoring the water at aggregation and breeding sites of migratory waterfowl, mainly wetland, is very important for early detection of avian influenza virus. The epidemiology investigation of avian influenza virus was performed in Dongting lake wetland which is an international important wetland.

Two surface glycoproteins of influenza virus, haemagglutinin (HA) and neuraminidase (NA), play opposite roles in terms of their interaction with host sialic acid receptors. HA attaches to sialic acid on host cell surface receptors to initiate virus infection while NA removes these sialic acids to facilitate release of progeny virions. This functional opposition requires a balance. To explore what might happen when NA of an influenza virus was replaced by one from another isolate or subtype, in this study, we generated three recombinant influenza A viruses in the background of A/PR/8/34 (PR8) (H1N1) and with NA genes obtained respectively from the 2009 pandemic H1N1 virus, a highly pathogenic avian H5N1 virus, and a lowly pathogenic avian H9N2 virus. These recombinant viruses, rPR8-H1N1NA, rPR8-H5N1NA, and rPR8-H9N2NA, were shown to have similar growth kinetics in cells and pathogenicity in mice. However, much more rPR8-H5N1NA and PR8-wt virions were released from chicken erythrocytes than virions of rPR8-H1N1NA and rPR8-H9N2NA after 1 h. In addition, in MDCK cells, rPR8-H5N1NA and rPR8-H9N2NA infected a higher percentage of cells, and induced cell-cell fusion faster and more extensively than PR8-wt and rPR8-H1N1NA did in the early phase of infection. In conclusion, NA replacement in this study did not affect virus replication kinetics but had different effects on infection initiation, virus release and fusion of infected cells. These phenomena might be partially due to NA proteins' different specificity to α2-3/2-6-sialylated carbohydrate chains, but the exact mechanism remains to be explored.

In 2003, severe acute respiratory syndrome (SARS) resulted in hundreds of infections and deaths globally. We aim to assess immunogenicity and protective efficacy of purified inactivated Vero-cell SARS vaccine in monkeys.

Maternal antibody is the major form of protection against disease in early life; however, its presence interferes with active immunization of offspring. In order to overcome the immunosuppression caused by maternal antibody, several immune strategies were explored in this paper using mouse model and influenza vaccines.

Vaccination against the SARS-CoV infection is an attractive means to control the spread of viruses in public. In this study, we employed a DNA vaccine technology with the levamisole, our newly discovered chemical adjuvant, to generate Th1 type of response. To avoid the enhancement antibody issue, genes encoding the nucleocapsid, membrane, and envelope protein of SARS-CoV were cloned and their expressions in mammalian cells were determined. After the intramuscular introduction into animals, we observed that the constructs of the E, M, and N genes could induce high levels of specific antibodies, T cell proliferations, IFN-gamma, DTH responses, and in vivo cytotoxic T cells activities specifically against SARS-CoV antigens. The highest immune responses were generated by the construct encoding the nucleocapsid protein. The results suggest that the N, M, and E genes could be used as the targets to prevent SARS-CoV infection in the DNA vaccine development.

PR domain zinc finger protein 14 (PRDM14) maintains stemness in embryonic stem cells via epigenetic mechanisms. Although PRDM14 is elevated in several cancers, it is unclear if and how PRDM14 confers stem cell-like properties and epigenetic changes to cancer cells. Here, we examined the phenotypic characteristics and epigenetic and gene expression profiles of cancer cells that differentially express PRDM14, and assessed the potential of PRDM14-targeted cancer therapy. PRDM14 expression was markedly increased in many different cancer types and correlated with poor survival of breast cancer patients. PRDM14 conferred stem cell-like phenotypes to cancer cells and regulated the expression of genes involved in cancer stemness, metastasis, and chemoresistance. PRDM14 also reduced the methylation of proto-oncogene and stemness gene promoters and PRDM14-binding regions were primarily occupied by histone H3 Lys-4 trimethylation (H3K4me3), both of which are positively correlated with gene expression. Moreover, strong PRDM14 binding sites coincided with promoters containing both H3K4me3 and H3K27me3 histone marks. Using calcium phosphate hybrid micelles as an RNAi delivery system, silencing of PRDM14 expression by chimera RNAi reduced tumor size and metastasis in vivo without causing adverse effects. Conditional loss of PRDM14 function also improved survival of MMTV-Wnt-1 transgenic mice, a spontaneous model of murine breast cancer. Our findings suggest that PRDM14 inhibition may be an effective and novel therapy for cancer stem cells.

Babesiosis is a socioeconomically important tick-borne disease of animals (including humans) caused by haemoprotozoan parasites. The severity of babesiosis relates to host and parasite factors, particularly virulence/pathogenicity. Although Babesia bovis is a particularly pathogenic species of cattle, there are species of Babesia of ruminants that have limited pathogenicity. For instance, the operational taxonomic unit Babesia sp. Xinjiang (abbreviated here as Bx) of sheep from China is substantially less virulent/pathogenic than B. bovis is in cattle. Although the reason for this distinctiveness is presently unknown, it is possible that Bx has a reduced ability to adhere to cells or evade/suppress immune responses, which might relate to particular proteins, such as the variant erythrocyte surface antigens (VESAs).

Theileriosis is an important tick-borne protozoosis that causes high morbidity and mortality in cattle. In this study, the pathological and clinical characteristics of cattle experimentally infected with Theileria annulata were investigated. The clinical findings revealed typical signs of bovine theileriosis, including fever, enlargement of superficial lymph nodes, anemia, and respiratory distress. The most common pathological features were petechial and ecchymotic hemorrhages on the mucosa and serosal surface, severe jaundice, pulmonary edema and emphysema, multifocal necrosis and numerous ulcerations in the abomasum, congestion and marble-like discoloration of the spleen, and severe intestinal ecchymotic hemorrhages. The main histological characteristics were proliferation and infiltration of lymphocytes, plasma cells, and macrophages in the lymph nodes, spleen, and lymph node mass. Macroschizonts were observed in the cytoplasm of lymphocytes and macrophages of the lymph nodes and spleen. This study has significance for basic research and the clinical detection and diagnosis of Theileria annulata infection and can aid the prevention and control of theileriosis and future studies of the pathogenic mechanisms.

DNA binding with One Finger (Dof) proteins are plant-specific transcription factors with highly conserved Dof domain, including C2-C2 type zinc finger motifs. In this study, we identified 45 PbDofs in pear (Pyrusbretschneideri). PbDofs were classified into eight subfamilies by phylogenetic analysis. Conserved motifs of PbDof proteins were analyzed by MEME. PbDofs in subfamily D1 werehomologous to CDFs in Arabidopsis. In this study, we showed that PbDof9.2 was regulated by both the circadian clock and photoperiod. PbDof9.2-GFP proteinwas localized in the nucleus. Overexpression of PbDof9.2 in Arabidopsis caused delayed flowering time. PbDof9.2 suppressed the flowering time regulator FT and could repress flowering time by promoting activity of PbTFL1a and PbTFL1b promoter. These results suggest that Doftranscription factors have conserved functions in plant development.

Immunotherapy shows remarkable efficacy in treating several types of cancer such as melanoma, leukemia, and lung carcinoma, but its therapeutic effect for most solid tumors is still limited. Various cancer therapies, such as chemotherapy, radiotherapy and phototherapy, kill solid tumors through non-inflammatory apoptosis or ablation, rather than making solid tumors immunogenic. As a highly-inflammatory programmed cell death (PCD), pyroptosis provides a great opportunity to alleviate immunosuppression and promote a systemic immune response in treating solid tumors. Herein, by fusing breast cancer membrane onto the poly(lactic-co-glycolic acid) polymeric core, we design a biomimetic nanoparticle (BNP) loaded with indocyanine green (ICG) and decitabine (DCT) for photo-activated cancer cell pyroptosis and solid tumor immunotherapy. The tumor-homing BNP effectively accumulate in tumor with low immunogenicity. ICG in BNP puncture cancer cell membranes induces a sharp cytoplasm Ca2+ concentration increase by low-dose NIR photo-activation, which promotes cytochrome c release followed by caspase-3 activation. DCT up-regulates GSDME expression synergistically via inhibiting DNA methylation, which enhances caspase-3 cleavage to GSDME and causes cancer cell pyroptosis. Finally, photo-activated pyroptosis mediated by BNP induces an impressive systemic antitumor immunity for inhibition of both primary tumor and distant tumors. Overall, pyroptosis-associated BNP shows a novel strategy for solid tumor immunotherapy with high compatibility and wide clinical applicability.

Downsizing nanocarriers is a promising strategy for systemically targeting fibrotic cancers, such as pancreatic cancer, owing to enhanced tissue permeability. We recently developed a small oligonucleotide nanocarrier called a unit polyion complex (uPIC) using a single oligonucleotide molecule and one or two molecule(s) of two-branched poly(ethylene glycol)-b-poly(l-lysine) (bPEG-PLys). The uPIC is a dynamic polyion-pair equilibrated with free bPEG-PLys, and thus, is highly stabilized in the presence of excess amounts of free bPEG-PLys in the bloodstream. However, the dynamic polyion-pairing behavior of uPICs needs to be further investigated for longevity in the bloodstream, especially under lower amounts of free bPEG-PLys. Herein, the polyion-pairing behavior of uPICs was investigated by highlighting oligonucleotide stability and negative charge number. To this end, small interfering RNA (siRNA) and antisense oligonucleotides (ASO) were chemically modified to acquire nuclease resistance, and the ASO was hybridized with complementary RNA (cRNA) to form a hetero-duplex oligonucleotide (HDO) with twice the negative charges. While all oligonucleotides similarly formed sub-20 nm-sized uPICs from a single oligonucleotide molecule, the association number of bPEG-PLys (ANbPEG-PLys) in uPICs varied based on the negative charge number of oligonucleotides (N-), that is, ANbPEG-PLys = ~2 at N- = ~40 (i.e., siRNA and HDO) and ANbPEG-PLys = ~1 at N- = 20 (i.e., ASO), presumably because of the balanced charge neutralization between the oligonucleotide and bPEG-PLys with a positive charge number (N+) of ~20. Ultimately, the uPICs prepared from the chemically modified oligonucleotide with higher negative charges showed considerably longer blood retention than those from the control oligonucleotides without chemical modifications or with lower negative charges. The difference in the blood circulation properties of uPICs was more pronounced under lower amounts of free bPEG-PLys. These results demonstrate that the chemical modification and higher negative charge in oligonucleotides facilitated the polyion-pairing between the oligonucleotide and bPEG-PLys under harsh biological conditions, facilitating enhanced blood circulation of uPICs.

Substantial progress has been made in understanding how tumors escape immune surveillance. However, few measures to counteract tumor immune evasion have been developed. Suppression of tumor antigen expression is a common adaptive mechanism that cancers use to evade detection and destruction by the immune system. Epigenetic modifications play a critical role in various aspects of immune invasion, including the regulation of tumor antigen expression. To identify epigenetic regulators of tumor antigen expression, we established a transplantable syngeneic tumor model of immune escape with silenced antigen expression and used this system as a platform for a CRISPR-Cas9 suppressor screen for genes encoding epigenetic modifiers. We found that disruption of the genes encoding either of the chromatin modifiers activating transcription factor 7-interacting protein (Atf7ip) or its interacting partner SET domain bifurcated histone lysine methyltransferase 1 (Setdb1) in tumor cells restored tumor antigen expression. This resulted in augmented tumor immunogenicity concomitant with elevated endogenous retroviral (ERV) antigens and mRNA intron retention. ERV disinhibition was associated with a robust type I interferon response and increased T-cell infiltration, leading to rejection of cells lacking intact Atf7ip or Setdb1. ATF7IP or SETDB1 expression inversely correlated with antigen processing and presentation pathways, interferon signaling, and T-cell infiltration and cytotoxicity in human cancers. Our results provide a rationale for targeting Atf7ip or Setdb1 in cancer immunotherapy.