RNA-Guided AsCas12a- and SpCas9-Catalyzed Knockout and Homology Directed Repair of the Omega-1 Locus of the Human Blood Fluke, Schistosoma mansoni.

The efficiency of the RNA-guided AsCas12a nuclease of Acidaminococcus sp. was compared with SpCas9 from Streptococcus pyogenes, for functional genomics in Schistosoma mansoni. We deployed optimized conditions for the ratio of guide RNAs to the nuclease, donor templates, and electroporation parameters, to target a key schistosome enzyme termed omega-1. Programmed cleavages catalyzed by Cas12a and Cas9 resulted in staggered- and blunt-ended strand breaks, respectively. AsCas12a was more efficient than SpCas9 for gene knockout, as determined by TIDE analysis. CRISPResso2 analysis confirmed that most mutations were deletions. Knockout efficiency of both nucleases markedly increased in the presence of single-stranded oligodeoxynucleotide (ssODN) template. With AsCas12a, ssODNs representative of both the non-CRISPR target (NT) and target (T) strands were tested, resulting in KO efficiencies of 15.67, 28.71, and 21.43% in the SpCas9 plus ssODN, AsCas12a plus NT-ssODN, and AsCas12a plus T-ssODN groups, respectively. Trans-cleavage against the ssODNs by activated AsCas12a was not apparent in vitro. SpCas9 catalyzed more precise transgene insertion, with knock-in efficiencies of 17.07% for the KI_Cas9 group, 14.58% for KI_Cas12a-NT-ssODN, and 12.37% for KI_Cas12a-T-ssODN. Although AsCas12a induced fewer mutations per genome than SpCas9, the phenotypic impact on transcription and expression of omega-1 was similar for both nucleases.

Relationship between salt-inducible kinase 2 (SIK2) and lymph node metastasis in colorectal cancer patients complicated with chronic schistosomiasis.

To investigate the relationship between salt-inducible kinase 2 (SIK2) and lymph node metastasis in colorectal cancer patients complicated with chronic schistosomiasis. Tissue specimens were collected from 363 patients who were diagnosed as colorectal cancer by clinical and pathological examination in Wuhu Second People's Hospital from June 2015 to June 2020. Fifty-six patients were colorectal cancer complicated with schistosomiasis (CRC-S) and 307 patients were colorectal cancer not complicated with schistosomiasis (CRC-NS). The clinical and pathological data of the patients were analyzed to explore the relationship between chronic schistosomiasis and colorectal cancer. Immunohistochemistry and Western blotting were used to detect the distribution and expression of SIK2 in colorectal cancer specimens. The relationship between SIK2 and lymph node metastasis of CRC-S was analyzed. The rate of lymph node metastasis in CRC-S group was significantly higher than that in CRC-NS group (62.5% vs. 47.2%, <0.05). In CRC-S patients with lymph node metastasis, schistosome eggs were distributed mainly in tumor tissues (25/35, 71.4%), while in patients with CRC-S without lymph node metastasis, schistosome eggs were distributed mainly in paracancerous tissues (17/21, 81.0%) (14.243, <0.01). The SIK2 was mainly located in cytosol, and its expression in tumor tissues was higher than that in paracancerous tissues. Compared with CRC-NS patients, the expression of SIK2 in CRC-S patients was significantly increased; the expression of SIK2 in patients with lymph node metastasis was higher than that in patients without lymph node metastasis; and the expression of SIK2 in patients with schistosome eggs in cancer tissues was higher than that in patients with schistosome eggs in paracancerous tissues (all <0.01). Lymph node metastasis is more likely to be occurred in colorectal cancer patients with schistosomiasis, especially in those with schistosome eggs in tumor tissues. The expression of SIK2 may be correlated with chronic schistosomiasis, egg distribution and lymphatic metastasis.

[Diagnosis and treatment of an imported case of schistosomiasis haematobium].

OBJECTIVE: To report the diagnosis and treatment of an imported case of schistosomiasis haematobium, including the pathological features of the disease and therapeutic efficacy of praziquantel. METHODS: The data of the patient with schistosomiasis haematobium were collected, and the pathological features of the bladder tissue were observed under a microscope. More-over, the patient was treated with praziquantel, and his urine was collected before and after the treatment. The eggs in the urine were examined by a microscope after sediment and the miracidia were hatched. RESULTS: The patient once worked in Angola for three months, and after returning home he had the symptoms of intermittent painless terminal hematuresis. It was ineffective after anti-inflammatory treatment in a number of hospitals. There were no sand spots discovered under the cystoscope. However, the inflammatory reaction to parasite with a lot of eosinophils infiltration in the bladder mucosa was found on the pathological sections under a microscope, and the egg structure was observed with individual characteristics. The eggs were detected in the urine and the miracidia were hatched before the praziquantel treatment. The hematuria symptoms disappeared after the praziquantel treatment. The eggs were still detected in the urine 7 days post-treatment, but the miracidium could not be hatched. One month and 6 months post-treatment, the eggs were not detected in the urine. CONCLUSIONS: The imported cases of schistosomiasis haematobium are often misdiagnosed, and therefore, it is necessary to strength the health education to the workers overseas and also to improve the ability of diagnosis in medical staff. For the case reported in this paper, there are typical structure of Schistosoma haematobium eggs and egg-granulomas on the pathological sections of bladder tissues. Praziquantel has satisfactory treatment results.

Corilagin Counteracts IL-13Rα1 Signaling Pathway in Macrophages to Mitigate Schistosome Egg-Induced Hepatic Fibrosis.

The IL-13Rα1 signaling pathway and M2 macrophages play crucial roles in schistosome egg-induced hepatic fibrosis via the expression of pro-fibrotic molecules. This study aims to investigate the inhibitory effect and mechanism of action of corilagin on schistosome egg-induced hepatic fibrosis via the IL-13Rα1 signaling pathway in M2 macrophages in vitro and in vivo. The mRNA and protein expression of IL-13Rα1, PPARγ, KLF4, SOCS1, STAT6, p-STAT6, and TGF-ß was measured in vitro with corilagin treatment after IL-13 stimulation and in vivo corilagin treatment after effectively killing the adult schistosomes in schistosome-infected mice. Histological analysis of liver tissue was assessed for the degree of hepatic fibrosis. The results revealed that corilagin significantly reduced the expression of PPARγ, KLF4, SOCS1, p-STAT6, and TGF-ß compared with model group and praziquantel administration (p < 0.01 or p < 0.05) in vivo and in vitro, which indicated a strong inhibitory effect of corilagin on IL-13Rα1 signaling pathway. As well, the inhibitory effect of corilagin showed a significant dose-dependence (p < 0.05). The area of fibrosis and distribution of M2 macrophages in mouse liver tissue were reduced significantly and dose-dependently with corilagin treatment compared to model group or praziquantel administration (p < 0.01 or p < 0.05), indicating that corilagin suppressed IL-13Rα1 signaling pathway and M2 macrophage polarization effectively in vivo. Furthermore, the anti-fibrogenic effect persisted even when IL-13Rα1 was up- or down-regulated in vitro. In conclusion, corilagin can suppress schistosome egg-induced hepatic fibrosis via inhibition of M2 macrophage polarization in the IL-13Rα1 signaling pathway.

Prospects for Vector-Based Gene Silencing to Explore Immunobiological Features of Schistosoma mansoni.

Schistosomiasis is a prevalent, socioeconomically important disease of humans caused by parasites of the genus Schistosoma (schistosomes or blood flukes). Currently, more than 200 million people worldwide are infected with schistosomes. Despite major research efforts, there is only one drug routinely used for effective treatment, and no vaccine is available to combat schistosomiasis. The purpose of the present article is to (1) provide a background on the parasites and different forms of disease; (2) describe key immunomolecular aspects of disease induced in the host; and (3) critically appraise functional genomic methods employed to explore parasite biology, parasite-host interactions and disease at the molecular level. Importantly, the article also describes the features and advantages of lentiviral delivery of artificial microRNAs to silence genes. It also discusses the first successful application of such an approach in schistosomes, in order to explore the immunobiological role of selected target proteins known to be involved in egg-induced disease. The lentiviral transduction system provides exciting prospects for future, fundamental investigations of schistosomes, and is likely to have broad applicability to other eukaryotic pathogens and infectious diseases. The ability to achieve effective and stable gene perturbation in parasites has major biotechnological implications, and might facilitate the development of radically new methods for the treatment and control of parasitic diseases.