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Ancylostoma ceylanicum is a common zoonotic nematode that inhabits the small intestine of humans, dogs, and cats. Saposin-like proteins (SLPs) have hemolytic and antibacterial activities and could be used as diagnostic or vaccine candidates. To explore the biological functions of Ancylostoma ceylanicum SLP (Ace-SLP-1), cDNA-encoding Ace-SLP-1 mature peptide was cloned into prokaryotic expression vector pET-28a and transformed into Escherichia coli BL21 (DE3) to induce expression. After incubation of canine red blood cell suspension with different concentrations of recombinant Ace-SLP-1, the supernatant was separated to measure OD value and calculate the hemolysis rate. The different concentrations of recombinant protein were co-cultured with E. coli and Enterococcus faecalis, and colony-forming units (CFU) were determined by the plate counting method. Peripheral blood mononuclear cells (PBMCs) from healthy dogs were incubated with different concentrations of recombinant Ace-SLP-1, and the cytokine expression was evaluated by relative quantitative PCR. Our results showed that the hemolytic activity of Ace-SLP-1 increased with the increase in protein concentration from 25 to 100 μg/mL. The recombinant protein had no antibacterial activity against the two kinds of bacteria but could stimulate the secretion of cytokines (IL-4, IL-10, IL-12, and IL-13) in canine PBMCs. These data suggest that Ace-SLP-1 is involved in hookworm blood-feeding and survival and has good immunogenicity, supporting its potential as a diagnostic and vaccine target molecule.

Ancylostoma ceylanicum is a zoonotic soil-derived nematode that parasitizes the intestine of humans and animals (dogs and cats), causing malnutrition and iron-deficiency anemia of the host. Studies have shown that the parasite can regulate or block the host's immune response by secreting calreticulin. However, no data are available on A. ceylanicum calreticulin. To study the biological function of A. ceylanicum calreticulin (Ace-CRT), we amplified the cDNA encoding Ace-CRT and constructed the prokaryotic expression vector pET28-Ace-CRT to express the target protein. The antigenicity of Ace-CRT was investigated by western blotting with canine serum. Mouse splenocytes and canine peripheral blood lymphocytes were stimulated with the recombinant protein in vitro to explore its proliferation activity and effect on the transcription level of cytokines. The recombinant protein was co-incubated with sensitized sheep erythrocytes to analyze the inhibitory effect on the complement-mediated hemolysis. Results showed that the rAce-CRT was abundantly expressed in Escherichia coli, with good antigenicity. The recombinant protein stimulated the proliferation of mouse splenocytes and canine peripheral blood mononuclear cells (PBMCs). Quantitative reverse-transcription polymerase chain reaction showed that rAce-CRT mainly promoted the expression of Th2 cytokines, especially IL-13, in canine peripheral blood lymphocytes. In vitro, rAce-CRT inhibited complement-mediated sheep erythrocytes hemolysis. These findings indicate that Ace-CRT has an immunomodulatory role and might be a promising candidate molecule for the hookworm vaccine.

We know little about the antigen bias in SARS-CoV-2 humoral response and the epitopes of spike recognized by the immune system in asymptomatic (AS) patients and symptomatic (S) patients. Here, we used a microarray to evaluate the humoral immune response in the sera collected from 33 COVID-19-recovered patients up to 1 year. We found that the levels of IgG and IgM induced by the 23 proteins differed significantly in the same patients, and were able to distinguish AS and S patients. The N- and S-specific antibodies were detected even at 12 months after onset. Five epitopes were identified to be associated with the clinical adverse events, and three peptides located in RBD. Overall, this study presents a systemic view of the SARS-CoV-2 specific IgG and IgM responses between AS and S recovered patients and provide insights to promote precise development of SARS-CoV-2 vaccines.

Background: The occurrence and development of colorectal cancer is related to the compositional and functional variation of gut microbiota. Compared with healthy people, gut microbiota of patients with colorectal cancer is in disorder. Most traditional Chinese Medicine is effective by oral administration and has both anti-tumor effect and enteric microecological regulative effect. However, whether the dysbiosis of gut microbiota under tumor burden affects the serum metabolome of human body that related to traditional Chinese medicine is unclear. In this study, Fufangchangtai (FFCT) was chosen to be the model prescription to explore the correlation between gut microbiota and the serum metabolism related to FFCT in anti colorectal cancer treatment. Results: The gut microbiota between colorectal tumor-bearing mice and healthy mice were determined by 16S rRNA gene sequencing, showing quite differences between the two groups and suggesting that Firmicutes , Deferribacteres , Bacteroidetes and Proteobacteria were marked differential intestinal bacteria. The alternations in serum metabolome in the FFCT-treating tumor-bearing mice and simple FFCT-treating mice were detected using Liquid chromatogragh-mass spectrometer (LC/MS), showing significant differences between the two groups as well. Metabolites of FFCT like Citric acid, （±）12-HEPE, Cycloartanyl ferulate were much more in simple FFCT-treating mice, indicating that the present of tumor could affect the absorption and metabolism of FFCT. Additionally, these differential metabolites of FFCT involved in multiple pathways including the Alanine, aspartate and glutamate metabolism, Central carbon metabolism in cancer, Biosynthesis of amino acids. Different doses of FFCT were given to the tumor-bearing mice through oral administration, and the results of gut microbiota 16S rRNA gene sequencing showing that FFCT-treating groups has higher abundance of Firmicutes , Turicibacter and Roseburia than tumor-bearing group, moreover, the abundance of these bacteria was positively correlated to the drug concentration. Firmicutes and Bacteroidetes in FFCT-treating groups showed a similar trend with Healthy group, indicating the modulation of FFCT on gut microbiota of colorectal tumor-bearing mice.Conclusions: Collectively, we concluded that the dysbiosis of gut microbiota in tumor-bearing mice could affect the serum metabolome of human body that related to FFCT, and FFCT could correct the gut microbiota of colorectal tumor-bearing mice. It was pointed out that the GM should be concerned during the therapy of FFCT. The more healthier intestinal microenvironment was conductive to the better clinical curative effect.

Ancylostoma ceylanicum is a zoonotic parasitic nematode that can cause iron-deficiency anemia and malnutrition in humans. A. ceylanicum hookworm platelet inhibitor (Ace-HPI) can inhibit platelet aggregation in the host to facilitate blood sucking, but whether it possesses platelet adhesion inhibitory activity or immunomodulatory role is yet unknown. To explore the effect of Ace-HPI on platelet adhesion, we expressed the recombinant protein in two competent cells, BL21 (DE3) and Rosetta-gami2 (DE3), and incubated this protein with canine platelets in a 96-well microplate. Ace-HPI was used to stimulate peripheral blood mononuclear cells (PBMC) in vitro to investigate the effect on PBMC proliferation and cytokine expression. Results showed that Ace-HPI expressed in Rosetta-gami2 (DE3) strain was mostly soluble. The inhibitory effect of this protein on platelet adhesion was relatively weak (7-8%). This protein stimulated the proliferation of PBMC and promoted the expression of Treg and Th2 cytokines, such as IL-10 and IL-13. These results lay a foundation for exploring the role of Ace-HPI in hookworm disease pathogenesis and as a candidate molecule for hookworm vaccines.

Ophidascaris species are parasitic roundworms that inhabit the python gut, resulting in severe granulomatous lesions or even death. However, the classification and nomenclature of these roundworms are still controversial. Our study aims to identify a snake roundworm from the Burmese python (Python molurus bivittatus) and analyze the mitochondrial genome. We identified this roundworm as Ophidascaris baylisi based on the morphology and cytochrome c oxidase subunit I (cox1) sequence. Ophidascaris baylisi complete mitochondrial genome was 14,784 bp in length, consisting of two non-coding regions and 36 mitochondrial genes (12 protein-coding genes, 22 tRNA genes, and two rRNA genes). The protein-coding genes used TTG, ATG, ATT, or TTA as start codons and TAG, TAA, or T as stop codons. All tRNA genes showed a TV-loop structure, except trnS1AGN and trnS2UCN revealed a D-loop structure. The mitochondrial large ribosomal subunit 16S (rrnL) and small ribosomal subunit 12S (rrnS) were 956 bp and 700 bp long, respectively. Phylogenetic analysis based on O. baylisi mitochondrial protein-coding genes demonstrated that O. baylisi clustered with the family Ascarididae members and was most closely related to Ophidascaris wangi. These results may enhance the nematode mitochondrial genome database and provide valuable molecular markers for further research on the taxonomy, phylogeny, and genetic relationships of Ophidascaris nematodes.

Aspartyl protease inhibitors (APIs) from parasitic intestinal nematodes are highly immunogenic and have been suggested as potential vaccine antigens. Ac-API-1 from Ancylostoma caninum showed strong immunogenicity and its polyclonal antibodies could specifically recognize the excretory/secretory products of adult worms. However, little is known about molecular characteristics and biological function of API from Ancylostoma ceylanicum (Ace-API). In this study, the Ace-API mature peptide coding sequence was cloned and expressed, and molecular characteristics of its full length sequence were analyzed. Ace-API cDNA was 684 bp in length, which encoded 228 amino acids. The similarity of the Ace-API amino acid sequence to Ac-API-1 and Adu-API-1 was 96.93% and 96.49%, respectively, and they clustered together in the phylogenetic tree. Escheria coli-expressed recombinant protein was mainly soluble in the supernatant of bacterial cell lysate. Western blot showed that Ace-API protein had good reactivity to the serum of infected dogs. Pepsin inhibition assay revealed that the recombinant protein had inhibitory activity on pepsin. Immunofluorescence results demonstrated that Ace-API was mainly localized to the epidermis, excretory glands, and pseudocoelomic fluid of the adult. Using the quantitative real-time PCR, the expression of Ace-api mRNA in adults was significantly higher than that in the third stage (L3) larvae. Together, these data indicate that Ace-API is secreted extracellularly by the parasite, and might play a role in protecting the parasite against the proteolytic digestion by the host proteases, which stimulate further studies to explore this protein as a potential hookworm vaccine candidate.