Functional characterization of three novel dense granule proteins in Neospora caninum using the CRISPR-Cas9 system.

Neospora caninum is an obligate intracellular parasite that infects a wide range of mammalian species, and particularly causes abortions in cattle and nervous system dysfunction in dogs. Dense granule proteins (GRAs) are thought to play an important role in the mediation of host-parasite interactions and facilitating parasitism. However, a large number of potential GRAs remain uncharacterized, and the functions of most of the identified GRAs have not been elucidated. Previously, we screened a large number GRAs including NcGRA27 and NcGRA61 using the proximity-dependent biotin identification (BioID) technique. Here, we identified a novel GRA protein NcGRA85 and used C-terminal endogenous gene tagging to determine its localization at the parasitophorous vacuole (PV) in the tachyzoite. We successfully disrupted three gra genes (NcGRA27, NcGRA61 and NcGRA85) of N. caninum NC1 strain using CRISPR-Cas9-mediated homologous recombination and phenotyped the single knockout strain. The NcGRA61 and NcGRA85 genes were not essential for parasite replication and growth in vitro and for virulence during infection of mice, as observed by replication assays, plaque assays and in vitro virulence assays in mice. Deletion of the NcGRA27 gene in the NC1 strain reduced the in vitro replication and growth of the parasite, as well as the pathogenicity of the NC1 strain in mice. In summary, our findings provide a basis for in-depth studies of N. caninum pathogenesis and demonstrate the importance of NcGRA27 in parasite growth and virulence, most likely a new virulence factor of N. caninum.

China's energy policy and sustainable energy transition for sustainable development: green investment in renewable technological paradigm.

It is generally accepted that China is a significant cause of global warming and other climate change consequences. This paper applies panel cointegration tests and autoregressive distributed lag (ARDL) techniques to investigate the interactions among energy policy, technological innovation, economic development, trade openness, and sustainable development using panel data from China from 1990 to 2020. Results explain that renewable energy policy and technology innovation are negatively associated with sustainable development. However, research shows that energy use significantly increases both short-term and long-term environmental damage. The findings show that economic growth has a lasting impact on the environment by distorting it. The findings recommend that politicians and government officials hold the key to attaining a green and clean environment by focusing on developing the proper energy policy mix, urban planning, and pollution prevention without compromising economic growth.

LncXIST Facilitates Iron Overload and Iron Overload-Induced Islet Beta Cell Injury in Type 2 Diabetes through miR-130a-3p/ALK2 Axis.

Iron overload is directly associated with diabetes mellitus, loss of islet beta cell, and insulin resistance. Likewise, long noncoding RNA (lncRNA) is associated with type 2 diabetes (T2D). Moreover, lncRNAs could be induced by iron overload. Therefore, we are going to explore the molecular mechanism of lncRNA XIST in iron overload-related T2D. Real-time quantitative PCR and Western blot were used to detect gene and protein levels, respectively. TUNEL and MTT assay were performed to examine cell survival. The glucose test strip, colorimetric analysis kit, ferritin ELISA kit, and insulin ELISA kit were performed to examine the levels of glycolic, iron, and total iron-binding capacity, ferritin, and insulin in serum. Fluorospectrophotometry assay was used to examine labile iron pool level. XIST was higher expressed in T2D and iron overload-related T2D rat tissues and cells, and iron overload-induced promoted XIST expression in T2D. Higher XIST expression was associated with iron overload in patients with T2D. Knockdown of XIST alleviated iron overload and iron overload-induced INS-1 cells injury. Further, we found that XIST can sponge miR-130a-3p to trigger receptor-like kinase 2 (ALK2) expression. Moreover, knockdown of ALK2 alleviated iron overload and iron overload-induced INS-1 cells injury by inhibiting bone morphogenetic protein 6 (BMP6)/ALK2/SMAD1/5/8 axis but reversed with XIST upregulation, which was terminally boosted by overexpression of miR-130a-3p. XIST has the capacity to promote iron overload and iron overload-related T2D initiation and development through inhibition of ALK2 expression by sponging miR-130a-3p, and that targeting this axis may be an effective strategy for treating patients with T2D.

Biotinylation of the Neospora caninum parasitophorous vacuole reveals novel dense granule proteins.

BACKGROUND: Neospora caninum is an obligate intracellular parasite that invades host cells and replicates within the parasitophorous vacuole (PV), which resists fusion with host cell lysosomal compartments. To modify the PV, the parasite secretes an array of proteins, including dense granule proteins (GRAs). The vital role of GRAs in the Neospora life cycle cannot be overestimated. Despite this important role, only a subset of these proteins have been identified, and most of their functions have not been elucidated. Our previous study demonstrated that NcGRA17 is specifically targeted to the delimiting membrane of the parasitophorous vacuole membrane (PVM). In this study, we utilize proximity-dependent biotin identification (BioID) to identify novel components of the dense granules. METHODS: NcGRA17 was BirA* epitope-tagged in the Nc1 strain utilizing the CRISPR/Cas9 system to create a fusion of NcGRA17 with the biotin ligase BirA*. The biotinylated proteins were affinity-purified for mass spectrometric analysis, and the candidate GRA proteins from BioID data set were identified by gene tagging. To verify the biological role of novel identified GRA proteins, we constructed the NcGRA23 and NcGRA11 (a-e) knockout strains using the CRISPR/Cas9 system and analyzed the phenotypes of these mutants. RESULTS: Using NcGRA17-BirA* fusion protein as bait, we have identified some known GRAs and verified localization of 11 novel GRA proteins by gene endogenous tagging or overexpression in the Nc1 strain. We proceeded to functionally characterize NcGRA23 and NcGRA11 (a-e) by gene knockout. The lack of NcGRA23 or NcGRA11 (a-e) did not affect the parasite propagation in vitro and virulence in vivo. CONCLUSIONS: In summary, our findings reveal that BioID is effective in discovering novel constituents of N. caninum dense granules. The exact biological functions of the novel GRA proteins are yet unknown, but this could be explored in future studies.

NcPuf1 Is a Key Virulence Factor in Neospora caninum.

BACKGROUND: Neospora caninum is an apicomplexan parasite that infects many mammals and particularly causes abortion in cattle. The key factors in its wide distribution are its virulence and ability to transform between tachyzoite and bradyzoite forms. However, the factors are not well understood. Although Puf protein (named after Pumilio from Drosophila melanogaster and fem-3 binding factor from Caenorhabditis elegans) have a functionally conserved role in promoting proliferation and inhibiting differentiation in many eukaryotes, the function of the Puf proteins in N. caninum is poorly understood. METHODS: The CRISPR/CAS9 system was used to identify and study the function of the Puf protein in N. caninum. RESULTS: We showed that N. caninum encodes a Puf protein, which was designated NcPuf1. NcPuf1 is found in the cytoplasm in intracellular parasites and in processing bodies (P-bodies), which are reported for the first time in N. caninum in extracellular parasites. NcPuf1 is not needed for the formation of P-bodies in N. caninum. The deletion of NcPuf1 (ΔNcPuf1) does not affect the differentiation in vitro and tissue cysts formation in the mouse brain. However, ΔNcPuf1 resulted in decreases in the proliferative capacity of N. caninum in vitro and virulence in mice. CONCLUSIONS: Altogether, the disruption of NcPuf1 does not affect bradyzoites differentiation, but seriously impairs tachyzoite proliferation in vitro and virulence in mice. These results can provide a theoretical basis for the development of attenuated vaccines to prevent the infection of N. caninum.

Cryptosporidium parvum upregulates miR-942-5p expression in HCT-8 cells via TLR2/TLR4-NF-κB signaling.

BACKGROUND: Micro (mi)RNAs are small noncoding RNA molecules that function in RNA silencing and post-transcriptional regulation of gene expression. This study investigated host miRNA activity in the innate immune response to Cryptosporidium parvum infection. METHODS: In vitro infection model adopts HCT-8 human ileocecal adenocarcinoma cells infected with C. parvum. The expression of miR-942-5p was estimated using quantitative real-time polymerase chain reaction (qPCR). The TLRs-NF-κB signaling was confirmed by qPCR, western blotting, TLR4- and TLR2-specific short-interfering (si)RNA, and NF-κB inhibition. RESULTS: HCT-8 cells express all known toll-like receptors (TLRs). Cryptosporidium parvum infection of cultured HCT-8 cells upregulated TLR2 and TLR4, and downstream TLR effectors, including NF-κB and suppressed IκBα (nuclear factor of kappa light polypeptide gene enhancer in B cells inhibitor, alpha). The expression of miR-942-5p was significantly upregulated at 4, 8, 12 and 24 h post-infection, and especially at 8 hpi. The results of TLR4- and TLR2-specific siRNA and NF-κB inhibition showed that upregulation of miR-942-5p was promoted by p65 subunit-dependent TLR2/TLR4-NF-κB pathway signaling. CONCLUSIONS: miR-942-5p of HCT-8 cells was significantly upregulated after C. parvum infection, especially at 8 hpi, in response to a p65-dependent TLR2/TLR4-NF-κB signaling. TLR4 appeared to play a dominant role.

GR silencing impedes the progression of castration-resistant prostate cancer through the JAG1/NOTCH2 pathway via up-regulation of microRNA-143-3p.

BACKGROUND: Despite notable progression from a therapeutic point of view, castration resistant prostate cancer (CRPC) remains a clinical significant stumbling block. The current study aimed to elucidate the functional role of the gene glucocorticoid receptor (GR) in CRPC, and identify the contributions of the GR gene in CRPC in connection with microRNA-143-3p (miR-143-3p)/Jagged1 (JAG1)/NOTCH2. METHODS: The expression of GR and miR-143-3p in CRPC tissues and cells as well as JAG1/NOTCH2 expression in CRPC tissues was initially determined by quantitative polymerase chain reaction and Western blot analyses. The relationship among GR, JAG1, NOTCH2 and miR-143-3p was subsequently verified using the dual-luciferase reporter gene assay. ChIP assay confirmed the binding of GR to miR-143-3p promoter. Gain- and loss-function approaches were applied to ascertain the role of GR and miR-143-3p in progression of CRPC. Additionally, xenograft tumor models in nude mice were established to further confirm our results. RESULTS: GR was found to be highly expressed while miR-143-3p was lowly expressed in the CRPC tissues and cells. Silencing GR reduced migration, invasion, proliferation and increased apoptosis of CRPC cells. GR was enriched in the miR-143-3p promoter region and could down-regulate miR-143-3p expression. The overexpression of miR-143-3p led to a reduction in the migration, invasion, proliferation and increased apoptosis of CRPC cells. JAG1 and NOTCH2 were the target genes of miR-143-3p, and GR up-regulated the JAG1/NOTCH2 expression by down-regulating miR-143-3p. Silencing JAG1/NOTCH2 inhibited epithelial-mesenchymal transition and CRPC progression in vitro. Furthermore, the in vitro findings were reproduced in the in vivo experiments. CONCLUSION: The key findings of the current study demonstrated that silencing GR suppressed the progression of CRPC through the JAG1/NOTCH2 pathway via up-regulation of miR-143-3p.

Knockout of MicroRNA-155 Ameliorates the Th17/Th9 Immune Response and Promotes Wound Healing.

MiRNAs are integral for maintaining immune homeostasis and self-tolerance. In this study, qPCR analyses were performed to determine which miRNAs play an important role in wound healing. Next, an experiment in a model of wound healing was performed, and histology, mRNA expression and T-cell subpopulations in wound tissue were analyzed. The accelerated experiments were performed by local injection of either rIL-17A and/or rIL-9 after wound healing. In vitro, the differentiation of Th17/Th9 in miR-155+/+ or miR-155-/- mice was investigated, and the target genes of miR155 were analyzed. From our findings, miR-155-/- in mice promoted wound healing and weakened T cell-mediated inflammation, especially in IL-17/IL-9, and less severe skin fibrosis developed in the mice. rIL-17A and/or rIL-9 could exacerbate inflammatory injury and delay wound healing. We also demonstrated that miR-155-/- led to a defect in the differentiation of Th17/Th9 in vitro, and this effect of IL-17/IL-9 might be related to the expression of C-maf, which is a target gene of miR155. MiR-155 regulated IL-17/IL-9-related inflammation in wound healing and might be a potential therapeutic target to attenuate the inflammatory response in wound tissue and promote the closure of wound injuries.

MicroRNA expression profile of HCT-8 cells in the early phase of Cryptosporidium parvum infection.

BACKGROUND: Cryptosporidium parvum is an important zoonotic parasitic disease worldwide, but the molecular mechanisms of the host-parasite interaction are not fully understood. Noncoding microRNAs (miRNAs) are considered key regulators of parasitic diseases. Therefore, we used microarray, qPCR, and bioinformatic analyses to investigate the intestinal epithelial miRNA expression profile after Cryptosporidium parvum infection. RESULTS: Twenty miRNAs were differentially expressed after infection (four upregulated and 16 downregulated). Further analysis of the differentially expressed miRNAs revealed that many important cellular responses were triggered by Cryptosporidium parvum infection, including cell apoptosis and the inflammatory and immune responses. CONCLUSIONS: This study demonstrates for the first time that the miRNA expression profile of human intestinal epithelium cells is altered by C. parvum infection. This dysregulation of miRNA expression may contribute to the regulation of host biological processes in response to C. parvum infection, including cell apoptosis and the immune responses. These results provide new insight into the regulatory mechanisms of host miRNAs during cryptosporidiosis, which may offer potential targets for future C. parvum control strategies.

An investigation of parasitic infections and review of molecular characterization of the intestinal protozoa in nonhuman primates in China from 2009 to 2015.

Parasites are a well-known threat to nonhuman primate (NHP) populations, and potentially cause zoonotic diseases in humans. In this study, the basic data was provided of the parasites in NHPs and the molecular characterization of the Enterocytozoon bieneusi, Giardia duodenalis, Cryptosporidium spp., and Entamoeba spp. were reviewed, which were found in these samples. A total of 3349 fecal samples were collected from 34 species reared at 17 districts in zoos, farms, free-range, or research laboratories, and examined microscopically. Eleven genera of intestinal parasites were detected: five genera of protozoans (Isospora spp., Entamoeba spp., Giardia sp., Cryptosporidium spp., and Cyclospora spp.) and six genera of helminths (Trichuris spp., Strongyloides spp., Ascaris spp., Physaloptera spp., Ancylostoma spp., and Enterobius spp.). The overall sample prevalence of parasitic infection was 54.1% (1811/3349). Entamoeba spp. was the most prevalent (36.4%, 1218/3349). The infection rate was the highest in free-range animals (73.0%, 670/918) (P < 0.01) and Guangxi Zhuang autonomous region (64.8%, 566/873). Mixed infections were mostly detected for Entamoeba spp., Trichuris spp., and Strongyloides spp.. Molecular characterization was reviewed of Enterocytozoon bieneusi, Giardia duodenalis, Cryptosporidium spp., and Entamoeba spp., as these are zoonotic species or genotypes. This parasitological data for NHPs in China, provides important information for veterinarians and public health authorities for the elimination of such parasites and monitor the potential transmission of zoonotic infections from NHPs.

Occurrence, molecular characterization and predominant genotypes of Enterocytozoon bieneusi in dairy cattle in Henan and Ningxia, China.

BACKGROUND: Enterocytozoon bieneusi is the most frequently diagnosed microsporidian species in humans and a wide range of animals. This study was conducted to assess the prevalence and molecular characteristics of E. bieneusi in dairy cattle in Henan Province of central China and the Ningxia Hui Autonomous Region of northwest China. FINDINGS: Of 879 fresh fecal specimens, 24.3 % (214/879) tested positive for E. bieneusi by nested polymerase chain reaction (PCR) based on the internal transcriber spacer (ITS) gene. The highest infection rate, 46.8 % (51/109, P < 0.0001), was observed in a group of dairy cattle with diarrhea, located in Ningxia. The age groups with higher infection rates were pre-weaned calves (29.3 %, 127/434, P < 0.0001) and post-weaned calves (23.9 %, 63/264, P = 0.006). Sequencing and phylogenetic analysis revealed 20 E. bieneusi ITS genotypes (15 known and five new), including members of Group 1 and Group 2. Genotypes I and J were detected in 64.5 % (138/214) of the E. bieneusi positive specimens. CONCLUSIONS: Genotypes I and J were the dominant genotypes in dairy cattle in the present study. The detection of zoonotic genotypes of E. bieneusi in dairy farms indicates that cattle may play an important role as a reservoir host for zoonotic infections.

Prevalence and molecular characterization of Cryptosporidium spp. and Giardia duodenalis in dairy cattle in Beijing, China.

822 fecal samples from cattle in six areas of Beijing were examined with microscopy for Cryptosporidium oocysts and Giardia cysts. The overall infection rates for Cryptosporidium spp. and Giardia duodenalis were 2.55% and 1.09%, respectively. Cryptosporidium was only detected in calves and heifers, whereas G. duodenalis was found in all age groups. Cryptosporidium spp. were characterized with a PCR-restriction fragment length polymorphism analysis and DNA sequence analysis of the small subunit (SSU) rRNA gene. Two Cryptosporidium species were identified: Cryptosporidium parvum (n=12) and Cryptosporidium andersoni (n=9). Six C. parvum isolates were successfully subtyped with the gp60 gene and three subtypes were detected: IIdA19G1 (n=1), IIdA17G1 (n=1), and IIdA15G1 (n=4). Subtype IIdA17G1 is reported for the first time in cattle worldwide. Nine G. duodenalis isolates were analyzed by sequencing the triosephosphate isomerase (tpi) gene, and only G. duodenalis assemblage E was identified. Therefore, the predominance of C. parvum detected in calves was identical to that found in the Xinjiang Uyghur and Ningxia Hui Autonomous Regions, but differed considerably from that in Henan, Heilongjiang, and Shannxi Provinces. In contrast, the predominance of G. duodenalis assemblage E was more or less similar to its predominance in other areas of China or countries. Our findings confirm the unique character of the C. parvum IId subtypes in China. More systematic studies are required to better understand the transmission of Cryptosporidium and G. duodenalis in cattle in China.

[Comparation of low-temperature plasm radiofrequency and high-intensity focused ultrasound therapy on allergic rhinitis patients with nasal septal deviation].

OBJECTIVE: To compare the therapeutic effects of low-temperature plasm radiofrequency and high-intensity focused ultrasound in treatment of perennial allergic rhinitis (PAR) complicated with nasal septum deviation. METHOD: A total of 224 cases of identified PAR who treated in our hospital were randomly divided into 2 matched groups: low-temperature plasm radiofrequency group (n = 140) and high-intensity focused ultrasound group (n = 84). The therapeutic affection were evaluated by clinical symptoms controlled counting-scores. Inferior turbinate mucosa were collected and examined pathologically before and at 15 days and 6 months after the therapy. RESULT: The follow-up data indicated the effective rate in 15 days was 97.1% in high-intensity focused ultrasound group, and 90.4% in low-temperature plasm radiofrequency group, in 6 months was 90.7% and 85.4% respectively. CONCLUSION: The treatment of high-intensity focused ultrasound in PAR is minimally invasive and safe with reliable efficacy.