The TCTP is essential for ovarian development and oviposition of Rhipicephalus haemaphysaloides.

Tick infestations transmit various infectious agents and result in significant socioeconomic consequences. Currently, the primary focus of tick control efforts is identifying potential targets for immune intervention. In a previous study, we identified a highly conserved protein abundant in tick haemolymph extracellular vesicles (EVs) known as translationally controlled tumour protein (TCTP). We have found that native TCTP is present in various tissues of the Rhipicephalus haemaphysaloides tick, including salivary glands, midgut, ovary, and fat body. Notably, TCTP is particularly abundant in the tick ovary and its levels increase progressively from the blood-feeding stage to engorgement. When the TCTP gene was knocked down by RNAi, there was a noticeable delay in ovarian development, and the reproductive performance, in terms of egg quantity and survival, was also hindered. Our investigations have revealed that the observed effects in ovary and eggs in dsRNA-treated ticks are not attributable to cell death mechanisms like apoptosis and autophagy but rather to the reduction in the expression of vitellogenin (Vg1, Vg2, and Vg3) and ferritin (ferritin 1 and ferritin 2) proteins crucial for ovarian development and embryo survival in ticks. Additionally, phylogenetic analysis and structural comparisons of RhTCTP and its orthologues across various tick species, vertebrate hosts, and humans have shown that TCTP is conserved in ticks but differs significantly between ticks and their hosts, particularly in the TCTP_1 and TCTP_2 domains. Overall, TCTP plays a vital role in tick reproductive development and presents itself as a potential target for tick control in both humans and animals.

Towards modelling tick-virus interactions using the weakly pathogenic Sindbis virus: Evidence that ticks are competent vectors.

Most tick-borne viruses (TBVs) are highly pathogenic and require high biosecurity, which severely limits their study. We found that Sindbis virus (SINV), predominantly transmitted by mosquitoes, can replicate in ticks and be subsequently transmitted, with the potential to serve as a model for studying tick-virus interactions. We found that both larval and nymphal stages of Rhipicephalus haemaphysaloides can be infected with SINV-wild-type (WT) when feeding on infected mice. SINV replicated in two species of ticks (R. haemaphysaloides and Hyalomma asiaticum) after infecting them by microinjection. Injection of ticks with SINV expressing enhanced Green Fluorescent Protein (eGFP) revealed that SINV-eGFP specifically aggregated in the tick midguts for replication. During blood-feeding, SINV-eGFP migrated from the midguts to the salivary glands and was transmitted to a new host. SINV infection caused changes in expression levels of tick genes related to immune responses, substance transport and metabolism, cell growth and death. SINV mainly induced autophagy during the early stage of infection; with increasing time of infection, the level of autophagy decreased, while the level of apoptosis increased. During the early stages of infection, the transcript levels of immune-related genes were significantly upregulated, and then decreased. In addition, SINV induced changes in the transcription levels of some functional genes that play important roles in the interactions between ticks and tick-borne pathogens. These results confirm that the SINV-based transmission model between ticks, viruses, and mammals can be widely used to unravel the interactions between ticks and viruses.

Proteomic analysis of extracellular vesicles from tick hemolymph and uptake of extracellular vesicles by salivary glands and ovary cells.

BACKGROUND: Extracellular vesicles (EVs) are a heterogeneous group of cell-derived membranous structures that are important mediators of intercellular communication. Arthropods transport nutrients, signaling molecules, waste and immune factors to all areas of the body via the hemolymph. Little is known about tick hemolymph EVs. METHODS: Hemolymph was collected from partially fed Rhipicephalus haemaphysaloides and Hyalomma asiaticum ticks by making an incision with a sterile scalpel in the middle (between the femur and metatarsus) of the first pair of legs, which is known as leg amputation. EVs were isolated from hemolymph by differential centrifugation and characterized by transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA). Proteins extracted from the hemolymph EVs were analyzed by 4D label-free proteomics. The EVs were also examined by western blot and immuno-electron microscopy analysis. Intracellular incorporation of PHK26-labeled EVs was tested by adding labeled EVs to tick salivary glands and ovaries, followed by fluorescence microscopy. RESULTS: In this study, 149 and 273 proteins were identified by 4D label-free proteomics in R. haemaphysaloides and H. asiaticum hemolymph EVs, respectively. TEM and NTA revealed that the sizes of the hemolymph EVs from R. haemaphysaloides and H. asiaticum were 133 and 138 nm, respectively. Kyoto Encyclopedia of Genes and Genomes and Gene Ontology enrichment analyses of identified proteins revealed pathways related to binding, catalytic and transporter activity, translation, transport and catabolism, signal transduction and cellular community. The key EV marker proteins RhCD9, RhTSG101, Rh14-3-3 and RhGAPDH were identified using proteomics and western blot. The presence of RhFerritin-2 in tick hemolymph EVs was confirmed by western blot and immuno-electron microscopy. We demonstrated that PKH26-labeled hemolymph EVs are internalized by tick salivary glands and ovary cells in vitro. CONCLUSIONS: The results suggest that tick EVs are secreted into, and circulated by, the hemolymph. EVs may play roles in the regulation of tick development, metabolism and reproduction.

Current Knowledge on Chemosensory-Related Candidate Molecules Potentially Involved in Tick Olfaction via Haller's Organ.

Ticks are obligatory hematophagous ectoparasites and vectors of many animal and human pathogens. Chemosensation plays a significant role in tick communication with their environment, including seeking out blood meal hosts. Studies on the structure and function of Haller's organ and its components have improved our understanding regarding tick olfaction and its chemical ecology. Compared with the knowledge on insect olfaction, less is known about the molecular basis of olfaction in ticks. This review focused on the chemosensory-related candidate molecules likely involved in tick olfaction. Members of the ionotropic receptor family and a new class of odorant-binding proteins are now known to be involved in tick olfaction, which appear to differ from that of insects. These candidate molecules are more closely related to those of mites and spiders than to other arthropods. The amino acid sequences of candidate niemann-pick type C2 and microplusin-like proteins in ticks exhibit features indicating their potential role as binding proteins. In the future, more comprehensive pertinent research considering the existing shortcomings will be required to fully understand the molecular basis of tick olfactory chemoreception. This information may contribute to the development of new molecular-based control mechanisms to reduce tick populations and related disease transmission.

Effect of Orthodontic Combined with Implant Repair on Aesthetic Effect and Gingival Crevicular Fluid Factor in Patients with Dentition Defect and Periodontitis.

In order to solve the effect of orthodontics combined with implant repair on the aesthetic effect and gingival crevicular fluid factor of patients with dentition defect and periodontitis, 60 patients who met the diagnostic criteria of chronic periodontitis were proposed. They were randomly divided into treatment group (taking Bushen Huoxue Guchi recipe for 3 courses while basic periodontal treatment) and control group (only basic periodontal treatment without taking any drugs). The experimental method of 30 cases in each group showed that PD, Al, and GI in the treatment group and control group decreased to varying degrees compared with those before treatment. The treatment group decreased significantly compared with the control group (P < 0.01). Chronic periodontitis is a common clinical periodontal disease, accounting for up to 95%, local stimulation, a variety of anaerobic bacteria infection, and periodontal plaque, and other factors may cause the occurrence of diseases. Routine treatment mainly includes upper gingival cleaning, lower curettage treatment, and equal root surface treatment. Although the clinical symptoms can be alleviated to a certain extent, the cause cannot be fundamentally excluded, leading to the disease progression in some patients and the formation of aggressive periodontitis and necrotizing periodontal disease.

Identification of the Bcl-2 and Bax homologs from Rhipicephalus haemaphysaloides and their function in the degeneration of tick salivary glands.

BACKGROUND: The salivary glands of female ticks degenerate rapidly by apoptosis and autophagy after feeding. Bcl-2 family proteins play an important role in the apoptosis pathways, but the functions of these proteins in ticks are unclear. We studied Bcl-2 and Bax homologs from Rhipicephalus haemaphysaloides and determined their functions in the degeneration of the salivary glands. METHODS: Two molecules containing conserved BH (Bcl-2 family homology) domains were identified and named RhBcl-2 and RhBax. After protein purification and mouse immunization, specific polyclonal antibodies (PcAb) were created in response to the recombinant proteins. Reverse transcription quantitative PCR (RT-qPCR) and western blot were used to detect the presence of RhBcl-2 and RhBax in ticks. TUNEL assays were used to determine the level of apoptosis in the salivary glands of female ticks at different feeding times after gene silencing. Co-transfection and GST pull-down assays were used to identify interactions between RhBcl-2 and RhBax. RESULTS: The RT-qPCR assay revealed that RhBax gene transcription increased significantly during feeding at all tick developmental stages (engorged larvae, nymphs, and adult females). Transcriptional levels of RhBcl-2 and RhBax increased more significantly in the female salivary glands than in other tissues post engorgement. RhBcl-2 silencing significantly inhibited tick feeding. In contrast, RhBax interference had no effect on tick feeding. TUNEL staining showed that apoptosis levels were significantly reduced after interference with RhBcl-2 expression. Co-transfection and GST pull-down assays showed that RhBcl-2 and RhBax could interact but not combine in the absence of the BH3 domain. CONCLUSIONS: This study identified the roles of RhBcl-2 and RhBax in tick salivary gland degeneration and finds that the BH3 domain is a key factor in their interactions.

Response of Leucine-Rich Repeat Domain-Containing Protein in Haemaphysalis longicornis to Babesia microti Infection and Its Ligand Identification.

Haemaphysalis longicornis is a blood-feeding hard tick known for transmitting a variety of pathogens, including Babesia How the parasites in the imbibed blood become anchored in the midgut of ticks is still unknown. Leucine-rich repeat domain (LRR)-containing protein, which is associated with the innate immune reaction and conserved in many species, has been detected in H. longicornis and has previously been indicated in inhibiting the growth of Babesia gibsoni However, the detailed mechanism is unknown. In this study, one of the ligands for LRR from H. longicornis (HlLRR) was identified in Babesia microti, designated BmActin, using glutathione transferase (GST) pulldown experiments and immunofluorescence assays. Moreover, RNA interference of HlLRR led to a decrease in the BmActin mRNA expression in the midgut of fully engorged ticks which fed on B. microti-infected mice. We also found that the expression level of the innate immune molecules in H. longicornis, defensin, antimicrobial peptides (AMPs), and lysozyme, were downregulated after the knockdown of HlLRR. However, subolesin expression was upregulated. These results indicate that HlLRR not only recognizes BmActin but may also modulate innate immunity in ticks to influence Babesia growth, which will further benefit the development of anti-Babesia vaccines or drugs.

Berberine Promotes the Proliferation and Osteogenic Differentiation of Alveolar Osteoblasts through Regulating the Expression of miR-214.

INTRODUCTION AND OBJECTIVE: Alveolar osteoblasts have critical functions during alveolar bone regeneration. Berberine (BBR) and microRNAs (miRNAs) are considered to play important roles in regulating osteoblast differentiation. The study aimed to investigate the role and mechanisms of BBR in osteogenic differentiation of human alveolar osteoblasts (HAOBs) and determine miR-214 expression in the process. METHODS: Healthy human alveolar bones were cultured in vitro and prepared for morphological observation and alkaline phosphatase (ALP) staining. The third generation of HAOBs was used for cell transfection and treated by different concentrations of BBR. Cell Counting Kit-8 was used to detect the effect of BBR and increased miR-214 on the proliferation of HAOBs. qRT-PCR and Western blot were used to detect the expression of osteogenic differentiation-related genes and miR-214 level, respectively. RESULTS: The ALP staining results were positive, indicating that cultured cells were HAOBs. Different concentrations of BBR significantly promoted the proliferation of HAOBs and increased the expression levels of ALP, osteocalcin (OCN), collagen type I alpha 1 (COL1A1), runt related transcription factor 2 (RUNX2), and osterix (OSX). Moreover, the expression of miR-214 was reduced as BBR concentrations increased, and the increase of miR-214 reversed the BBR-induced proliferation and osteogenic differentiation of HAOBs. CONCLUSION: BBR could promote the proliferation and osteogenic differentiation of HAOBs through downregulating the expression of miR-214.

A family of serine protease inhibitors (serpins) and its expression profiles in the ovaries of Rhipicephalus haemaphysaloides.

Serpins are evolutionarily conserved serine protease inhibitors found in many organisms. In arthropods, serpins are involved in feeding, development, oviposition, anti-coagulation and innate immune responses. We characterized of 11 serpins in the tick Rhipicephalus haemaphysaloides. These serpins have orthologous genes in other ticks, as indicated by phylogenetic analysis. Analysis of the reactive center loop and hinge regions of the protein sequences indicated that RHS7 encodes proteins that may lack proteinase inhibitor activity. All R. haemaphysaloides serpins had high amino acid sequence identities to Rhipicephalus microplus serpins. Tissue and temporal transcriptional profiling of eight R. haemaphysaloides serpins located in the ovaries demonstrated that they are transcribed during feeding and oviposition. These suggested their participation in the regulation of tick physiology. Immune serum from rabbits repeatedly infested with larvae, nymphs and adults of R. haemaphysaloides can recognize multiple recombinant serpins, respectively. After gene silencing, the blood feeding to repletion time was significantly longer and the 24 h attachment rate was significantly lower in the RHS3 and RHS7 knock down groups. The RHS9 and RHS11 silenced ticks had significant reduction in repletion time and egg-laying rate. Egg hatchability was significantly decreased in RHS4, RHS5 and RHS9 silenced ticks. All groups had significant reductions in engorged body weight. This study increases information on the serpins of R. haemaphysaloides and suggests that some RHSs are potential targets for development of tick vaccines.

Inclusion of PD-L1 into a recombinant profilin antigen enhances immunity against Babesia microti in a murine model.

Pathogens and cancer cells employ the programmed cell death-Ligand 1 (PD-L1)/ programmed cell death-1 (PD-1) signaling pathway to inhibit the immune response. Hence, blockade of PD-L1/PD-1 recognition through monoclonal antibodies enhances the immune response. Antibodies that block PD-L1 and PD-1 binding have been used for the prevention and therapy of human pathogenic diseases, but have not yet been evaluated for the treatment of infectious diseases of livestock. In the present study, a recombinant vaccine named PROF-PDL1E, was designed comprising the Babesia microti-derived vaccine candidate profilin and the host PD-L1 protein, and its effect on immunization against murine B. microti infection was evaluated. PD-L1-specific antibodies generated after vaccination blocked PD-L1 and PD-1 binding as shown by in vitro assays. PROF-PDL1E reduced the burden of B. microti in a mouse model and decreased PD-1 expression in T cells. Furthermore, no tissue damage could be observed after PROF-PDL1E vaccination as verified by hematoxylin and eosin tissue staining of essential organs. In conclusion, vaccines targeting immune checkpoints seem to be a promising strategy for anti-Babesia vaccine development.

A serpin from the tick Rhipicephalus haemaphysaloides: Involvement in vitellogenesis.

Tick serpins are involved in enzyme activity, food digestion, blood-feeding, immune response and anticoagulation. Little is known about the potential roles of serpins in tick reproduction. RHS8, a serpin from the tick Rhipicephalus haemaphysaloides, has an open reading frame 1212 bp long and encodes a protein that has 404 amino acids and a predicted molecular weight of 45 kDa. RHS8 exhibits 89.58 % amino acid identity with RmS15 in Rhipicephalus microplus. RHS8 was expressed primarily in larvae and nymphs. RHS8 mRNA expression in the ovaries, fat bodies and salivary glands were up-regulated from feeding to ovipositing ticks. RNAi results showed that RHS8 dsRNA-injected ticks had a lower body weight, longer feeding time, fewer eggs laid and lower egg hatchability. Tick reproduction, such as egg laying and hatching, was disrupted by RNAi. Compared with the control group, ovaries of the RHS8 interference group were light brown color, indicating a reduction in yolk granule accumulation. Western blot results showed that the expression of RHVg3 and RHVg4 proteins in ovaries was reduced in the RHS8 dsRNA-injected group. These results indicate that RHS8 is related to tick reproduction and its interference affects vitellogenesis.

Immunomodulatory effects of Rhipicephalus haemaphysaloides serpin RHS2 on host immune responses.

BACKGROUND: Rhipicephalus haemaphysaloides is a widespread tick species in China and other South East Asian countries, where it is the vector of many pathogens. The objective of this study was to study the role of serpin (serine protease inhibitor) during the tick-host interaction. METHODS: The differentiation of bone marrow-derived dendritic cells (BMDC) was induced in vitro, and the effect of RHS2 on the maturation of DCs was evaluated. The effects of RHS2 on T cell activation and cytotoxic T lymphocytes' (CTLs) activity were analyzed by flow cytometry. Antibody subtypes after immunization of mice with RHS2 and OVA were determined. RESULTS: RHS2 can inhibit the differentiation of bone marrow-derived cells into DCs and promote their differentiation into macrophages. RHS2 can inhibit the maturation of DCs and the expression of CD80, CD86 and MHCII. The number of CD3+CD4+ and CD3+CD8+ T cells secreting IFN-γ, IL-2 and TNF-α was decreased, and the number of CD3+CD4+ T cells secreting IL-4 was increased, indicating that RHS2 can inhibit the activation of CD4 T cells and CD8 T cells, leading to inhibition of Th1 immune response. RHS2 inhibits the elimination of target cells by cytotoxic T lymphocytes. After immunization of mice with RHS2 and OVA, serum IgG2b was significantly reduced and IgM was increased. CONCLUSIONS: The results show that RHS2 has an inhibitory effect on the host immune response. Ticks have evolved various ways to circumvent adaptive immunity. Their serpin inhibits BMDC differentiation to reduce immune responses.

Immunosuppressive effects of tick protein RHcyst-1 on murine bone marrow-derived dendritic cells.

BACKGROUND: Ticks, as blood-feeding arthropod vectors, have evolved their own unique mechanism to suppress host immune responses and evade immune defenses in order to complete blood-feeding. The immunoregulatory effect of tick bioactive molecules on hosts has been widely reported, and the cystatin family has been identified as one of the major immunomodulators. In previous studies, we obtained a novel tick salivary bioactive protein named RHcyst-1, which belongs to the type 1 cystatin family. Here, we demonstrated the effects of RHcyst-1 on the host immune response mainly on dendritic cell (DC) function. Understanding the function of tick-derived bioactive molecule may help to clarify the mechanisms of how ticks escape the host immune response and help to control ticks and tick-borne disease transmission. METHODS: Bone marrow-derived DCs (BMDCs) were generated and induced by GM-CSF and IL-4 with or without RHcyst-1 addition. Flow cytometry was used to analyze the differentiation and maturation of BMDCs and T cell cytokine production. Quantitative real-time PCR (qRT-PCR) and western blot were used to measure changes in expression within STAT and p38 MAPK signaling pathways. RESULTS: Flow cytometry analysis revealed that RHcyst-1 inhibited the differentiation of BMDCs, but had no effect on the maturation of BMDCs. T cells co-cultured with DCs treated with RHcyst-1 produced significantly less TNF-α, IFN-γ and IL-2 than the control group. Further analysis showed that the mRNA level and phosphorylation of p38, ERK and STAT were significantly changed after RHcyst-1 added to bone marrow monocytes during the differentiation stage. CONCLUSIONS: Our results suggest that RHcyst-1 is one of the major immunosuppressive proteins of BMDC function from blood-feeding ticks.

A Tick Cysteine Protease Inhibitor RHcyst-1 Exhibits Antitumor Potential.

BACKGROUND/AIMS: We previously identified a potent and tight-binding inhibitor of cysteine proteases from Rhipicephalus haemaphysaloides, RHcyst-1, which belongs to the cystatin type 1 family. Cathepsins, which are members of the cysteine protease family, participate in various pathological processes, including the initiation and development of cancers. The present study aimed to investigate the antitumor effects of RHcyst-1 and to explore the underlying mechanism of these effects. METHODS: Different tumor cells were treated with RHcyst-1 in vitro. Proliferation activity was evaluated using Cell Counting Kit-8, and migration and invasion were determined by wound healing and Transwell® invasion assays. In addition, a mouse tumor therapy model was established by inoculating the left forelimb of mice with B16-F10 cells, and tumor progression was evaluated by assessing tumor volume and survival. Flow cytometry was conducted to evaluate myeloid-derived suppressor cells (MDSCs), CD4+, and CD8+ T cell levels in PBMCs and spleens. Immunohistochemistry was performed to analyze immune cell infiltration and angiogenesis in the tumors. RESULTS: RHcyst-1 significantly inhibited the proliferation, migration, and invasion of all four different tumor cells in vitro. Additionally, it inhibited tumor growth and improved survival in vivo. A decrease and an increase in MDSCs levels were observed in PBMCs and in the spleen, respectively, after RHcyst-1 application. CONCLUSIONS: Tick RHcyst-1 has potential antitumor efficacy, and the observed antitumor activities may be partly attributable to changes in cathepsin expression and MDSCs levels in the PBMCs and spleens. The findings of the present study suggest that RHcyst-1 may have the potential to be utilized in cancer treatment.

[Analysis of influencing factors for curative effect of maxillofacial fractures].

OBJECTIVE: To explore the influencing factors for curative effect of maxillofacial fractures. METHOD: Retrospective analysis of the 86 patients for maxillofacial fractures from Jan. 2008 to Dec. 2010 in our hospital, to observe data, sex, type, reason, associated injury, methods of treatment, and so on. RESULT: The success rate of curing was 95.35%. The length of stay for the 86 patients was from 7 days to 28 days, average 16. 8 days. The type, reason, associated injury, methods of treatment were the influencing factors for curative effect of for maxillofacial fracture. The success rate of curing for different ways of operation were different. Recovery rate of operation was 6.097%, it of expectant treatment was 75%, they were statistical different significantly (P < 0.05). CONCLUSION: The type, reason, associated injury, methods of treatment were the influencing factors for curative effect of for maxillofacial fracture.

[Comparison of nitrifying populations between a lab-scale system and an industrial system treating coking wastewater].

Molecular analysis of community structure can help diagnose problems in malfunctioning full-scale wastewater treatment system. A lab-scale A1-A2-O fixed biofilm system with highly efficient nitrification (NH3-N removal at 95. 2%) was set up as a reference for an industrial system with poor performance of nitrification (NH3-N removal efficiency at -106%) for treating coking wastewater. Composition of nitrifying bacteria of biofilm samples in aerobic reactors of the two systems was compared by 16S rDNA clone library analysis. The composition of clone library of the lab-scale system indicate Nitrosomonas europaea-Nitrosoccus mobilis cluster and sublineage I of Nitrospira genus are dominant ammonia and nitrite oxidizers in this process respectively. However, there were no clones related to nitrifying bacteria in clone library of the industrial system which suggested low abundance of nitrifying bacteria in this system. Further, Real-Time PCR with Taqman probe revealed that 16S rDNA copy number of Nitrospira in aerobic reactor of the lab-scale system was 3.4 x 10(6)/microg DNA of biofilm which is 300 times higher than that in the industrial system. The absence of Nitrosomonas and Nitrospira in the industrial system leads to poor performance of nitrification. Building up of a high population level of Nitrosomonas and Nitrospira in aerobic reactor is the key to improve efficiency of nitrification of the industrial system.

Cloning of environmental genomic fragments as physical markers for monitoring microbial populations in coking wastewater treatment system.

The association between community functional shift and dynamics of genomic DNA composition can be used to identify functionally relevant populations as indicator organisms for systems monitoring. In this work, fingerprinting-based community DNA hybridization was used to monitor community structural dynamics and identify genomic fragments whose abundance shifts were concomitant to changes in COD removal capacity in a reactor. A laboratory-scale anaerobic-anoxic-oxic fixed biofilm system treating coking wastewater was operated with (LR mode) or without effluent recirculation (LNR mode). The contribution to total chemical oxygen demand (COD) removal by the anoxic reactor increased from 4% in LNR mode to 26% in LR mode. Long primer RAPD (randomly amplified polymorphic DNA) community fingerprints of the anoxic reactor also changed most significantly from the one similar to the anaerobic reactor to one similar to the oxic reactor. DNA hybridization revealed one signature band of 2.1 kb shared by the anoxic and oxic reactors in LR, but not LNR mode. Clone library profiling of this band resulted in one predominant 2.1-kb genomic fragment (B3) with no homologous sequences in GenBank. Real-time polymerase chain reaction indicated that copy numbers of B3 in the anoxic reactor under LR mode were 69 times higher than that under LNR mode, concomitant to a significant increase in COD removal capacity in this reactor. The different patterns of distribution of B3 in the laboratory system and a comparable malfunctioning industrial system demonstrated the potential of this genomic fragment as physical markers in systems monitoring. In addition, this genomic fragment may allow sequence-guided isolation of the host microbe.