Comparative analysis of the anticoagulant activities and immunogenicity of HSC70 and HSC70TKD of Haemaphysalis flava.

BACKGROUND: Haemaphysalis flava is a hematophagous ectoparasite that acquires the nutrition needed for development and reproduction by sucking blood and digesting the blood meal. During blood-sucking and blood-meal digestion, the prevention of blood coagulation is important for this tick. Previous studies have shown that heat shock cognate 70 (HSC70) protein has certain anticoagulant activities, but its immunogenicity remains unclear. Also, whether the mutation of individual bases of the TKD-like peptide of HSC70 through the overlap extension method can change its anticoagulant activities and immunogenicity remains to be investigated. METHODS: The gene encoding the HSC70 protein was cloned from a complementary DNA library synthesized from H. flava. The coding gene of the TKD-like peptide of HSC70 was mutated into a TKD peptide coding gene (HSC70TKD) using the overlap extension method. Escherichia coli prokaryotic expression plasmids were constructed to obtain the recombinant proteins of HSC70 (rHSC70) and HSC70TKD (rHSC70TKD). The purified rHSC70 and rHSC70TKD were evaluated at different concentrations for anticoagulant activities using four in vitro clotting assays. Emulsifying recombinant proteins with complete and incomplete Freund's adjuvants were subcutaneously immunized in Sprague Dawley rats. The serum antibody titers and serum concentrations of interferon-gamma (IFN-γ) and interleukin-4 (IL-4) were detected using an indirect enzyme-linked immunosorbent assay to assess the immunogenicity of rHSC70 and rHSC70TKD. RESULTS: The open reading frame of HSC70 was successfully amplified and found to have a length of 1958 bp. The gene encoding the TKD-like peptide of HSC70 was artificially mutated, with the 1373-position adenine (A) of the original sequence mutated into guanine (G), the 1385-position cytosine (C) mutated into G and the 1386-position G mutated into C. rHSC70 and rHSC70TKD that fused with His-tag were obtained using the expression plasmids pET-28a-HSC70 and pET-28a-HSC70TKD, respectively. rHSC70 and rHSC70TKD prolonged the thrombin time (TT) and reduced the fibrinogen (FIB) content in the plasma, but did not affect the prothrombin time (PT) or activated partial thromboplastin time (APTT) when compared to the negative control. Interestingly, the ability of rHSC70TKD to prolong the TT and reduce the FIB content in the plasma was better than that of rHSC70. The specific antibody titers of both rHSC70 and rHSC70TKD in rat serum reached 1:124,000 14 days after the third immunization. The serum concentration of IFN-γ in the rHSC70TKD group was higher than that in the rHSC70 group. The rHSC70 group has the highest serum concentration of IL-4, and the serum concentration of IL-4 in the rHSC70TKD group was higher than that in the negative group. CONCLUSIONS: rHSC70 and rHSC70TKD exhibited anticoagulant activities by prolonging the TT and reducing the FIB content in vitro. rHSC70TKD had better anticoagulant activities than rHSC70. Both rHSC70 and rHSC70TKD had good immunogenicity and induced humoral and cellular immunity.

Microbiome analysis of the midguts of different developmental stages of Argas persicus in China.

Argas persicus is an ectoparasite of poultry. The bacterial community structure and the pathogenic bacteria associated with different developmental stages of A. persicus have implications for control. Argas persicus were collected from chickens in the city of Jiuquan in Gansu, China. Bacterial DNA was extracted from the midgut contents of blood engorged larvae, nymphs and adult females. The V3-V4 hypervariable regions of 16S rRNA genes were sequenced using the IonS5™XL platform. Identification of Rickettsia spp. and detection of Coxiella burnetii were performed using PCR on target genes. The bacterial diversity within larvae was the highest and the bacterial diversity within nymphs was greater than that of adults. At different classification levels, seven bacterial phyla were common phyla, 27 genera were common genera, and 18 species were common species in the three samples. At the phylum level, Proteobacteria showed a marked predominance in all samples. Rickettsia, Stenotrophomonas, Spiroplasma, and Coxiella were the dominant bacteria at the genus level. The Rickettsia species in A. persicus was identified as Rickettsia hoogstraalii and the Coxiella species was identified as a Coxiella-like endosymbiont. Additionally, some bacterial species such as Pseudomonas geniculata, Sphingomonas koreensis, and Acinetobacter haemolyticus were reported here for the first time in A. persicus.

CCL2 facilitates spinal synaptic transmission and pain via interaction with presynaptic CCR2 in spinal nociceptor terminals.

Previous studies have shown that CCL2 may cause chronic pain, but the exact mechanism of central sensitization is unclear. In this article, we further explore the presynaptic role of CCL2. Behavioral experiments show that intervertebral foramen injection CCR2 antagonists into dorsal root ganglion (DRG) can inhibit the inflammatory pain caused by CCL2 in spinal cord. We raised the question of the role of presynaptic CCR2 in the spinal dorsal horn. Subsequent electron microscopy experiments showed that CCR2 was expressed in the presynaptic CGRP terminal in the spinal dorsal horn. CCL2 can enhance presynaptic calcium signal. Whole-cell patch-clamp recordings showed that CCL2 can enhance NMDAR-eEPSCs through presynaptic effects, and further application of glutamate sensor method proved that CCL2 can act on presynaptic CCR2 to increase the release of presynaptic glutamate. In conclusion, we suggest that CCL2 can directly act on the CCR2 on presynaptic terminals of sensory neurons in the spinal dorsal horn, leading to an increase in the release of presynaptic glutamate and participate in the formation of central sensitization.