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Eimeria maxima possesses integral families of immunogenic constituents that promote differentiation of immune cells during host-parasite interactions. Dendritic cells (DCs) have an irreplaceable role in the modulation of the host immunity. However, the selection of superlative antigen with immune stimulatory efficacies on host DCs is lacking. In this study, 5 recombinant proteins of E. maxima (Em), including Em14-3-3, rhomboid family domain containing proteins (ROM) EmROM1 and EmROM2, microneme protein 2 (EmMIC2), and Em8 were identified to stimulate chicken splenic derived DCs in vitro. The cultured populations were incubated with recombinant proteins, and typical morphologies of stimulated DCs were obtained. DC-associated markers major histocompatibility complex class II, CD86, CD11c, and CD1.1, showed upregulatory expressions by flow cytometry assay. Immunofluorescence assay revealed that recombinant proteins could bind with the surface of chicken splenic derived DCs. Moreover, quantitative real-time PCR results showed that distinct gene expressions of Toll-like receptors and Wnt signaling pathway were upregulated after the coincubation of recombinant proteins with DCs. The ELISA results indicated that the DCs produced a significant higher level of interleukin (IL)-12 and interferon-γ secretions after incubation with recombinant proteins. While transforming growth factor-β was significantly increased with rEmROM1, rEmROM2, and rEmMIC2 as compared to control groups, and IL-10 did not show significant alteration. Taken together, these results concluded that among 5 potential recombinant antigens, rEm14-3-3 could promote immunogenic functions of chicken splenic derived DCs more efficiently, which might represent an effective molecule for inducing the host Th1-mediated immune response against Eimeria infection.

Abstract Dendritic cells (DCs) play a pivotal role to amplify antigen-specific immune responses. Antigens that sensitize T cells via antigen-presentation by DCs could enhance the capacity of host immunity to fight infections. In this study, we tested the immunogenic profiles of chicken DCs towards Glyceraldehyde-3-phosphate dehydrogenase from Eimeria acervulina (EaGAPDH). Immunoblot analysis showed that recombinant EaGAPDH (rEaGAPDH) protein was successfully recognized by rat sera generated against rEaGAPDH. Interaction and internalisation of rEaGAPDH by chicken splenic-derived DCs (chSPDCs) was confirmed by immunofluorescence analysis. Flow cytometry revealed that chSPDCs upregulated MHCII, CD1.1, CD11c, CD80, and CD86 cell-surface markers. Moreover, mRNA expressions of DC maturation biomarkers (CCL5, CCR7, and CD83) and TLR signalling genes (TLR15 and MyD88) were also upregulated whereas those of Wnt signalling were non-significant compared to negative controls. rEaGAPDH treatment induced IL-12 and IFN-γ secretion in chSPDCs but had no effect on IL-10 and TGF-β. Likewise, DC-T cell co-culture promoted IFN-γ secretion and the level of IL-4 was unaffected. Proliferation of T cells and their differentiation into CD3+/CD4+ T cells were triggered in chSPDCs-T cells co-culture system. Taken together, rEaGAPDH could promote Th1 polarization by activating both host DCs and T cells and sheds new light on the role of this important molecule which might contribute to the development of new DCs-based immunotherapeutic strategies against coccidiosis.

Abstract Background Modulation of the host immune response by nematode parasites has been widely reported. Rhodaneses (thiosulfate: cyanide sulfurtransferases) are present in a wide range of organisms, such as archaea, bacteria, fungi, plants and animals. Previously, it was reported that a rhodanese homologue could be bound by goat peripheral blood mononuclear cells (PBMCs) in vivo. Methods In the present study, we cloned and produced a recombinant rhodanese protein originating from Haemonchus contortus (rHCRD), a parasitic nematode of small ruminants. rHCRD was co-incubated with goat PBMCs to assess its immunomodulatory effects on proliferation, apoptosis and cytokine secretion. Results We verified that the natural HCRD protein localized predominantly to the bowel wall and body surface of the parasite. We further demonstrated that serum produced by goats artificially infected with H. contortus successfully recognized rHCRD, which bound to goat PBMCs. rHCRD suppressed proliferation of goat PBMCs stimulated by concanavalin A but did not induce apoptosis in goat PBMCs. The production of TNF-α and IFN-γ decreased significantly, whereas secretion of IL-10 and TGF-β1 increased, in goat PBMCs after exposure to rHCRD. rHCRD also inhibited phagocytosis by goat monocytes. Moreover, rHCRD downregulated the expression of major histocompatibility complex (MHC)-II on goat monocytes in a dose-dependent manner, but did not alter MHC-I expression. Conclusions These results propose a possible immunomodulatory target that may help illuminate the interactions between parasites and their hosts at the molecular level and reveal innovative protein species as candidate drug and vaccine targets.

Abstract Background Gastrointestinal nematodes could release excretory-secretory (ES) proteins into the host environment to ensure their survival. These ES proteins act as immunomodulators to suppress or subvert the host immune response via the impairment of immune cell functions, especially in chronic infections. In our preliminary study, Haemonchus contortus adhesion-regulating molecule 1 (HcADRM1) was identified from H. contortus ES proteins (HcESPs) that interacted with host T cells via liquid chromatography-tandem mass spectrometry analysis. However, little is known about HcADRM1 as an ES protein which may play a pivotal role at the parasite-host interface. Methods Based on bioinformatics approaches, multiple amino acid sequence alignment was conducted and the evolutionary relationship of HcADRM1 with ADRM1 orthologues was extrapolated. Employing RT-qPCR and immunohistochemistry assays, temporal transcriptional and spatial expression profiles of HcADRM1 were investigated. Using immunostaining approaches integrated with immunological bioassays, the immunomodulatory potentials of HcADRM1 on goat T cells were assessed. Results We hereby demonstrated that HcADRM1 with immunodiagnostic utility was a mammalian ADRM1 orthologue abundantly expressed at all developmental stages of H. contortus. Given the implications of ADRM1 proteins in cell growth, survival and development, we further investigated the immunomodulatory property of HcADRM1 as an individual ES protein acting at the parasite-host interface. The rHcADRM1 stimuli notably suppressed T cell viability, promoted intrinsic and extrinsic T cell apoptosis, inhibited T cell proliferation and induced cell cycle arrest at G1 phase. Simultaneously, rHcADRM1 stimuli exerted critical controls on T cell cytokine secretion profiles, predominantly by restraining the secretions of interleukin (IL)-4, IL-10 and interferon-gamma. Conclusions Importantly, HcADRM1 protein may have prophylactic potential for anti-H. contortus vaccine development. Together, these findings may contribute to the clarification of molecular and immunomodulatory traits of ES proteins, as well as improvement of our understanding of parasite immune evasion mechanism in H. contortus-host biology.

Abstract Avian coccidian parasites exhibit a high degree of site specificity in different Eimeria species. Although the underlying mechanism is unclear, an increasing body of evidence suggests that site specificity is due to the interaction between microneme proteins (MICs) and their receptors on the surface of target host cells. In this study, the binding ability of E. tenella MICs (EtMICs) to different intestinal tissue was observed by immunofluorescence to identify the key surface molecule on the parasite responsible for the site specificity. Subsequently, we identified the corresponding host-cell receptors by yeast two-hybrid screening and glutathione-S-transferase pull-down experiments and the distribution of these receptors was observed by immunofluorescence in chicken intestinal tissues. Finally, we evaluated the efficacy of receptor antiserum against the infection of E. tenella in chickens. The results showed that EtMIC3 could only bind to the caecum while EtMIC1, EtMIC2, and EtAMA1 did not bind to any other intestinal tissues. Anti-serum to EtMIC3 was able to block the invasion of sporozoites with a blocking rate of 66.3%. The receptors for EtMIC3 were BCL2-associated athanogene 1 (BAG1) and Endonuclease polyU-specific-like (ENDOUL), which were mainly distributed in the caecum. BAG1 and ENDOUL receptor antiserum reduced weight loss and oocyst output following E. tenella infection, showing partial inhibition of E. tenella infection. These data elucidate the mechanism of site specificity for Eimeria infection and reveal a potential therapeutic avenue.

Abstract Haemonchus contortus has evolved highly integrated and sophisticated mechanisms to promote coexistence with hosts. The excretory-secretory (ES) products generated by this parasite contribute to the regulation of the host immune response to facilitate immune evasion and induce chronicity, but the proteins responsible for this process and the exact cellular mechanisms have yet to be defined. In this study, we identified 114 H. contortus ES proteins (HcESPs) interacting with host T cells and 15 T cell binding receptors via co-immunoprecipitation and shotgun liquid chromatography-tandem mass spectrometry analysis. Based on bioinformatics analysis, we demonstrated that HcESPs could inhibit T cell viability, induce cell apoptosis, suppress T cell proliferation and cause cell cycle arrest. Furthermore, the stimulation of HcESPs exerted critical control effects on T cell cytokine production profiles, predominantly promoting the secretion of interleukin (IL)-10, IL-17A and transforming growth factor-β1 and inhibiting IL-2, IL-4 and interferon-γ production. Collectively, these findings may provide insights into the interaction between ES proteins and key host effector cells, enhancing our understanding of the molecular mechanism underlying parasite immune evasion and providing new clues for novel vaccine development.

Abstract Background RNA interference (RNAi) is an important tool to determine the role of genes. RNAi has been widely used to downregulate target molecules, resulting in the reduction of mRNA for protein expression. Matrix metalloprotease 12A (MMP-12) is known to have important roles during embryonic development, organ morphogenesis and pathological processes in animals. However, MMP-12 from Haemonchus contortus has not been characterized. Methods Haemonchus contortus MMP-12 gene was cloned and recombinant protein of MMP-12 (rHc-MMP-12) was expressed. Binding activities of rHc-MMP-12 to goat peripheral blood mononuclear cells (PBMCs) were assessed by immunofluorescence assay (IFA) and the immuno-regulatory effects of rHc-MMP-12 on cell proliferation and nitric oxide production were observed by co-incubation of rHc-MMP-12 with goat PBMCs. Furthermore, a soaking method was used to knockdown the expression of Hc-MMP12 gene using three siRNA, targeting different regions of the gene and infectivity of effective siRNA on the development of H. contortus was evaluated in goat. Results rHc-MMP-12 was successfully expressed in an expression vector as well as the tissues of the cuticle of adult H. contortus worms and a successful binding with PBMCs surface were observed. Increased cellular proliferation and nitric oxide production by goat PBMCs was observed in a dose-dependent manner. Quantitative real time PCR (qRT-PCR) results confirmed the successful silencing of Hc-MMP-12 gene in siRNA of 1, 2 and 3 treated third-stage larvae (L3) of H. contortus in vitro. The most efficient qRT-PCR-identified siRNA template was siRNA-2, with a 69% suppression rate compared to the control groups. Moreover, in an in vivo study, silencing of the Hc-MMP-12 gene by siRNA-2 reduced the number of eggs (54.02%), hatchability (16.84%) and worm burden (51.47%) as compared to snRNA-treated control group. In addition, a shorter length of worms in siRNA-2-treated group was observed as compared to control groups. Conclusions Our results indicate that siRNA-mediated silencing of Hc-MMP-12 gene in H. contortus significantly reduce the egg counts, larval hatchability, and adult worm counts and sizes. The findings of the present study demonstrate important roles of Hc-MMP-12 in the development of H. contortus.

Abstract Background Methyltransferases (MTFs) are broad range of enzymes, which are ubiquitously expressed in diverse organisms ranging from bacteria to animals. MTFs proteins have been associated with various biological/cellular processes including transcriptional regulation, subcellular protein and RNA localization, signal transduction and DNA-damage repair. However, the role of MTFs in immune mechanism during host–parasite interaction has not been addressed yet. Results An open reading frame (764 bp) of methyltransferase-type 12 gene of H. contortus denoted as HcMTF-12, was successfully cloned using reverse transcriptase-polymerase chain reaction (RT-PCR) followed by prokaryotic expression in Escherichia coli BL21 (DE3 strain). The recombinant HcMTF-12 protein (rHcMTF-12) was about 47 kDa along with a fusion vector protein of 18 kDa. Immunoblot results identified the native protein MTF-12 with antibodies produced in rats against rHcMT-12, whereas rHcMTF-12 protein was recognized with sera of goat experimentally infected with H. contortus. Immunohistochemical analysis revealed that the native MTF-12 protein was mainly located in the periphery (cuticle) of parasite sections as well as within the pharynx and intestinal region. An immunofluorescence assay validated that rHcMTF-12 attached to the surface of goat PBMCs. Furthermore, the cytokines transcription of IL-2, IFN-γ and IL-4 transcripts of PBMCs incubated with rHcMTF-12 were enhanced in a dose-dependent manner. The secretion of TGF-β1 and IL-10 was significantly decreased. However, IL-6 production was not significantly different as compared to the control groups. Moreover, the migration activity and nitric oxide (NO) production by PBMCs were induced considerably, whereas the proliferation of PBMCs cells was negatively affected when incubated with the rHcMTF-12 protein. Conclusions Our findings suggest that HcMTF-12 significantly mediated the functions of PBMCs, and it might be a potential candidate for therapeutic interventions against haemonchosis.

Abstract Background Haemonchus contortus (H. contortus) is one of the most important parasites that cause huge economic losses to small ruminant industry worldwide. Effective prognosis and treatment depend upon the early diagnosis of H. contortus infection. To date, no widely-approved methods for the identification of prepatent H. contortus infection are available to identify prepatent H. contortus infection properly. The aim of this study was to evaluate the diagnostic potential of recombinant cold shock H. contortus protein (rHc-CS) during early and late infections of H. contortus in goat. Results Purified rHc-CS exhibited a clear band, with a molecular weight about 38 kDa. H. contortus eggs were not detected by fecal egg count technique from feces collected at 0 to 14 days post infection (D.P.I). However, eggs were detected at 21, 28 and 35 D.P.I. Hence, results of immunoblotting assay showed specific anti rHc-CS antibody detection in all goat sera collected at early stage (14 D.P.I) and late stage (21–103 D.P.I) of H. contortus infection. Furthermore, no cross reactivity was observed against Trichinella spiralis, Fasciola hepatica and Toxoplasma gondii or uninfected goats. Among several evaluated rHc-CS indirect-ELISA format variables, favorable antigen coating concentration was found 0.28 μg/well at 37 °C 1 h and overnight at 4 °C. Moreover, optimum dilution ratio of serum and rabbit anti-goat IgG was recorded as 1:100 and 1:4000, respectively. The best blocking buffer was 5% Bovine Serum Albumin (BSA) while the best time for blocking, serum incubation and TMB reaction were recorded as 60, 120 and 10 min, respectively. The cut-off value for positive and negative interpretation was determined as 0.352 (OD450). The diagnostic specificity and sensitivity of the rHc-CS, both were recorded as 100%. Conclusion These results validated that rHc-CS is a potential immunodiagnostic antigen to detect the specific antibodies during early and late H. contortus infections in goat.

Caused by Toxoplasma gondii, toxoplasmosis has aroused great threats to public health around the world. So far, no effective vaccine or drug is commercially available, and the demands for a safe and effective therapeutic strategy have become more and more urgent. In the current study, we constructed a DNA vaccine encoding T. gondii ribosomal P2 protein (TgP2) and denoted as TgP2-pVAX1 plasmid. To improve the immunoprotection, nanomaterial poly-lactic-co-glycolic acid (PLGA) and chitosan were used as the delivery vehicle to construct TgP2-pVAX1/PLGA and TgP2-pVAX1/CS nanospheres. Before vaccinations in BALB/c mice, TgP2-pVAX1 plasmids were transiently transfected into Human Embryonic Kidney (HEK) 293-T cells, and the expression of the eukaryotic plasmids was detected by laser confocal microscopy and Western blotting. Then the immunoprotection of naked DNA plasmids and their two nano-encapsulations were evaluated in the laboratory animal model. According to the investigations of antibody, cytokine, dendritic cell (DC) maturation, molecule expression, splenocyte proliferation, and T lymphocyte proportion, TgP2-pVAX1 plasmid delivered by two types of nanospheres could elicit a mixed Th1/Th2 immune response and Th1 immunity as the dominant. In addition, TgP2-pVAX1/PLGA and TgP2-pVAX1/CS nanospheres have great advantages in enhancing immunity against a lethal dose of T. gondii RH strain challenge. All these results suggested that TgP2-pVAX1 plasmids delivered by PLGA or chitosan nanomaterial could be promising vaccines in resisting toxoplasmosis and deserve further investigations and applications.

Trehalose phosphate synthase (TPS), a key enzyme in trehalose synthesis, is not present in mammals but critical to the viability of a wide range of lower organisms. However, almost nothing is known about the function of Hc-TPS (GT1-TPS structural domain protein from Haemonchus contortus). In this study, Hc-TPS gene was cloned and the recombinant protein (rHc-TPS) was expressed and purified. The quantitative real-time PCR (qPCR) results showed that Hc-TPS was transcribed at different stages of H. contortus, with higher levels of transcription at the molting and embryo stages. Immunofluorescence analysis showed that Hc-TPS was widely distributed in adults, but the expression was mainly localized on the mucosal surface of the intestine as well as in the embryos of female worms. The impacts of rHc-TPS on peripheral blood mononuclear cell (PBMC) proliferation, nitric oxide (NO) generation, transcriptional expression of cytokines, and related pathways were examined by co-incubating rHc-TPS with goat PBMCs. The results showed that rHc-TPS significantly inhibited PBMC proliferation and NO secretion in a dose-dependent manner. We also found that rHc-TPS activated the interleukin (IL)-10/signal transducer and activator of transcription 3/suppressor of cytokine signaling 3 (IL-10/STAT3/SOCS3) axis and significantly promoted SOCS3 expression, while inhibiting interferon-gamma (INF-γ), IL-4, IL-9, and IL-2 pathways. Our findings may contribute to understanding the immune evasion mechanism for the parasite during host–parasite interactions and also help to provide ideas for discovering new drug targets.

Zoysia japonica is an important warm-season turfgrass used worldwide. The decreased aesthetic quality and functionality during leaf senescence hamper its further utilization. However, information about the transcriptional mechanism and genes involved in leaf senescence in Z. japonica needs to be more extensive. Therefore, to better understand leaf senescence in Z. japonica, we investigated the integrated analysis of chlorophyll fluorescence test (JIP-test) and RNA sequencing (RNA-seq) of mature and senescent leaves. First, we identified 22,049 genes, of which 4038 were differentially expressed genes (DEGs). The results for gene expression profiles were evaluated using quantitative real-time PCR. A total of 2515 genes have homologous genes in other plants. The matched known-function SAGs are mainly involved in chlorophyll degradation and plant hormone response. A total of 539 differentially expressed transcription factor genes, including AP2/ERF-ERF, NAC, WRKY, bHLH, and MYB, were identified to be associated with leaf senescence. Next, senescence represses chlorophyll biosynthesis while upregulating chlorophyll degradation. Senescence harms the integrity and functionality of PSII, PSI, and the intersystem electron transport chain. In addition, IAA biosynthesis was inhibited, whereas ABA and ET biosynthesis were activated in leaf senescence, and senescence activates signal transduction of IAA, ABA, and ET. These findings add to our understanding of the regulatory mechanism of leaf senescence. The senescence-associated genes are candidate targets for providing new insight into leaf senescence modeling in Z. japonica. They provided a theoretical foundation to reveal the functions of senescence-associated genes and chlorophyll catabolic genes involved in leaf senescence.

The quasi-zero stiffness (QZS) isolator shows excellent characteristics of low-frequency vibration isolation. However, the jump behavior caused by the strong nonlinearity is a primary reason for the failure of QZS isolators. In order to grasp the effective frequency range and failure mechanism of a horizontal QZS isolator comprehensively, the dynamics of the isolator were studied in the following two cases. In the first case, the isolator is subject to a base displacement excitation; in the second case, the isolator is installed on a linear structure that is subject to a harmonic force. The nonlinear algebraic equations describing the steady-state response of the two systems were derived via the complexification-averaging method, and the results obtained using the derived expressions were verified by comparing the results of the complexification-averaging method and the Runge–Kutta method. The effective frequency ranges of the isolator were then obtained, and the jump phenomena in the response amplitude induced by the strong nonlinearity of the isolator were analyzed. The results show that when the excitation amplitude is small, the vibration isolation system does not exhibit jumping behavior and the effective frequency range is relatively wide. With increases in the excitation amplitude, the system can exhibit jumping behavior when an additional impact load is considered, and this phenomenon leads to a narrowing of the effective frequency range. The characteristics of the jump phenomena produced in the two cases were analyzed, and the differences in the jump behaviors were elucidated. Furthermore, the effect of the isolator parameters on the effective frequency range was investigated.

This study proposed a novel experimental platform to conduct dynamic loading tests of a truncated model steel catenary riser (SCR) within the touchdown zone (TDZ). The facilities of the platform, including a soil tank, a loading system, and a soil stirring system, were introduced in detail. The parameters of the test were determined through the simulation of an in situ riser. A steel pipe was adopted as the model riser, with its outer diameter equaling that of the prototype SCR. Before executing the dynamic loadings, the model riser developed its static configuration under the submerged weight and applied bending moment. Subsequently, cyclic vertical and lateral displacement loads were applied to the truncated point. The test results showed that when the vertical loading amplitude increased from 200 mm to 300 mm, the stress ranges at the front of the model riser increased by more than 100%, whereas the stress range only differed by less than 5% under different loading periods. Numerical models of the SCR were built based on the vector form intrinsic finite element (VFIFE) method. High similarities between the test and simulation results proved the reliability of the nonlinear soil model and the numerical model. During the test, a seabed trench was developed with a depth of 0.71 D and a width of 0.48 D, and its shape was similar to the in situ trench.

Pheophytinase (PPH), the phytol hydrolase, plays important roles in chlorophyll degradation. Nevertheless, little attention has been paid to the PPHs in warm-season grass species; neither its detailed function in photosynthesis has been systematically explored to date. In this study, we isolated ZjPPH from Zoysia japonica, an excellent warm-season turfgrass species. Quantitative real-time PCR analysis and promoter activity characterization revealed that the expression of ZjPPH could be induced by senescence, ABA, and dark induction. Subcellular localization observation proved that ZjPPH was localized in the chloroplasts. Overexpression of ZjPPH accelerated the chlorophyll degradation and rescued the stay-green phenotype of the Arabidopsis pph mutant. Moreover, ZjPPH promoted senescence with the accumulation of ABA and soluble sugar contents, as well as the increased transcriptional level of SAG12 and SAG14. Transmission electron microscopy investigation revealed that ZjPPH caused the decomposition of chloroplasts ultrastructure in stable transformed Arabidopsis. Furthermore, chlorophyll a fluorescence transient measurement analysis suggested that ZjPPH suppressed photosynthesis efficiency by mainly suppressing both photosystem II (PSII) and photosystem I (PSI). In conclusion, ZjPPH plays an important role in chlorophyll degradation and senescence. It could be a valuable target for genetic editing to cultivate new germplasms with stay-green performance and improved photosynthetic efficiency.

Gliomas exhibit high intra-tumoral histological and molecular heterogeneity. Introducing stereotactic biopsy, we achieved a superior molecular analysis of glioma using O-(2-18F-fluoroethyl)-L-tyrosine (FET)-positron emission tomography (PET) and diffusion-weighted magnetic resonance imaging (DWI). Patients underwent simultaneous DWI and FET-PET scans. Correlations between biopsy-derived tumor tissue values, such as the tumor-to-background ratio (TBR) and apparent diffusion coefficient (ADC)/exponential ADC (eADC) and histopathological diagnoses and those between relevant genes and TBR and ADC values were determined. Tumor regions with human telomerase reverse transcriptase (hTERT) mutation had higher TBR and lower ADC values. Tumor protein P53 mutation correlated with lower TBR and higher ADC values. α-thalassemia/mental-retardation-syndrome-X-linked gene (ATRX) correlated with higher ADC values. 1p/19q codeletion and epidermal growth factor receptor (EGFR) mutations correlated with lower ADC values. Isocitrate dehydrogenase 1 (IDH1) mutations correlated with higher TBRmean values. No correlation existed between TBRmax/TBRmean/ADC/eADC values and phosphatase and tensin homolog mutations (PTEN) or O6-methylguanine-DNA methyltransferase (MGMT) promoter methylation. Furthermore, TBR/ADC combination had a higher diagnostic accuracy than each single imaging method for high-grade and IDH1-, hTERT-, and EGFR-mutated gliomas. This is the first study establishing the accurate diagnostic criteria for glioma based on FET-PET and DWI.

Trichinella spiralis is mammalian skeletal muscles parasite which may cause trichinellosis in animals and humans. Gamma interferon inducible lysosomal thiol reductase (GILT) is a widespread superfamily which plays key role in processing and presentation of MHC class II restricted antigen by catalyzing disulfide bond reduction. There are no reports about GILT in T. spiralis. In present study, GILT from T. spiralis (Tsp-GILT) was cloned, analyzed by multiple-sequence alignment, and predicted by 3D structure model. Recombinant Tsp-GILT (about 46 kDa) was efficiently expressed in Escherichia coli and thiol reductase activity suggested that in acidic environment the addition of a reducing agent is needed. Soaking method was used to knockdown expression of Tsp-GILT using small interference RNA (siRNA). Immunofluorescence assay confirmed the transformation of siRNA into muscle larva (ML) and new born larva (NBL). Quantitative real time-PCR (QRT-PCR) analysis revealed that transcription level of Tsp-GILT mRNA can be up-regulated by stimulation of mouse IFN-γ and down-regulated by siRNA2 in vitro. NBLs soaked with siRNA2 showed 32.3% reduction in the generation of MLs. MLs soaked with siRNA2 showed 26.2% reduction in the next generation of MLs, but no significant effect was observed on adult worms or NBLs. These findings concluded that GILT may play important roles in the development of T. spiralis parasite.

Avian Eimeria species vary in their replication location, fecundity, and pathogenicity. They are required to complete the development within the limited space of host intestines, and some synergistic or antagonistic effects occur among different Eimeria species. This study evaluated the impact of Eimeria mitis on the outcome of Eimeria necatrix or Eimeria tenella challenge infection. The severity of E. mitis/E. necatrix and E. mitis/E. tenella mixed infections were quantified by growth performance evaluation, survival rate analysis, lesion scoring, blood stool scoring, and oocyst output counting. The presence of E. mitis exacerbated the outcome of co-infection with E. tenella, causing high mortality, intestinal lesion score, and oocyst production. However, E. mitis/E. tenella co-infection had little impact on the body weight gain compared to individual E. tenella infection. In addition, the presence of E. mitis appeared not to enhance the pathogenicity of E. necatrix, although it tends to inhibit the growth of challenged birds and facilitate oocyst output and mortality in an E. mitis/E. necatrix co-infection model. Collectively, the results suggested a synergistic relationship between E. mitis and E. tenella/E. necatrix when sharing the same host. The presence of E. mitis contributed to disease pathology induced by E. tenella and might also advance the impact of E. necatrix in co-infections. These observations indicate the importance of accounting for differences in the relationships among different Eimeria species when using mixed infection models.

Toxoplasma gondii (T. gondii) is an intracellular parasitic protozoan that can cause serious public health problems. However, there is no effectively preventive or therapeutic strategy available for human and animals. In the present study, we developed a DNA vaccine encoding T. gondii oxidoreductase from short-chain dehydrogenase/reductase family (TgSDRO-pVAX1) and then entrapped in chitosan and poly lactic-co-glycolic acid (PLGA) to improve the efficacy. When encapsulated in chitosan (TgSDRO-pVAX1/CS nanospheres) and PLGA (TgSDRO-pVAX1/PLGA nanospheres), adequate plasmids were loaded and released stably. Before animal immunizations, the DNA vaccine was transfected into HEK 293-T cells and examined by western blotting and laser confocal microscopy. Th1/Th2 cellular and humoral immunity was induced in immunized mice, accompanied by modulated secretion of antibodies and cytokines, promoted the maturation and MHC expression of dendritic cells, and enhanced the percentages of CD4+ and CD8+ T lymphocytes. Immunization with TgSDRO-pVAX1/CS and TgSDRO-pVAX1/PLGA nanospheres conferred significant immunity with lower parasite burden in the mice model of acute toxoplasmosis. Furthermore, our results also lent credit to the idea that TgSDRO-pVAX1/CS and TgSDRO-pVAX1/PLGA nanospheres are substitutes for each other. In general, the current study proposed that TgSDRO-pVAX1 with chitosan or PLGA as the delivery vehicle is a promising vaccine candidate against acute toxoplasmosis.

Glioblastoma is the most common primary brain malignancy with limited treatment options. EphA2 is a tumor-associated-antigen overexpressed in glioblastoma. Pre-clinical studies have demonstrated the promise of EphA2-redirected CAR T-cells against glioblastoma. We conduct the first-in-human trial of EphA2-redirected CAR T-cells in patients with EphA2-positive recurrent glioblastoma and report the results of three patients enrolled as the first cohort receiving the starting dosage (1×106 cells/kg). A single infusion of EphA2-redirected CAR T-cells was administrated intravenously, with the lymphodepletion regimen consisting of fludarabine and Cyclophosphamide. In two patients, there was grade 2 cytokine release syndrome accompanied by pulmonary edema, which resolved completely with dexamethasone medication. Except that, there was no other organ toxicity including neurotoxicity. In both the peripheral blood and cerebral-spinal-fluid, we observed the expansion of CAR T-cells which persisted for more than four weeks. In one patient, there was a transit diminishment of the tumor. Among these three patients, one patient reported SD and two patients reported PD, with overall survival ranging from 86 to 181 days. At the tested dose level (1×106 cells/kg), intravenously infusion of EphA2-rediretected CAR T-cells were preliminary tolerable with transient clinical efficacy. Future study with adjusted dose and infusion frequency of CAR T-cells is warranted.Trial Registration NumbersNCT 03423992

Abstract Haemonchus contortus (H. contortus) has evolved sophisticated evasion mechanisms to ensure their survival, including generating excretion and secretion products (ESPs) to regulate the secretion of host cytokines. Interleukin 4 (IL4) is a classic T-helper cell type 2 (Th2)-type cytokine that plays an irreplaceable role against nematode infection. In this study, three proteins, glutathione S-transferase domain containing protein (HcGST), transthyretin domain containing protein (HcTTR) and calponin actin-binding domain containing protein (HcCab), were identified to bind to goat IL4 by co-immunoprecipitation (Co-IP) assays and yeast two-hybrid screening. Additionally, cell proliferation analysis showed that HcTTR blocked the IL4-induced proliferation of peripheral blood mononuclear cells in goats, while HcGST and HcCab did not. In addition, HcTTR could also downregulate the transcription of candidate genes in the IL4-induced JAK/STAT pathway. These results indicated that HcTTR is a novel antagonist against goat IL4 from HcESPs, and this information could improve our understanding of the relationship between host cytokines and parasite infections.

Abstract Background Hepatocellular carcinoma-associated antigen 59 (HCA59), which is one of the most important excretory/secretory products of Haemonchus contortus (HcESPs), is known to have antigenic functions. However, its immunomodulatory effects on host cells are poorly understood. Methods Here, we cloned the HCA59 gene and expressed the recombinant protein of HCA59 (rHCA59). Binding activities of rHCA59 to goat peripheral blood mononuclear cells (PBMCs) and dendritic cells (DCs) were checked by immunofluorescence assay (IFA) and the immunoregulatory effects of rHCA59 on cytokine secretions, cell migration, cell proliferation, nitric oxide production, and changes in expression of genes in related pathways were observed by co-incubation of rHCA59 with goat PBMCs and DCs. Monocyte phagocytosis and characterization of goat blood DC subsets were detected by flow cytometry. Results The IFA results revealed that rHCA59 could bind to PBMCs and DCs. Treatment of PBMCs with rHCA59 significantly increased cellular proliferation and NO production in a dose–dependent manner, while cell migration was vigorously blocked. Treatment with rHCA59 significantly suppressed monocytes phagocytosis. The quantity of surface marker CD80 on DCs increased significantly after rHCA59 treatment. In addition, the expression of genes included in the WNT pathway was related to the differentiation and maturation of DCs, and the production of IL-10 and IL-17 produced by PBMCs was altered. Conclusions Our findings illustrated that rHCA59 could enhance host immune responses by regulating the functions of goat PBMCs and DCs, which would benefit our understanding of HCA59 from parasitic nematodes contributing to the mechanism of parasitic immune evasion.

In this paper, we propose a weakly supervised landmark-region-based convolutional neural network (LR-CNN) framework to detect facial component and landmark simultaneously. Most of the existing course-to-fine facial detectors fail to detect landmark accurately without lots of fully labeled data, which are costly to obtain. We can handle the task with a small amount of finely labeled data. First, deep convolutional generative adversarial networks are utilized to generate training samples with weak labels, as data preparation. Then, through weakly supervised learning, our LR-CNN model can be trained effectively with a small amount of finely labeled data and a large amount of generated weakly labeled data. Notably, our approach can handle the situation when large occlusion areas occur, as we localize visible facial components before predicting corresponding landmarks. Detecting unblocked components first helps us to focus on the informative area, resulting in a better performance. Additionally, to improve the performance of the above tasks, we design two models as follows: 1) we add AnchorAlign in the region proposal networks to accurately localize components and 2) we propose a two-branch model consisting classification branch and regression branch to detect landmark. Extensive evaluations on benchmark datasets indicate that our proposed approach is able to complete the multi-task facial detection and outperforms the state-of-the-art facial component and landmark detection algorithms.

Collecting a large dataset of real infrared (IR) images is expensive, time-consuming, and even unavailable in some specific scenarios. With recent progress in machine learning, it has become more feasible to replace real IR images with qualified synthetic IR images in learning-based IR systems. However, this alternative may fail to achieve the desired performance, due to the gap between real and synthetic IR images. Inspired by adversarial learning for image-to-image translation, we propose the Synthetic IR Refinement Generative Adversarial Network (SIR-GAN) to narrow this gap. By learning the bidirectional mappings between two unpaired domains, the realism of the simulated IR images generated from the IR Simulator are significantly improved, where the source domain contains a large number of simulated IR images, where the target domain contains a limited quantity of real IR images. Specifically, driven by the idea of transferring infrared characteristic and protect target semantic information simultaneously, we propose a SIR refinement loss to consider an infrared loss and a structure loss further to the adversarial loss and the consistency loss. To further reduce the gap, stabilize training, and avoid artefacts, we modify the proposed algorithm by developing a training strategy, adding the U-net in the generators, using the dilated convolution in the discriminators and invoking the N-Adam acts as the optimizer. Qualitative, quantitative, and ablation study experiments demonstrate the superiority of the proposed approach compared with the state-of-the-art techniques in terms of realism and fidelity. In addition, our refined IR images are evaluated in the context of a feasibility study, where the accuracy of the trained classifier is significantly improved by adding our refined data into a small real-data training set

Eimeria tenella is a protozoan parasite endemic in chickens and is one of the causative agents of avian coccidiosis. The aim of this research was to determine if poly (D, L-lactide-co-glycolide) (PLGA) nanoparticles carrying recombinant TA4 protein of E. tenella (rEtTA4) could improve the level of protective immunity against E. tenella challenge. Recombinant TA4 protein was expressed and purified. Poly (D, L-lactide-co-glycolide) loaded with rEtTA4 (PLGA-rEtTA4) nanoparticles was prepared and was delivered to 2-week-old layer chickens via intramuscular inoculation. Chickens injected with PBS and PLGA nanoparticles were served as control groups. The rEtTA4 and PLGA-rEtTA4 nanoparticles induced changes of serum cytokines, IgY levels, and T lymphocytes subpopulation, and the protective efficacy against E. tenella challenge was evaluated. Results showed that both rEtTA4 and PLGA-rEtTA4 vaccination groups induced significantly higher levels of specific EtTA4 IgY antibody and IL-17 and higher proportion of CD8+ T lymphocytes. However, no significant differences were observed in the proportion of CD4+ T lymphocytes compared with the PBS control. Chickens immunized with rEtTA4 and PLGA-rEtTA4 prominently increased the BW gains and decreased oocyst output compared with chickens immunized with PBS and PLGA after oral challenge with E. tenella. Poly (D, L-lactide-co-glycolide) encapsulated rEtTA4 nanoparticles–immunized chickens significantly induced higher levels of interferon gamma, IL-6, and IL-17 and a little bit higher proportion of CD8+ T lymphocytes compared with rEtTA4 subunit vaccine–immunized chickens. Thus, PLGA encapsulated rEtTA4 nanoparticles appeared to have great potential to enhance the immune response and improved the protective efficacy against E. tenella infection. Our results provided available protective subunit vaccine rEtTA4 and PLGA loaded with rEtTA4 nanoparticles against coccidiosis and suggested that PLGA nanoparticles could be an effective adjuvant to enhance the protective efficacy of rEtTA4 subunit vaccine.

Avian coccidiosis brings tremendous economic loss to the poultry industry worldwide. The third generation vaccine, including subunit and DNA vaccines, exhibited promising developmental prospects. In a previous study, we found rhomboid-like protein 3 of Eimeria maxima (EmROM3) was involved in infections by Eimeria species. However, the protective efficacy of EmROM3 against Eimeria maxima (E. maxima) remains unknown. In this study, chickens were intramuscularly immunized with the recombinant protein EmROM3 (rEmROM3) or pVAX1-EmROM3 to determine the EmROM3-induced immune response. The induced humoral immune response was determined by measuring serum IgG antibody levels in immunized chickens. The induced cellular immune response was detected by measuring the transcription level of immune related cytokines and the proportion of T cell subsets of the immunized chickens. Finally, the protective efficacy of the EmROM3 vaccine against E. maxima was evaluated by immunization-challenge trials. Results revealed that the purified rEmROM3 reacted with chicken anti-E. maxima serum. The recombinant plasmid of pVAX1-EmROM3 was transcribed and translated in the injected muscle from the vaccinated chickens. In experimental groups, the IgG titers, proportions of CD4+ and CD8+ T cells, and transcription level of splenic cytokines were significantly increased compared with the control groups. The immunization-challenge trial revealed that immunization with rEmROM3 or pVAX1-EmROM3 led to restored weight gain, alleviated enteric lesion, decreased oocyst output as well as the higher anticoccidial index (ACI), indicating partial protection against E. maxima. These results indicate that EmROM3 is an effective candidate antigen for developing novel vaccines against infection by E. maxima.

Kentucky bluegrass (Poa pratensis L.) is an excellent cool-season turfgrass utilized widely in Northern China. However, turf quality of Kentucky bluegrass declines significantly due to drought. Ethephon seeds-soaking treatment has been proved to effectively improve the drought tolerance of Kentucky bluegrass seedlings. In order to investigate the effect of ethephon leaf-spraying method on drought tolerance of Kentucky bluegrass and understand the underlying mechanism, Kentucky bluegrass plants sprayed with and without ethephon are subjected to either drought or well watered treatments. The relative water content and malondialdehyde conent were measured. Meanwhile, samples were sequenced through Illumina. Results showed that ethephon could improve the drought tolerance of Kentucky bluegrass by elevating relative water content and decreasing malondialdehyde content under drought. Transcriptome analysis showed that 58.43% transcripts (254,331 out of 435,250) were detected as unigenes. A total of 9.69% (24,643 out of 254,331) unigenes were identified as differentially expressed genes in one or more of the pairwise comparisons. Differentially expressed genes due to drought stress with or without ethephon pre-treatment showed that ethephon application affected genes associated with plant hormone, signal transduction pathway and plant defense, protein degradation and stabilization, transportation and osmosis, antioxidant system and the glyoxalase pathway, cell wall and cuticular wax, fatty acid unsaturation and photosynthesis. This study provides a theoretical basis for revealing the mechanism for how ethephon regulates drought response and improves drought tolerance of Kentucky bluegrass.

Early diagnosis of cancer is of paramount significance for the therapeutic intervention of cancers. Although the detection of circulating cell-free DNA (cfDNA) has emerged as a promising, minimally invasive approach for early cancer diagnosis, there is an urgent need to develop a highly sensitive and rapid method to precisely identify plasma cfDNA from clinical samples. Herein, we report a robust fluorescent “turn-on” clutch probe based on non-emissive QDs-Ru complexes to rapidly recognize EGFR gene mutation in plasma cfDNA from lung cancer patients. In this system, the initially quenched emission of QDs is recovered while the red emission of Ru(II) complexes is switched on. This is because the Ru(II) complexes can specifically intercalate into the double-stranded DNA (dsDNA) to form Ru-dsDNA complexes and simultaneously liberate free QDs from the QDs-Ru complexes, which leads to the occurrence of an overlaid red fluorescence. In short, the fluorescent “turn-on” clutch probe offers a specific, rapid, and sensitive paradigm for the recognition of plasma cfDNA biomarkers from clinical samples, providing a convenient and low-cost approach for the early diagnosis of cancer and other gene-mutated diseases.

Electrothermal micromirrors have become an important type of micromirrors due to their large angular scanning range and large linear motion. Typically, electrothermal micromirrors do not have a torsional bar, so they can easily generate linear motion. In this paper, electrothermal micromirrors based on different thermal actuators are reviewed, and also the mechanisms of those actuators are analyzed, including U-shape, chevron, thermo-pneumatic, thermo-capillary and thermal bimorph-based actuation. Special attention is given to bimorph based-electrothermal micromirrors due to their versatility in tip-tilt-piston motion. The exemplified applications of each type of electrothermal micromirrors are also presented. Moreover, electrothermal micromirrors integrated with electromagnetic or electrostatic actuators are introduced.

The α/β-hydrolase domain (ABHD) proteins belonging to α/β-hydrolase (ABH) superfamily are ubiquitously distributed throughout all the organisms, and their functional roles have been implicated in energy metabolism, cell signaling, growth and development. In our preliminary work, we identified a novel ABHD protein derived from Haemonchus contortus excretory-secretory (ES) proteins (HcESPs) that interacted with host T cells. Here, we demonstrated that H. contortus ABHD (HcABHD) protein, expressed in all life-cycle stages of H. contortus, is a mammalian ABHD17 homolog with immunodiagnostic utility and lipase activity. Given its catalytic activities and immunomodulatory potentials, we further investigated the functional diversity of HcABHD as an individual ES protein in parasite-host interactions. HcABHD protein may serve as depalmitoylase or thioesterase to suppress cell viability, inhibit cell proliferation, induce intrinsic and extrinsic T cell apoptosis, and cause cell cycle arrested at G1 phase. Moreover, recombinant HcABHD stimuli exerted critical controls on T cell cytokine production profiles, predominantly by inhibiting the secretions of interleukin (IL)-4, interferon-gamma (IFN-γ) and transforming growth factor-beta (TGF-β) 1, and promoting IL-10 production. As the immunomodulator acting at the parasite-host interface, HcABHD protein may have potential applications for the vaccine development of therapeutic intervention. Together, these findings may help illuminate the molecular and particularly immunomodulatory aspects of ES proteins and contribute to an enhanced understanding of parasite immune evasion in H. contortus-host biology.

Summary: Background: Adolescents and young adults might play a key role in the worldwide spread of Coronavirus Disease 2019 (COVID-19) because they are more likely to be involved in overseas study, business, work, and travel. However, the epidemiological and clinical characteristics remain unknown. Methods: We collected demographic, epidemiological, and clinical data from 46 confirmed COVID-19 patients aged 10 to 35 years from the Chongqing Three Gorges Central Hospital. Several key epidemiological parameters, asymptomatic cases, transmission to family members, and clinical characteristics at admission and during treatment were summarized. Results: Of 46 confirmed patients, 14 patients (30.4%) were aged between 10 and 24 years, and 24 (52.2%) patients were male. The estimated mean incubation period was 6.6 days (95% confidence interval [CI] 4.4–9.6). The median serial interval was 1.9 days (95% CI 0.4–6.2). Three of the asymptomatic cases showed transmission to their family members. Only one patient was identified as a severe case at admission. The common symptoms at admission were dry cough (34, 81.0%) and fever (29, 69.1%). Nearly 60% of the patients showed ground-glass opacity on chest computed tomography. Three patients developed acute kidney injury during treatment. Most of the patients (78.3%) recovered and were discharged by the end of the follow-up. Conclusions: This single-center study with a relatively small sample size showed that adolescent and young adult patients with COVID-19 had a long incubation period and a short serial interval. The transmission occurred from asymptomatic cases to family members. Fewer patients developed complications during treatment.

CD4+ T cells play critical roles in mediating adaptive immunity to a variety of pathogens. Recently, new subset of CD4+T named as T helper 9 cells that express the prototypical interleukin-9 (IL-9) cytokine have been recognized in human and mice models during different parasitic infections. Haemonchus contortus is a gastrointestinal nematode of small ruminants which cause high mortality in young animals. During infection, Excretory and Secretary Products (ESPs) are released in the host body. No other study has reported yet on immunomodulatory dynamics of H. contortus ESPs on Th9 immune response in vitro or in vivo. In this study, immunomodulatory effects of ESPs (5, 10, 20, 40, 80; μg/mL) incubated with goat PBMCs on Th9 cells, IL-9 immune response and TGF-β/Smad signaling regulator were evaluated in vitro. Moreover, for in vivo study, goats were infected with different doses (P-800, P-2400, and P-8000) of H. contortus infective larva (L3) and immunomodulatory effects on Th9 cells, IL-9 immune response and TGF-β/Smad signaling regulator were evaluated at 7, 10, 14, 18, 21, 28 Days Post Infection (DPI). Flow cytometry was performed to evaluate the effects on Th9 cells and quantitative real time polymerase chain reaction was performed to evaluate the IL-9 cytokine transcription level. Additionally, fecal egg counting was also performed in parallel to confirm the infection. All goats were dewormed at 29 DPI and all experiments were also performed at 35 DPI, one week post deworming. The finding indicated that 10, 20, 40, 80 μg/mL concentration of ESPs incubated with goat PBMCs showed significant increase in the production of Th9 cells, signature cytokine IL-9 and expression of TGF-β/Smad signaling regulator as compared to control group in vitro.All infected groups showed significant increase in production of Th9 cells and IL-9 cytokine and expression of TGF-β/Smad key genes at 18, 21, and 28 DPI as compared to control group. Likewise, at 14 DPI, P-2400 and P-8000 groups showed significant increase in production of Th9 cells, IL-9 cytokine and expression of TGF-β/Smad key genes. While at 10 DPI, production of Th9 cells and IL-9 was significantly increased in P-2400 & P-8000 groups, and at 7 DPI only P-8000 showed significantly increase in IL-9 production. No immunomodulatory effects were observed at 0 and 3 DPI. Additionally, significant gradually up-regulated key genes expression of TGF-β/Smad signaling regulator in all infected groups confirmed the above results. After deworming, production of Th9 cells, associated immune response and expression of signaling regulator in each group were significantly decreased. Based on this study, it is concluded that Th9 immune response was induced during H. contortus infection in goat by up-regulation of TGF-β/Smad signaling key genes.

Zoysia japonica is a warm-season turfgrass with a good tolerance and minimal maintenance requirements. However, its use in Northern China is limited due to massive chlorophyll loss in early fall, which is the main factor affecting its distribution and utilization. Although ethephon treatment at specific concentrations has reportedly improved stress tolerance and extended the green period in turfgrass, the potential mechanisms underlying this effect are not clear. In this study, we evaluated and analyzed chlorophyll changes in the physiology and transcriptome of Z. japonica plants in response to cold stress (4 °C) with and without ethephon pretreatment. Based on the transcriptome and chlorophyll content analysis, ethephon pretreatment increased the leaf chlorophyll content under cold stress by affecting two processes: the stimulation of chlorophyll synthesis by upregulating ZjMgCH2 and ZjMgCH3 expression; and the suppression of chlorophyll degradation by downregulating ZjPAO, ZjRCCR, and ZjSGR expression. Furthermore, ethephon pretreatment increased the ratio of chlorophyll a to chlorophyll b in the leaves under cold stress, most likely by suppressing the conversion of chlorophyll a to chlorophyll b due to decreased chlorophyll b synthesis via downregulation of ZjCAO. Additionally, the inhibition of chlorophyll b synthesis may result in energy redistribution between photosystem II and photosystem I.

This paper proposed a compact design of the subsea cobalt-rich crust mining vehicle with a general purpose support vessel for subsea resource exploration, sample collection, and research. The necessary functions were considered in the concept design, including walk, crushing/mining, sample collection, cutter head adaptation, vehicle orientation, crust texture measurement, awareness, positioning, and navigation. The prototype was tested in both tank and subsea environment. The sea trials were carried out with the support of a general purpose support vessel. The track design worked well in both the tank and subsea environment and the mining vehicle walked smoothly in the sea trial. The crust was crushed to the size of 2 mm and 10 mm with different cutting parameters and successfully collected by the jet pump, 6 kg in total. The crust texture was measured by the onboard sonar successfully and can be used for cutting parameter selection. The cameras captured the images of the subsea environment, but the actions of crushing and sample collection produced plumes, which blocked the camera vision. In the situation, the front image sonar can be used to keep the vehicle away from big rocks. The mining vehicle is not limited to the mining and sampling of subsea cobalt-rich crust. Most of the subsea solid resources on the seabed can be considered to use the compact mining vehicle for sampling and related research. The only issues to be considered are the crushing ability and sample size required.

Abstract Background Red clover (Trifolium pratense L.) is an important cool-season legume plant, which is the most widely planted forage legume after alfalfa. Although a draft genome sequence was published already, the sequences and completed structure of mRNA transcripts remain unclear, which limit further explore on red clover. Results In this study, the red clover transcriptome was sequenced using single-molecule long-read sequencing to identify full-length splice isoforms, and 29,730 novel isoforms from known genes and 2194 novel isoforms from novel genes were identified. A total of 5492 alternative splicing events was identified and the majority of alter spliced events in red clover was corrected as intron retention. In addition, of the 15,229 genes detected by SMRT, 8719 including 186,517 transcripts have at least one poly(A) site. Furthermore, we identified 4333 long non-coding RNAs and 3762 fusion transcripts. Conclusions We analyzed full-length transcriptome of red clover with PacBio SMRT. Those new findings provided important information for improving red clover draft genome annotation and fully characterization of red clover transcriptome.

Abstract Background Avian coccidiosis is often caused by co-infection with several species of Eimeria worldwide. Developing a multivalent vaccine with an antigen common to multiple Eimeria species is a promising strategy for controlling clinical common co-infection of Eimeria. In the previous study, 14–3-3 was identified as one of the immunogenic common antigen in E. tenella, E. acervulina and E. maxima. The aim of the present study was to evaluate the immunogenicity and protective efficacy of Ea14–3-3 in the form of DNA vaccine against infection with three species of Eimeria both individually and simultaneously. Results After vaccination with pVAX-Ea14–3-3, the Ea14–3-3 gene was transcribed and expressed in the injected muscles. Vaccination with pVAX-Ea14–3-3 significantly increased the proportion of CD4+ and CD8+ T lymphocytes and produced a strong IgY response in immunized chickens. Similarly, pVAX-Ea14–3-3 stimulated the chicken’s splenocytes to produce high levels of Th1-type (IFN-γ, IL-2) and Th2-type (IL-4) cytokines. The vaccine-induced immune response was responsible to increase weight gain, decreased the oocyst output, and alleviated enteric lesions significantly in immunized chickens as compared to control group, in addition to induce moderate anti-coccidial index (ACI). Conclusion These results indicate that Ea14–3-3 is highly immunogenic and capable to induce significant immune responses. Furthermore, Ea14–3-3 antigen can provide effective protection against infection with Eimeria tenella, Eimeria acervulina, Eimeria maxima both individually and in combination with three Eimeria species. Significant outcomes of our study provide an effective candidate antigen for developing a multivalent Eimeria vaccine against mixed infection with various Eimeria species under natural conditions.

Abstract Background Coccidiosis is recognised as a major parasitic disease in chickens. Eimeria maxima is considered as a highly immunoprotective species within the Eimeria spp. family that infects chickens. In the present research, the surface antigen gene of E. maxima (EmSAG) was cloned, and the ability of EmSAG to stimulate protection against E. maxima was evaluated. Methods Prokaryotic and eukaryotic plasmids expressing EmSAG were constructed. The EmSAG transcription and expression in vivo was performed based on the RT-PCR and immunoblot analysis. The expression of EmSAG in sporozoites and merozoites was detected through immunofluorescence analyses. The immune protection was assessed based on challenge experiments. Flow cytometry assays were used to determine the T cell subpopulations. The serum antibody and cytokine levels were evaluated by ELISA. Results The open reading frame (ORF) of EmSAG gene contained 645 bp encoding 214 amino acid residues. The immunoblot and RT-PCR analyses indicated that the EmSAG gene were transcribed and expressed in vivo. The EmSAG proteins were expressed in sporozoite and merozoite stages of E. maxima by the immunofluorescence assay. Challenge experiments showed that both pVAX1-SAG and the recombinant EmSAG (rEmSAG) proteins were successful in alleviating jejunal lesions, decreasing loss of body weight and the oocyst ratio. Additionally, these experiments possessed anticoccidial indices (ACI) of more than 170. Higher percentages of CD4+ and CD8+ T cells were detected in both EmSAG-inoculated birds than those of the negative control groups (P

Abstract Background Eimeria maxima initiates infection by invading the jejunal epithelial cells of chicken. However, the proteins involved in invasion remain unknown. The research of the molecules that participate in the interactions between E. maxima sporozoites and host target cells will fill a gap in our understanding of the invasion system of this parasitic pathogen. Methods In the present study, chicken jejunal epithelial cells were isolated and cultured in vitro. Western blot was employed to analyze the soluble proteins of E. maxima sporozoites that bound to chicken jejunal epithelial cells. Co-immunoprecipitation (co-IP) assay was used to separate the E. maxima proteins that bound to chicken jejunal epithelial cells. Shotgun LC-MS/MS technique was used for proteomics identification and Gene Ontology was employed for the bioinformatics analysis. Results The results of Western blot analysis showed that four proteins bands from jejunal epithelial cells co-cultured with soluble proteins of E. maxima sporozoites were recognized by the positive sera, with molecular weights of 70, 90, 95 and 130 kDa. The co-IP dilutions were analyzed by shotgun LC-MS/MS. A total of 204 proteins were identified in the E. maxima protein database using the MASCOT search engine. Thirty-five proteins including microneme protein 3 and 7 had more than two unique peptide counts and were annotated using Gene Ontology for molecular function, biological process and cellular localization. The results revealed that of the 35 annotated peptides, 22 (62.86%) were associated with binding activity and 15 (42.86%) were involved in catalytic activity. Conclusions Our findings provide an insight into the interaction between E. maxima and the corresponding host cells and it is important for the understanding of molecular mechanisms underlying E. maxima invasion.

Abstract Background This study not only evaluated the clinical effects of treatment using haploidentical hematopoietic stem cells (haplo-HSCs) combined with human umbilical cord mesenchymal stem cells (UC-MSCs) in patients with severe aplastic anemia (SAA), but also investigated the factors related to graft versus host disease (GVHD). Methods Cotransplantation of haplo-HSCs and UC-MSCs was performed in 24 SAA patients. The conditioning regimens consisted of rabbit anti-human T-lymphocyte immunoglobulin (ATG), cyclophosphamide, and fludarabine with or without busulfan. GVHD was prevented using cyclosporine A, ATG, anti-CD25 monoclonal antibody, and mycophenolate material. Results The incidence of acute GVHD was 50%. The incidence of severe acute GVHD was not related to gender, age, donor-recipient relations, and patient/donor pair, while patient/donor pair (r = 0.541, P = 0.022) was significantly correlated with incidence of chronic GVHD. Upon follow-up for a median of 13 months, 5 of the 24 patients (20.8%) were dead. The survival rates at 3 and 6 months in all patients were 87.5% (21/24) and 83.3% (20/24), respectively. Conclusion Cotransplantation of haplo-HSCs combined with UC-MSCs was an effective and safe approach for the treatment of patients with SAA. The appropriate conditioning regimen and early treatment for infection also played a critical role in the success of HSCT.

Eimeria maxima (E. maxima) is one of the most prevalent species that causes chicken coccidiosis on chicken farms. During apicomplexan protozoa invasion, rhomboid-like proteins (ROMs) cleave microneme proteins (MICs), allowing the parasites to fully enter the host cells, which suggests that ROMs have the potential to be candidate antigens for the development of subunit or DNA vaccines against coccidiosis. In this study, a recombinant protein of E. maxima ROM5 (rEmROM5) was expressed and purified and was used as a subunit vaccine. The eukaryotic expression plasmid of pVAX–EmROM5 was constructed and was used as a DNA vaccine. Chickens who were two weeks old were vaccinated with the rEmROM5 and pVAX–EmROM5 vaccines twice, with a one-week interval separating the vaccination periods. The transcription and expression of pVAX–EmROM5 in the injected sites were detected through reverse transcription PCR (RT-PCR) and Western blot (WB) assays. The cellular and humoral immune responses that were induced by EmROM5 were determined by detecting the proportion of CD4+ and CD8+ T lymphocytes, the cytokine levels, and the serum antibody levels. Finally, vaccination-challenge trials were conducted to evaluate the protective efficacy of EmROM5 in forms of the recombinant protein (rEmROM5) and in the DNA plasmid (pVAX–EmROM5) separately. The results showed that rEmROM5 was about 53.64 kDa, which was well purified and recognized by the His-Tag Mouse Monoclonal antibody and the chicken serum against E. maxima separately. After vaccination, pVAX–EmROM5 was successfully transcribed and expressed in the injected sites of the chickens. Vaccination with rEmROM5 or pVAX–EmROM5 significantly promoted the proportion of CD4+/CD3+ and CD8+/CD3+ T lymphocytes, the mRNA levels of the cytokines IFN-γ, IL-2, IL-4, IL-17, TNF SF15, and IL-10, and specific IgG antibody levels compared to the control groups. The immunization also significantly reduced the weight loss, oocyst production, and intestinal lesions that are caused by E. maxima infection. The anticoccidial index (ACI)s of the vaccinated groups were beyond 160, showing moderate protection against E. maxima infection. In summary, EmROM5 was able to induce a robust immune response and effective protection against E. maxima in chickens in the form of both a recombinant protein and DNA plasmid. Hence, EmROM5 could be used as a candidate antigen for DNA vaccines and subunit vaccines against avian coccidiosis.

The pathogen of toxoplasmosis, Toxoplasma gondii (T. gondii), is a zoonotic protozoon that can affect the health of warm-blooded animals including humans. Up to now, an effective vaccine with completely protection is still inaccessible. In this study, the DNA vaccine encoding T. gondii histone deacetylase SIR2 (pVAX1-SIR2) was constructed. To enhance the efficacy, chitosan and poly (d, l-lactic-co-glycolic)-acid (PLGA) were employed to design nanospheres loaded with the DNA vaccine, denoted as pVAX1-SIR2/CS and pVAX1-SIR2/PLGA nanospheres. The pVAX1-SIR2 plasmids were transfected into HEK 293-T cells, and the expression was evaluated by a laser scanning confocal microscopy. Then, the immune protections of pVAX1-SIR2 plasmid, pVAX1-SIR2/CS nanospheres, and pVAX1-SIR2/PLGA nanospheres were evaluated in a laboratory animal model. The in vivo findings indicated that pVAX1-SIR2/CS and pVAX1-SIR2/PLGA nanospheres could generate a mixed Th1/Th2 immune response, as indicated by the regulated production of antibodies and cytokines, the enhanced maturation and major histocompatibility complex (MHC) expression of dendritic cells (DCs), the induced splenocyte proliferation, and the increased percentages of CD4+ and CD8+ T lymphocytes. Furthermore, this enhanced immunity could obviously reduce the parasite burden in immunized animals through a lethal dose of T. gondii RH strain challenge. All these results propose that pVAX1-SIR2 plasmids entrapped in chitosan or PLGA nanospheres could be the promising vaccines against acute T. gondii infections and deserve further investigations.

Trichinellosis is a foodborne zoonotic disease caused by Trichinella spp., including Trichinella spiralis. In the present study, T. spiralis membrane-associated progesterone receptor component-2 (Ts-MAPRC2) gene was cloned and characterized using protein sequencing analysis. Furthermore, the expression, purification, immunoblot assay, binding ability with progesterone antibody, and immunofluorescence assay were performed. A direct effect of progesterone (P4) and mifepristone (RU486) on the Ts-MAPRC2 gene was determined using in vitro cell culture that showed different expression levels at all developmental stages (muscle larvae (ML), female adult worm (F-AL), male adult worm (M-AL), and newborn larvae (NBL)). Subsequently, the in vitro phenotypic effects of P4, RU486, and rTs-MAPRC2-Ab on F-AL and ML stages were measured. Later, the in vivo phenotypic effect and relative mRNA expression of mifepristone on the F-AL stage were studied. Our results revealed that the Ts-MAPRC2 gene is critical to maintaining pregnancy in the female adult worm (F-AL) of T. spiralis. The 300 ng/mL of P4 and 100 ng/mL of RU486 showed downregulation of the Ts-MAPRC2 gene in F-AL (p ≤ 0.05). This plays an important role in abortion and possibly decreases the worm burden of T. spiralis in the host. Only 30 ng/mL P4 showed significant upregulation in F-AL (p ≤ 0.05). The current study provides new insights regarding the antihormone (P4 and RU486) drug design and vaccine therapy of recombinant (rTs-MAPRC2) protein as well as their combined effects to control T. spiralis infection.

Abstract Background Haemonchosis is a disease of the small ruminant caused by a nematode parasite Haemonchus contortus, and it is most important and alarming challenges to the small ruminant’s production. The infection of the H. contortus could cause high economic losses worldwide. H. contortus is a blood feeding parasite which penetrates into the abomasal mucosa to feed the blood of the host and causing the anemia and decreased total plasma protein. Modulation and suppression of the immune response of the host by nematode parasites have been reported extensively, and the cysteine protease inhibitor (cystatin) is identified as one of the major immunomodulators. Methods The recombinant protein of HCcyst-3 was expressed in a histidine-tagged fusion soluble form in Escherichia coli, and its inhibitory activity against cathepsin L, B, as well as papain, were identified by fluorogenic substrate analysis. Native HCcyst-3 protein was localized by an Immunohistochemical test. The immunomodulatory effects of HCcyst-3 on cytokine secretion, MHC molecule expression, NO production and phagocytosis were observed by co-incubation of rHCcyst-3 with goat monocytes. Results We cloned and produced recombinant cystatin protein from H. contortus (rHCcyst-3) and investigated its immunomodulatory effects on goat monocyte. The rHCcyst-3 protein is biologically functional as shown by its ability to inhibit the protease activity of cathepsin L, cathepsin B, and papain. The immunohistochemical test demonstrated that the native HCcyst-3 protein was predominantly localized at the body surface and internal surface of the worm’s gut. We demonstrated that rHCcyst-3 could be distinguished by antisera from goat experimentally infected with H. contortus and could uptake by goat monocytes. The results showed that the engagement of rHCcyst-3 decreased the production of TNF-α, IL-1β and IL-12p40. However, it significantly increased the secretion of IL-10 and TGF-β1 in goat monocytes. After rHCcyst-3 exposure, the expression of MHC-II on goat monocytes was restricted. Moreover, rHCcyst-3 could upregulate LPS induced NO production of goat monocytes. Phagocytotic assay by FITC-dextran internalization showed that rHCcyst-3 inhibited the phagocytosis of goat monocytes. Conclusions Our results suggested that the recombinant cystatin from H. contortus (rHCcyst-3) significantly modulated goat monocyte function in multiple aspects.

Abstract Background Hco-gal-m is a tandem-repeat galectin isolated from the adult worm of Haemonchus contortus. A growing body of studies have demonstrated that Hco-gal-m could exert its immunomodulatory effects on host peripheral blood mononuclear cells (PBMC) to facilitate the immune evasion. Our previous work revealed that C-terminal and N-terminal carbohydrate recognition domains (CRD) of Hco-gal-m had different sugar binding abilities. However, whether different domains of Hco-gal-m account differently for its multiple immunomodulatory functions in the host-parasite interaction remains to be elucidated. Results We found that the N-terminal CRD of Hco-gal-m (MNh) and the C-terminal CRD (MCh) could bind to goat peripheral blood mononuclear cells by distinct receptors: transmembrane protein 63A (TMEM63A) was a binding receptor of MNh, while transmembrane protein 147 (TMEM147) was a binding receptor of MCh. In addition, MCh was much more potent than MNh in inhibiting cell proliferation and inducing apoptosis, while MNh was much more effective in inhibiting NO production. Moreover, MNh could suppress the transcription of interferon-γ (IFN-γ), but MCh not. Conclusions Our data suggested that these two CRDs of Hco-gal-m bind to distinct receptors and contributed differently to its ability to downregulate host immune response. These results will improve our understanding of galectins from parasitic nematodes contributing to the mechanism of parasitic immune evasion and continue to illustrate the diverse range of biological activities attributable to the galectin family.

About 0.4 s after the Laser Interferometer Gravitational-Wave Observatory (LIGO) detected a transient gravitational-wave (GW) signal GW150914, the Fermi Gamma-ray Burst Monitor (GBM) also found a weak electromagnetic transient (GBM transient 150914). Time and location coincidences favor a possible association between GW150904 and GBM transient 150914. Under this possible association, we adopt Fermi's electromagnetic (EM) localization and derive constraints on possible violations of the Weak Equivalence Principle (WEP) from the observations of two events. Our calculations are based on four comparisons: (1) The first is the comparison of the initial GWs detected at the two LIGO sites. From the different polarizations of these initial GWs, we obtain a limit on any difference in the parametrized post-Newtonian (PPN) parameter Δγ≲10−10. (2) The second is a comparison of GWs and possible EM waves. Using a traditional super-Eddington accretion model for GBM transient 150914, we again obtain an upper limit Δγ≲10−10. Compared with previous results for photons and neutrinos, our limits are five orders of magnitude stronger than those from PeV neutrinos in blazar flares, and seven orders stronger than those from MeV neutrinos in SN1987A. (3) The third is a comparison of GWs with different frequencies in the range [35 Hz, 250 Hz]. (4) The fourth is a comparison of EM waves with different energies in the range [1 keV, 10 MeV]. These last two comparisons lead to an even stronger limit, Δγ≲10−8. Our results highlight the potential of multi-messenger signals exploiting different emission channels to strengthen existing tests of the WEP.

Abstract Background Arginine kinase (AK), an important member of phosphagen kinase family has been extensively studied in various vertebrates and invertebrates. Immunologically, AKs are important constituents of different body parts, involved in various biological and cellular functions, and considered as immune-modulator and effector for pro-inflammatory cytokines. However, immunoregulatory changes of host cells triggered by AK protein of Haemonchus contortus, a parasitic nematode of ruminants, are still unknown. The current study was focused on cloning and characterisation of Hc-AK, and its regulatory effects on cytokines level, cell migration, cell proliferation, nitric oxide production and apoptosis of goat peripheral blood mononuclear cells (PBMCs) were observed. Methods The full-length sequence of the Hc-AK gene was amplified by reverse transcription-polymerase chain reaction (RT-PCR) and sub-cloned into the prokaryotic expression vector pET-32a. The biochemical characteristics of recombinant protein Hc-AK, which was purified by affinity chromatography, were performed based on the enzymatic assay. Binding of rHc-AK with PBMCs was confirmed by immunofluorescence assay (IFA). Immunohistochemical analysis was used to detect localisation of Hc-AK within adult worms sections. The immunoregulatory effects of rHc-AK on cytokine secretions, cell proliferation, cell migration, nitric oxide production and apoptosis were determined by co-incubation of rHc-AK with goat PBMCs. Results The full-length ORF (1080 bp) of the Hc-AK gene was successfully cloned, and His-tagged AK protein was expressed in the Escherichia coli strain BL21. The recombinant protein of Hc-AK (rHc-AK) was about 58.5 kDa together with the fused vector protein of 18 kDa. The biochemical assay showed that the protein encoded by the Hc-ak exhibited enzymatic activity. Western blot analysis confirmed that the rHc-AK was recognised by the sera from rat (rat-antiHc-AK). The IFA results showed that rHc-AK could bind on the surface of goat PBMCs. Immunohistochemically, Hc-AK was localised at the inner and outer membrane as well as in the gut region of adult worms. The binding of rHc-AK to host cells increased the levels of IL-4, IL-10, IL-17, IFN-γ, nitric oxide (NO) production and cell apoptosis of goat PBMCs, whereas, TGF-β1 levels, cell proliferation and PBMCs migration were significantly decreased in a dose dependent manner. Conclusions Our findings suggested that rHc-AK is an important excretory and secretory (ES) protein involved in host immune responses and exhibit distinct immunomodulatory properties during interaction with goat PBMCs.

Abstract Background Aspartyl protease inhibitor (API) was thought to protect intestinal parasitic nematodes from their hostile proteolytic environment. Studies on Ostertagia ostertagi, Ascaris suum and Brugia malayi indicated that aspins might play roles in nematode infection. In a recent study, proteins differentially expressed between free-living third-stage larvae (L3) and activated L3 (xL3) of Haemonchus contortus were identified by 2D-DIGE. API was found downregulated in xL3 when compared with L3. However, there was no report about the functions of H. contortus API in the parasite-host interaction. In this study, the gene encoding API from H. contortus was cloned, expressed, and part of its biological characteristics were studied. Results A DNA fragment of 681 bp was amplified by RT-PCR. Ninety one percent of the amino acid sequence was similar with that for aspin from O. ostertagi. The recombinant API protein was fusion-expressed with a molecular weight of 48 × 103. Results of Western blot showed that the recombinant API could be recognized by serum from goat infected with H. contortus. It was found that API was localized exclusively in the subcutaneous tissue and epithelial cells of the gastrointestinal tract in adult H. contortus. qRT-PCR suggested that the API gene was differentially transcribed in different life-cycle stages, with the lowest level in female adults and the highest in free-living L3 larvae. Enzyme inhibition assay indicated that the recombinant API can inhibit the activity of pepsin significantly, and the optimal reaction pH and temperature were 4.0 and 37–50 °C respectively. In vitro study showed that the recombinant API could induce goat PBMCs to express IFN-γ, IL-4 and IL-10. Conclusions A new aspartyl protease inhibitor was cloned from H. contortus and its characteristics were studied for the first time. The results indicate that API may regulate the immune response of the host and play roles in the infection.

Carbon fiber felt (CFF)/microfine cement (MC)/silica fume (SF) composite was fabricated by using vacuum infusion impregnation method to infiltrate mechanically stirred mixing slurry of MC, SF, and water into CFF. MC, SF, and water were mixed, with a water-cement ratio of 0.5, SF was used to in place of MC in percentages of 0%, 5%, 10%, 15%, 20%, 25%, 30% and 35%. Drainage method was used to determine the density of CFF/MC/SF composite. The bending strength and compressive strength of CFF/MC/SF composite were characterized by universal testing machine. The microstructure of the fracture surface of CFF/MC/SF composite was studied by scanning electron microscopy. Toughening mechanism of CFF/MC/SF composite was also analyzed. The results show that the density and mechanical strength of CFF/MC/SF composites first increases and then decreases as SF content increases. Crack propagation process includes several toughening and reinforcing mechanisms, such as, fiber/matrix debonding, fiber bridging, fiber friction, crack deflection, and fiber pull-out. At 20% SF content, the compactness and mechanical strength of the CFF/MC/SF composite are optimum, with density, flexural strength, and compressive strength at 1.53 g/cm3, 51.08 MPa, and 53.56 MPa, respectively. Keywords: Carbon fiber felt, Microfine cement, Silica fume, Vacuum infusion impregnation method, Mechanical property

A high-brightness single-end pumping monolithic fiber laser with 10.6 kW output power has been demonstrated through laser diode pumping schemes in traditional step-index large mode area double cladding fiber for the first time, and the configuration of the fiber laser has been detailed reported. To obtain a high power output by means of single-end pumping, a cascade-end pump configuration has been proposed based on a 30/900 μm double cladding fiber, in which the maximal pump power can be increased significantly, and the requirement of thermal management has been reduced. To enable cascade-end pump configuration, a signal/pump combiner has been developed, the signal port of which can pass signal and pump light simultaneously. In experiment, this monolithic fiber laser achieves 10.6 kW output power at a center wavelength of 1080 nm with an optic-to-optic efficiency of 82.6% and a βFL beam quality of 1.95. This single-end pumped fiber laser manifests good thermal management ability, and is promising for higher power scaling. Keywords: Fiber laser, Cascade-end pump, Fiber device

Nacre-like composite films with different ratios of graphene oxide (GO), polyvinyl alcohol (PVA) and carboxylated single-walled carbon nanotubes (SWCNTs) were prepared by solvent evaporation and chemical reduction. Changes in the microstructure, mechanical properties, electrical properties and thermal stability of the composite films were studied. It was found that the cross-section of the composite films had a regular inter-layer alternating structure. Meanwhile, introduction of SWCNTs not only improved the mechanical properties but also the in-plane (σp) and through-thickness (σt) conductivities of the composite films. The tensile strength of the composite film reached 62.8 MPa. The σp and σt of the composite film reached 806 S/m and 479 S/m, respectively, 300 times and 490 times greater than the values obtained prior to the addition of the SWCNTs. Furthermore, the thermal stability of the composite film was also improved. Keywords: Nacre-like structure, Graphene oxide, Polyvinyl alcohol, Single-walled carbon nanotubes