circPTN Promotes the Progression of Non-Small Cell Lung Cancer through Upregulation of E2F2 by Sponging miR-432-5p.

Background: Non-small cell lung cancer (NSCLC) is one of the most prevalent cancers, accounting for around 80% of total lung cancer cases worldwide. Exploring the function and mechanism of circRNAs could provide insights into the diagnosis and treatment for NSCLC. Methods: In this study, we collected tumor tissues and adjacent normal tissues from NSCLC patients to detect the expression level of circPTN and analyzed the association of its expression level with the clinicopathological parameter of NSCLC patients. Moreover, the functional engagement of circPTN in NSCLC cells was examined by cell counting kit-8 (CCK-8) cell proliferation assay, transwell migration and invasion assays, and tube formation assay. Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting (WB) analysis were used to detect gene and protein expression, respectively. The molecular targets of cicrPTN were predicted using starBase online resources, which was validated by RNA immunoprecipitation (RIP) and dual-luciferase reporter assay. Results: Compared with adjacent normal tissues, there was a remarkable increase of the circPTN levels in NSCLC tissues. A high level of circPTN expression was associated with more lymph node metastasis (LNM) and advanced TNM stages. Functionally, circPTN knockdown inhibited the proliferation, migration, and invasion and tube formation ability of NSCLC cells. We further demonstrated that circPTN regulated the malignant phenotype of NSCLC cells through targeting the miR-432-5p/E2F2 axis. Conclusion: Together, our results suggest that circPTN, which is upregulated in NSCLC tissues, could serve as a prognostic marker for NSCLC patients. circPTN regulates the malignant progression of NSCLC cells through targeting the miR-432-5p/E2F2 axis, which may be employed as a potential strategy for the management of NSCLC.

Comparative Characterization of CpCDPK1 and CpCDPK9, Two Potential Drug Targets Against Cryptosporidiosis.

As the invasion, egress, and growth of Cryptosporidium spp. are regulated by the calcium ion, calcium-dependent protein kinases (CDPKs) are considered potential drug targets against these pathogens. In this study, we expressed CpCDPK1 of Cryptosporidium parvum encoded by the cgd3_920 gene and CpCDPK9 encoded by the the cgd7_1260 gene in Escherichia coli, and we conducted some comparative studies with quantitative PCR, immunofluorescence staining, and in vitro neutralization assays. By immunofluorescence microscopy, CpCDPK1 was expressed over the entirety of the sporozoites, while CpCDPK9 was mainly expressed in the apical region. The expression of the cgd3_920 gene was the highest at 12 h of the in vitro culture, whereas the expression of the cgd7_1260 gene peaked between 2 h and 6 h. Polyclonal antibodies against these two CpCDPK proteins had similar neutralization efficiency on C. parvum growth, reaching approximately 40%. Of the 50 candidate compounds from the molecular docking of CpCDPK1, 10 had significant in vitro anti-cryptosporidial effects, but only one inhibited enzyme activity. For CpCDPK9, five of the forty-five candidate compounds showed significant in vitro anti-cryptosporidial effects. Results obtained from this study suggest that CpCDPK1 and CpCDPK9 might function differently in C. parvum infection.

Characterization of Calcium-Dependent Protein Kinases 3, a Protein Involved in Growth of Cryptosporidium parvum.

Calcium-dependent protein kinases (CDPKs) are considered promising targets for pharmaceutical intervention of cryptosporidiosis. Whole-genome sequencing has revealed the presence of several CDPKs (CpCDPKs) in Cryptosporidium parvum. In this study, we expressed recombinant CpCDPK3 encoded by the cgd5_820 gene in Escherichia coli. The biologic characteristics and functions of CpCDPK3 were examined using qRT-PCR, immunofluorescence microscopy, and in vitro neutralization assay. The expression of the cgd5_820 gene peaked in merozoites during in vitro culture while the CpCDPK3 protein was expressed in both sporozoites and merozoites. Polyclonal antibodies against CpCDPK3 showed no significant inhibitory effects on host invasion by the parasites. We assessed the inhibitory effects of 46 candidate compounds from molecular docking of CpCDPK3 on both C. parvum development and CpCDPK3 enzyme activities. One compound was identified to be effective. Results of these analyses suggest that CpCDPK3 might play an important role in the growth of C. parvum.

Characterization of Three Calcium-Dependent Protein Kinases of Cryptosporidium parvum.

In Cryptosporidium spp., calcium-dependent protein kinases (CDPKs) are considered promising targets for the development of pharmaceutical interventions. Whole-genome sequencing has revealed the presence of 11 CDPKs in Cryptosporidium parvum (CpCDPKs). In this study, we expressed recombinant CpCDPK4, CpCDPK5, and CpCDPK6 in Escherichia coli. The biological characteristics and functions of these CpCDPKs were examined by using quantitative reverse transcription PCR (qRT-PCR), immunofluorescence microscopy, and an in vitro neutralization assay. The expression of the CpCDPK4 gene peaked at 12 h post-infection, the CpCDPK5 gene peaked at 12 and 48 h, and the CpCDPK6 gene peaked at 2-6 h. CpCDPK4 protein was located in the anterior and mid-anterior regions of sporozoites, and CpCDPK5 protein was located over the entire sporozoites, while CpCDPK6 protein was expressed in a spotty pattern. Immune sera of CpCDPK4 and CpCDPK6 exhibited significant inhibitory effects on host cell invasion, while the immune sera of CpCDPK5 had no effects. These differences in protein localization, gene expressions, and neutralizing capacities indicated that the CpCDPK proteins may have different roles during the lifecycle of Cryptosporidium spp.

Different distribution of Cryptosporidium species between horses and donkeys.

Few studies have been conducted on the distribution of Cryptosporidium species and subtypes in equine animals. In this study, 878 stool specimens were collected during 2015-2019 from 551 donkeys and 327 horses in Shandong, Xinjiang, and Inner Mongolia, China and screened for Cryptosporidium spp. by PCR analysis of the small subunit rRNA gene. The Cryptosporidium species presented were identified by sequence analysis of the PCR products and subtyped by sequence analysis of the 60 kDa glycoprotein gene. The infection rates of Cryptosporidium spp. in horses and donkeys were 3.1% (10/327) and 14.5% (80/551), respectively. Four Cryptosporidium species/genotypes were identified, including C. parvum (in 5 horses), C. hominis (in 75 donkeys), Cryptosporidium horse genotype (in 5 horses and 4 donkeys) and a new genotype that is genetically related to Cryptosporidium mink genotype (in 1 donkey). All C. parvum isolates were subtyped as IIdA19G1, C. hominis as IkA16G1, and horse genotype as VIaA15G4. Data from this study indicate that four Cryptosporidium species are circulating in horses and donkeys in the study areas, with C. hominis as a dominant Cryptosporidium species in only donkeys. Attention should be paid to reduce the transmission of these zoonotic Cryptosporidium spp.

Differential Expression of Three Cryptosporidium Species-Specific MEDLE Proteins.

Cryptosporidium parvum and Cryptosporidium hominis share highly similar proteomes, with merely ~3% divergence in overall nucleotide sequences. Cryptosporidium-specific MEDLE family is one of the major differences in gene content between the two species. Comparative genomic analysis indicated that MEDLE family may contribute to differences in host range among Cryptosporidium spp. Previous studies have suggested that CpMEDLE-1 encoded by cgd5_4580 and CpMEDLE-2 encoded by cgd5_4590 are potentially involved in the invasion of C. parvum. In this study, we expressed in Escherichia coli, the C. hominis-specific member of the MEDLE protein family, ChMEDLE-1 encoded by chro.50507, and two C. parvum-specific members, CpMEDLE-3 encoded by cgd5_4600 and CpMEDLE-5 encoded by cgd6_5480. Quantitative PCR, immunofluorescence staining and in vitro neutralization assay were conducted to assess their biologic characteristics. The expression of the cgd5_4600 gene was high during 12-48 h of the in vitro culture, while the expression of cgd6_5480 was the highest at 2 h. ChMEDLE-1 and CpMEDLE-3 proteins were mostly located in the anterior and mid-anterior region of sporozoites and merozoites, whereas CpMEDLE-5 was expressed over the entire surface of these invasive stages. Polyclonal antibodies against MEDLE proteins had different neutralization efficiency, reaching approximately 50% for ChMEDLE-1 and 60% for CpMEDLE-3, but only 20% for CpMEDLE-5. The differences in protein and gene expression and neutralizing capacity indicated the MEDLE proteins may have different roles during Cryptosporidium invasion and growth.

Characterization of MEDLE-1, a protein in early development of Cryptosporidium parvum.

BACKGROUND: Cryptosporidium spp. are important diarrhea-causing pathogens in humans and animals. Comparative genomic analysis indicated that Cryptosporidium-specific MEDLE family proteins may contribute to host adaptation of Cryptosporidium spp., and a recent study of one member of this family, CpMEDLE-2 encoded by cgd5_4590, has provided evidence supporting this hypothesis. In this study, another member of the protein family, CpMEDLE-1 of Cryptosporidium parvum encoded by cgd5_4580, which is distinct from CpMEDLE-2 and has no signature motif MEDLE, was cloned, expressed and characterized to understand its function. METHODS: CpMEDLE-1 was expressed in Escherichia coli and polyclonal antibodies against the recombinant CpMEDLE-1 protein were prepared in rabbits. Quantitative PCR was used to analyze the expression profile of cgd5_4580 in C. parvum culture. Immunofluorescence staining was used to locate CpMEDLE-1 expression in life-cycle stages, and in vitro neutralization assay with antibodies was adopted to assess the role of the protein in C. parvum invasion. RESULTS: The results indicated that cgd5_4580 had a peak expression at 2 h of C. parvum culture. CpMEDLE-1 was located in the mid-anterior region of sporozoites, probably within the dense granules. The neutralization efficiency of anti-CpMEDLE-1 antibodies was approximately 40%. CONCLUSIONS: The differences in protein and gene expression profiles between CpMEDLE-1 and CpMEDLE-2 suggest that MEDLE proteins have different subcellular locations, are developmentally regulated, could be potentially involved in the transcriptional regulation of the expression of parasite or host proteins and may exert their functions in different stages of the invasion and development process.

Preliminary Characterization of MEDLE-2, a Protein Potentially Involved in the Invasion of Cryptosporidium parvum.

Cryptosporidium spp. are important causes of diarrhea in humans, ruminants, and other mammals. Comparative genomic analysis indicated that genetically related and host-adapted Cryptosporidium species have different numbers of subtelomeric genes encoding the Cryptosporidium-specific MEDLE family of secreted proteins, which could contribute to differences in host specificity. In this study, a Cryptosporidium parvum-specific member of the protein family MEDLE-2 encoded by cgd5_4590 was cloned and expressed in Escherichia coli. Immunofluorescent staining with antibodies generated from the recombinant protein showed the expression of the protein in sporozoites and development stages. In vitro neutralization assay with the antibodies partially blocked the invasion of sporozoites. These results support the potential involvement of MEDLE-2 in the invasion of host cells.