CRISPR/Cas12a-Responsive Smart DNA Hydrogel for Sensitive Electrochemiluminescence Detection of the Huanglongbing Outer Membrane Protein Gene.

Citrus Huanglongbing (HLB) is known as the cancer of citrus, where Candidatus Liberibacter asiaticus (CLas) is the most prevalent strain causing HLB. In this study, we report a novel electrochemiluminescence (ECL) biosensor for the highly sensitive detection of the CLas outer membrane protein (Omp) gene by coupling rolling circle amplification (RCA) with a CRISPR/Cas12a-responsive smart DNA hydrogel. In the presence of the target, a large number of amplicons are generated through RCA. The amplicons activate the trans-cleavage activity of CRISPR/Cas12a through hybridizing with crRNA, triggering the response of smart DNA hydrogel to release the encapsulated AuAg nanoclusters (AuAg NCs) on the electrode and therefore leading to a decreased ECL signal. The ECL intensity change (I0 - I) is positively correlated with the concentration of the target in the range 50 fM to 5 nM, with a limit of detection of 40 fM. The performance of the sensor has also been evaluated with 10 samples of live citrus leaves (five HLB negative and five HLB positive), and the result is in excellent agreement with the gold standard qPCR result. The sensing strategy has expanded the ECL versatility for detecting varying levels of dsDNA or ssDNA in plants with high sensitivity.

Effect of macrophage polarization on parasitic protection against type 1 diabetes mellitus.

Type 1 diabetes mellitus is a chronic disease caused by the destruction of pancreatic beta cells. Based on the hygiene hypothesis, a growing body of evidence suggests a negative association between parasitic infections and diabetes in humans and animal models. The mechanism of parasite-mediated prevention of type 1 diabetes mellitus may be related to the adaptive and innate immune systems. Macrophage polarization is a new paradigm for the treatment of type 1 diabetes mellitus, and different host macrophage subsets play various roles during parasite infection. Proinflammatory cytokines are released by M1 macrophages, which are important in the development of type 1 diabetes mellitus. Parasite-activated M2 macrophages prevent the development of type 1 diabetes mellitus and can influence the development of adaptive immune responses through several mechanisms, including Th2 cells and regulatory T cells. Here, we review the role and mechanism of macrophage polarization in parasitic protection against type 1 diabetes mellitus.

The effects of helminth infections against type 2 diabetes.

Type 2 diabetes mellitus (T2D) is a prevalent inflammation-related disease characterized by insulin resistance and elevated blood glucose levels. The high incidence rate of T2D in Western societies may be due to environmental conditions, including reduced worm exposure. In human and animal models, some helminths, such as Schistosoma, Nippostrongylus, Strongyloides, and Heligmosomoides, and their products reportedly ameliorate or prevent T2D progression. T2D induces adaptive immune pathways involved in the inhibition of type 1 immune responses, promotion of type 2 immune responses, and expansion of regulatory T cells and innate immune cells, such as macrophages, eosinophils, and group 2 innate lymphoid cells. Among immune cells expanded in T2DM, type 2 immune cells and macrophages are the most important and may have synergistic effects. The stimulation of host immunity by helminth infections also promotes interactions between the innate and adaptive immune systems. In this paper, we provide a comprehensive review of intestinal helminths' protective effects against T2D.

Effects of programmed cell death protein 10 on fecundity in Schistosoma japonicum.

Programmed cell death protein 10 (PCDP10) is widely distributed in animal tissues and exerts extensive biological effects. This study aimed to investigate the effect of Schistosoma japonicum PCDP10 (SjPCDP10) on the fecundity of schistosomes. We performed real-time PCR to assess Sjpcdp10 expression levels at different developmental stages of S. japonicum. Immunoprotection against S. japonicum was assessed in vivo in mice, and Sjpcdp10 expression was inhibited via RNA interference (RNAi) to determine its role in fecundity. Real-time PCR analysis revealed that Sjpcdp10 mRNA was expressed during different developmental stages in S. japonicum, reaching maximum and minimum levels in female worms and lung-stage schistosomula, respectively. Recombinant SjPCDP10 had a molecular weight of approximately 28 kDa, displaying good immunogenicity but poor immunoprotection. SjPCDP10 was primarily localized in the egg, eggshell, epiphragm of adult worms, and especially the vitelline glands of female worms. RNAi-mediated knockdown of Sjpcdp10 by greater than 90%, and the protein expression decreased by 73%, reduced the number of eggs per female worm significantly more than RNAi-mediated knockdown of Egfp (negative control) (P < 0.05). The present results indicate that Sjpcdp10 knockdown affects the fecundity of schistosomes and may play a vital role in oogenesis.

Immunization with recombinant schistosome adenylate kinase 1 partially protects mice against Schistosoma japonicum infection.

Highly effective and safe prophylactic vaccines are urgently needed to sustainably control schistosomiasis, one of the most serious endemic zoonoses in China. In this study, we characterized adenylate kinase 1 from Schistosoma japonicum (SjAK1), a phosphotransferase that regulates cellular energy and metabolism, and evaluated its potential as a recombinant vaccine. Based on real-time quantitative PCR, western blot, and immunolocalization, SjAK1 is active throughout the life of the worm, although its expression is higher in 21-day-old schistosomula, adult worms, and eggs deposited in the host liver. Further, the enzyme accumulates in the eggshell, intestinal epithelium, integument of adult worms and in the vitellaria tissue in female worms. A 594-bp full-length complementary DNA (cDNA) encoding SjAK1 was synthesized from total RNA of 3-day-old schistosomes, and immunization with recombinant SjAK1 reduced worm burden by 50%, decreased the density of eggs deposited in the host liver by 40%, and reduced the area of granulomas in the host liver by 56%. ELISA results showed that recombinant SjAK1 also stimulated Th1 cytokines such as IL-2 and IFN-γ, but not IL-5 and IL-4. Collectively, a recombinant form of the enzyme SjAK1 elicits partial protective immunity against Schistosoma japonicum infection and the induction of Th1 cytokines. Thus, the enzyme has potential as a component of a multivalent vaccine against schistosomiasis.

The effect of regulatory T cells in Schistosoma-mediated protection against type 2 diabetes.

In western societies, the prevalence of type 2 diabetes (T2D) is related to the hygiene hypothesis, which implies that reduced exposure to infectious factors results in a loss of the immune stimulation necessary to form the immune system during development. In fact, it has been reported that parasites, such as Schistosoma, can improve or prevent the development of T2D, which may be related to the activity of immune cells, including regulatory T cells (Tregs). Hence, Schistosoma, Tregs, and T2D share a close relationship. Schistosoma infection and the molecules released can lead to an increase in Tregs, which play an important role in the suppression of T2D. In this review, we provide an overview of the role of Tregs in the response to Schistosoma infection and the protective mechanism of Schistosoma-related molecular products against T2D.

LCDM medium supports the derivation of bovine extended pluripotent stem cells with embryonic and extraembryonic potency in bovine-mouse chimeras from iPSCs and bovine fetal fibroblasts.

Cattle have emerged as one of the most important domestic animals widely used for meat, milk, and fur. Derivation of bovine pluripotent stem cells (PSCs) can be applied in drug selecting and human disease modeling and facilitated agriculture-related applications such as production of genetically excellent cattle by gene editing. Extended PSCs (EPSCs), capable of differentiating into embryonic and extraembryonic parts, have been generated in mouse, human, and pig. Whether bovine EPSCs could be generated, and their chimeric competency remains unclear. This study focused on derivation of bovine EPSCs using LCDM medium and exploring the characteristics of EPSCs among different species, including bovine, mouse, and human EPSCs. Here, using LCDM medium (consisting of hLIF, CHIR99021, (S)-(+)-dimethindene maleate, and minocycline hydrochloride) enables the derivation of bovine EPSCs from induced PSCs (iPSCs) and bovine fetal fibroblasts (BFF) with stable morphology, pluripotent marker expression, and in vitro differentiation ability. Notably, bovine EPSCs exhibited interspecies chimeric contribution to embryonic and extraembryonic tissues in pre-implantation blastocysts and postimplantation bovine-mouse chimeras. Transcriptome analysis revealed the unique molecular characteristics of bovine EPSCs compared with iPSCs. The similarities and differences in molecular features across bovine, human, and mouse EPSCs were also described by transcriptome analysis. Taken together, the LCDM culture system containing chemical cocktails can be used for the establishment and long-term passaging of bovine EPSCs with embryonic and extraembryonic potency in bovine-mouse chimeras. Our findings lay the foundation of generating PSCs in domestic animals and open avenues for basic and applied research in biology, medicine, and agriculture. DATABASE: Gene expression data of bovine EPSCs and bovine iPSCs are available in the GEO databases under the accession number PRJNA693452.

Effects of programmed cell death protein 10 on the Schistosoma japonicum female reproductive system.

This study aimed to investigate the effects of programmed cell death protein 10 (PCDP10) on the female reproductive system of Schistosoma japonicum, one of the major infectious agents of schistosomiasis. We found that PCDP10 was widely distributed in the integument, the worm parenchymal area, and the vitellarium of the female worm, but was localized to a lesser extent in the ovary and testicles. RNAi experiments successfully achieved gene knockdown, and the ultrastructural morphology of the adult reproductive organs was observed. The results demonstrated that, compared with those of the negative control group, the number of cortical granules around oocytes decreased and the number of immature oocyte cells increased. Fusion of yolk globules occurred, and the number and the diameter of yolk droplets decreased significantly. Real-time PCR showed that the expression of yolk glands reached its peak before ovulation and then decreased. The TUNEL assay results showed that apoptosis in the RNAi group was significantly higher than that in the negative control group. These results suggested that SjPCDP10 plays an important role in the female reproductive system. In conclusion, PCD10 is involved in oocyte growth and development, especially in eggshell formation, which may provide a reference for further elucidating the molecular mechanism of PCDP10 involved in egg formation and embryo development in Schistosoma japonicum.

Role of adenylate kinase 1 in the integument development of Schistosoma japonicum schistosomula.

Schistosomula antigens play an important role in the growth and development of Schistosoma japonicum. We investigated the role of S. japonicum adenylate kinase 1 (SjAK1) in the growth and development of schistosomula. Quantitative real-time PCR showed that SjAK1 mRNA was expressed in all schistosomula stages, but increased gradually with the development of S. japonicum schistosomula. Using immunohistochemical techniques, the AK1 protein was found to be mainly distributed in the tegument and in some parenchymal tissues of the schistosomula. Double-stranded RNA-mediated knockdown of AK1 reduced AK1 mRNA transcripts by more than 90%; western blot analysis demonstrated that AK1 protein expression decreased by 66%. Scanning electron microscopy following RNA-mediated AK1 knockdown demonstrated that the sensory papillae degenerated significantly. Transmission electron microscopy demonstrated that the mean thickness of the tegument in the SjAK1 interference group was lower than that in the negative control group. Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) suggested that, compared with the negative control group, apoptosis increased in the interference group. These results show that AK1 may be involved in the growth and development of S. japonicum schistosomula, and thus may be a target when developing treatments for schistosomiasis.

Role of regulatory T cells in Schistosoma-mediated protection against type 1 diabetes.

The prevalence of T1D in developed societies is partly based on the hygiene hypothesis, that is, the loss of exposure to infectious agents accompanies the loss of immune stimuli shaping the immune system during development. Indeed, the components of parasites, such as Schistosoma, have been reported to ameliorate or prevent the development of T1D, which might be associated with immune cell activity especially that of regulatory T cells (Tregs). Schistosoma infection can lead to the expansion of Treg. Herein, we provide a comprehensive overview of the involvement of Tregs in the response against Schistosoma infection and the mechanism of Schistosoma-associated host protection against T1D.

[Secretory expression of recombinant porcine zona pellucida glycoprotein-3alpha (rpZP3alpha) in Pichia pastoris].

To obtain the recombinant pZP3alpha protein for the study of the contraceptive vaccines, the DNA sequence (446-1423) encoding purified pZP3alpha was inserted into a vector--pPICZalphaA. The recombinant plasmid pPICZalphaA-pZP3alpha was linearized and then transformed into Pichia pastoris GS115 by electroporation. Engineering strains were attained by screening with zeocin and induced to produce rpZP3alpha in high-density fermentation. Then rpZP3alpha was purified by Cu2+ metal affinity column chromatography from the separated and concentrated fermentative supernatants. The purified rpZP3alpha was identified by SDS-PAGE and Western blot, and the quantity, purity and rate of recovery of the rpZP3alpha were analyzed by Quantity One software. One male rabbit was immunized with the Cu-NTA-purified rpZP3alpha. The antibody responses against rpZP3alpha and porcine ZP were detected by ELISA and the indirect immunofluorescence. Engineering strains expressing rpZP3alpha in secretion were constructed. A 46kD component named rpZP3alpha which can react with anti-pZP3 antibody was purified from fermentative supernatants of engineering strains and the average yield of purified rpZP3alpha obtained from fermentative supernatants was 8mg/L. The purity and the rate of recovery were up to 92% and 63% respectively. The anti-rpZP3alpha antiserum was prepared by immunization of a male rabbit with purified rpZP3alpha. This anti-rpZP3alpha antiserum could react with rpZP3alpha and purified pZP3 in ELISA and bind to porcine zona pellucida which produced bright green fluorescence in the indirect immunofluorescence. The rpZP3alpha (46kD) protein could be successfully expressed in the Pichia pastoris expression system. And this protein retained the immunogenic activity of natural pZP3.