csi-miR-96-5p delivered by Clonorchis sinensis extracellular vesicles promotes intrahepatic cholangiocarcinoma proliferation and migration via the ferroptosis-related PTEN/SLC7A11/GPX4 axis.

BACKGROUND: Clonorchis sinensis (CS) is classified as a group 1 carcinogen and can cause intrahepatic cholangiocarcinoma (ICC). CS extracellular vesicles (CsEVs) play important roles in mediating communication between parasitic helminths and humans. Ferroptosis is a novel cell death mechanism that is mainly induced by lipid peroxidation and iron overload. However, the role of CsEVs in the regulation of ferroptosis in ICC remains unclear. This study aimed to explore the role of CS-secreted miR-96-5p (csi-miR-96-5p) delivered by CsEVs in ICC progression and ferroptosis. METHODS: Tissue samples were collected from ICC patients with CS infection (CS-ICC) or without CS infection (NC-ICC). The levels of csi-miR-96-5p and PTEN gene were determined by quantitative polymerase chain reaction (qPCR) and western blotting, and survival analysis was performed. CsEVs were isolated and identified by ultracentrifugation and transmission electron microscopy. Lentiviruses were used to establish stable cell lines with csi-miR-96-5p mimic expression, PTEN overexpression (PTEN-EXO) and PTEN CRISPR/Cas9-based knockout (PTEN-KO) and their respective negative controls. Cell proliferation was assessed by performing Cell Counting Kit-8 assays in vitro and in a tumor xenograft model in vivo, and cell migration was assessed by performing Transwell assays. Erastin is used to induce ferroptosis. Ferroptosis levels were evaluated using biomarkers. RESULTS: High csi-miR-96-5p and low PTEN expression was observed in CS-ICC tissues and was associated with poor overall survival. csi-miR-96-5p was highly enriched in CsEVs and was taken up by ICC cells. csi-miR-96-5p mimics or PTEN-KO significantly promoted the growth and migration of ICC cells in vitro and in vivo, whereas PTEN-EXO exerted the opposite effect. Mechanistically, csi-miR-96-5p mimics or PTEN-KO inhibited erastin-induced ferroptosis, including reducing the accumulation of Fe2+, lipid reactive oxygen species, and malondialdehyde, increasing the GSH/GSSG ratio and levels of SLC7A11 and GPX4, whereas PTEN-EXOs exerted the opposite effect. CONCLUSIONS: csi-miR-96-5p delivered by CsEVs reduced ferroptosis by regulating the expression of the PTEN/SLC7A11/GPX4 axis, thereby promoting ICC proliferation and migration. For the first time to our knowledge, we found that CS miRNAs could promote tumor development through ferroptosis.

EGR1 functions as a new host restriction factor for SARS-CoV-2 to inhibit virus replication through the E3 ubiquitin ligase MARCH8.

IMPORTANCE: Emerging vaccine-breakthrough severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants highlight an urgent need for novel antiviral therapies. Understanding the pathogenesis of coronaviruses is critical for developing antiviral drugs. Here, we demonstrate that the SARS-CoV-2 N protein suppresses interferon (IFN) responses by reducing early growth response gene-1 (EGR1) expression. The overexpression of EGR1 inhibits SARS-CoV-2 replication by promoting IFN-regulated antiviral protein expression, which interacts with and degrades SARS-CoV-2 N protein via the E3 ubiquitin ligase MARCH8 and the cargo receptor NDP52. The MARCH8 mutants without ubiquitin ligase activity are no longer able to degrade SARS-CoV-2 N proteins, indicating that MARCH8 degrades SARS-CoV-2 N proteins dependent on its ubiquitin ligase activity. This study found a novel immune evasion mechanism of SARS-CoV-2 utilized by the N protein, which is helpful for understanding the pathogenesis of SARS-CoV-2 and guiding the design of new prevention strategies against the emerging coronaviruses.

A Single-Pass Type I Membrane Protein from the Apicomplexan Parasite Cryptosporidium parvum with Nanomolar Binding Affinity to Host Cell Surface.

Cryptosporidium parvum is a globally recognized zoonotic parasite of medical and veterinary importance. This parasite mainly infects intestinal epithelial cells and causes mild to severe watery diarrhea that could be deadly in patients with weakened or defect immunity. However, its molecular interactions with hosts and pathogenesis, an important part in adaptation of parasitic lifestyle, remain poorly understood. Here we report the identification and characterization of a C. parvum T-cell immunomodulatory protein homolog (CpTIPH). CpTIPH is a 901-aa single-pass type I membrane protein encoded by cgd5_830 gene that also contains a short Vibrio, Colwellia, Bradyrhizobium and Shewanella (VCBS) repeat and relatively long integrin alpha (ITGA) N-terminus domain. Immunofluorescence assay confirmed the location of CpTIPH on the cell surface of C. parvum sporozoites. In congruence with the presence of VCBS repeat and ITGA domain, CpTIPH displayed high, nanomolar binding affinity to host cell surface (i.e., Kd(App) at 16.2 to 44.7 nM on fixed HCT-8 and CHO-K1 cells, respectively). The involvement of CpTIPH in the parasite invasion is partly supported by experiments showing that an anti-CpTIPH antibody could partially block the invasion of C. parvum sporozoites into host cells. These observations provide a strong basis for further investigation of the roles of CpTIPH in parasite-host cell interactions.

MicroRNA profiling of Neospora caninum tachyzoites (NC-1) using a high-throughput approach.

Neospora caninum is an important pathogen commonly causing spontaneous abortion in livestock. The parasite is known to remain in cysts in an inactive state; or it can undergo expansive development within an intermediate host. However, the mechanisms that trigger the proliferation of N. caninum have not been thoroughly elucidated. For various organisms, it has been demonstrated that microRNAs (miRNAs) can act as important endogenous regulatory factors in gene regulation during cell differentiation and development. However, miRNAs and their function have not been studied in N. caninum. In this study, small RNA libraries from N. caninum tachyzoites (NC-1 strain) were analyzed using a high-throughput RNA sequencing technology combined with systematic bioinformatics analysis. A considerable number of novel miRNAs from N. caninum NC-1 strain tachyzoites were identified. Of the 300 miRNAs found by bioinformatics analysis, 10 were conserved miRNAs belonging to 10 metazoan miRNA families, while 290 were novel miRNAs. The expression of 13 novel miRNAs was verified by real-time quantitative PCR (qRT-PCR). Data from this study provided and identified authentic miRNAs for the first time in N. caninum. The study also introduces a framework for further investigations of RNAi-dependent regulatory mechanisms of the parasite and provides data for further understanding of N. caninum development.

Low-viscosity sodium alginate combined with TiO2 nanoparticles for improving neuroblastoma treatment.

Titanium dioxide (TiO2) nanoparticles have been explored to prevent various cancer developments but it may cause oxidation, inflammation and high cytotoxicity. Alginate has nontoxic, anti-inflammatory, and antioxidant effects. We aimed to explore the effects of alginate-TiO2 temozolomide (TMZ) nanoparticles on neuroblastoma. A neuroblastoma model was established with neuroblastoma cells and alginate-TiO2 TMZ nanoparticles were made by spraying low-viscosity sodium alginate (250-360 kDa). The morphology of nanoparticles was observed via scanning electron microscope (SEM). The crystallinity values were analyzed via X-ray diffraction (XRD) and Fourier transform infrared (FTIR) spectroscopic study. Neuroblastoma mice were treated with saline solution, TMZ, TiO2-TMZ and alginate-TiO2-TMZ nanoparticles. Anti-oxidant, anti-inflammatory, and anti-tumor properties and the mouse survival rates were measured. The spectrometric profiles of alginate-TiO2 were consistent with those of TiO2 and alginate. Alginate-TiO2 TMZ nanoparticles had higher cytotoxicity toward neuroblastoma cells and less inhibitory activity toward normal neuronal cells. The combined nanoparticles increased antioxidant, anti-inflammatory and antitumor activities and prolonged the survival time of the neuroblastoma model (P < 0.05). On the other hand, Alginate-TiO2 TMZ nanoparticles reduced the levels of mitogen-activated protein kinases (MAPKs) and nuclear factor-κB (NF-κB). The combined nanoparticles improved neuroblastoma treatment by affecting NF-κB and MAPK signals.

Chitosan Oligosaccharides Show Protective Effects in Coronary Heart Disease by Improving Antioxidant Capacity via the Increase in Intestinal Probiotics.

We explored the effects of chitosan oligosaccharides (COS) on coronary heart disease (CHD) patients. The component of COS was measured by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). CHD patients were evenly assigned into the COS group (COG) and the placebo group (CG). The duration of treatment was 6 months and therapeutic results were explored by measuring left ventricular ejection fraction (LVEF) value, Lee scores, quality of life (QOL), blood urea nitrogen, and serum creatinine. The intestinal flora were determined by 16s rDNA sequencing. The circulating antioxidant levels and lipid profiles were compared between two groups. There were 7 different degrees of polymerization (DP4-10) in COS. Lee scores, QOL scores, and LVEF values in the COG group were higher than those in the CG group (P < 0.05). COS treatment improved blood urea nitrogen and serum creatinine when compared with controls (P < 0.05). Circulating antioxidant levels were higher in the COG group than in the CG group. COS consumption increased the serum levels of SOD and GSH and reduced the levels of ALT and AST (P < 0.05). Meanwhile, lipid profiles were improved in the COG group. COS consumption increased the abundance of Faecalibacterium, Alistipes, and Escherichia and decreased the abundance of Bacteroides, Megasphaera, Roseburia, Prevotella, and Bifidobacterium (P < 0.05). On the other hand, COS consumption increased the probiotic species Lactobacillus, Lactococcus, and Phascolarctobacterium. The increased species have been reported to be associated with antioxidant properties or lipid improvement. COS had similar effects with chitohexaose on the growth rate of these species. Therefore, COS ameliorate the symptoms of CHD patients by improving antioxidant capacities and lipid profiles via the increase of probiotics in the intestinal flora.

Oral Administration of Prunella vulgaris L Improves the Effect of Taxane on Preventing the Progression of Breast Cancer and Reduces Its Side Effects.

We aimed to explore the efficacy and safety of Prunella vulgaris L (PVL) combined with taxane for treatment of patients with breast cancer (BC). The main ingredients of PVL were analyzed by high-performance liquid chromatography (HPLC). In the experiment, 424 patients with BC were evenly assigned into two groups: experimental group (EG, oral administration of PVL and taxane) and control group (CG, oral administration of placebo and taxane). The primary endpoint was pathologic complete response (pCR), which was evaluated using Miller and Payne system. The secondary endpoints included adverse events (AE) and overall survival (OS), which were evaluated by Common Terminology Criteria for Adverse Event version and Kaplan-Meier curves, respectively. Response Evaluation Criteria in Solid Tumors was used to evaluate the clinical efficacy of PVL. Estrogen receptor (ER) status was also measured. The main side effects were compared between the two groups. The main ingredients of PVL were caffeic acid and rosmarinic acid, which both exert anti-tumor properties. The average follow-up time was 41 months. Eighteen and 31 patients dropped out from EG and CG, respectively. Overall, pCRs were detected in 94 cases (25.1%), comprising 61 cases (31.4%) from EG and 33 cases (18.2%) from CG (P < 0.05). PVL treatment improved the pCR rate and OS time compared with those in CG (P < 0.05). The 3-year OS rates were 86.5 and 77.2% in patients from EG and CG, respectively (P < 0.05). Moreover, ER status was associated with pCR rate and could be an independent prognostic factor in BC. Moreover, treatment with PVL prevented side effects, namely, neutrophil-reduced fever and anemia caused by chemotherapy. Hence, chemotherapy using PVL and taxane could be a safe and effective treatment for patients with BC. PVL may be a potential adjuvant medicine for BC treatment.

MiR-29a-5p inhibits proliferation and invasion and induces apoptosis in endometrial carcinoma via targeting TPX2.

This study was aimed to explore the effects of miR-29a-5p expression and its target gene TPX2 (target protein for Xenopus kinesin-like protein 2) on endometrial cancer (EC) devel on EC development and to assess the prognostic impacts of TPX2. Microarray-based GEO and TCGA (the Cancer Genome Atlas) EC expression data were used to identify differentially expressed miRNAs and mRNAs. The observed potential target relationship between miR-29a-5p and TPX2 was verified using TargetScan and luciferase reporter assays. The mRNA and protein expression levels of miR-29a-5p and TPX2 were confirmed by qRT-PCR and western blot, respectively. Associations between TPX2 expression and patient prognosis were assessed using Kaplan-Meier and log-rank assays. Changes in EC-derived cell proliferation, invasion and apoptosis after exogenous miR-29a-5p and TPX2 over-expression and/or silencing were assessed using CCK-8 (cell counting kit-8), colony formation, Transwell and flow cytometry assays, respectively. We found that in primary EC tissues the expression of miR-29a-5p was down-regulated and the expression of TPX2 was up-regulated. We also found that low expression of TPX2 were associated with a better prognosis, and vice versa. Subsequent exogenous miR-29a-5p over-expression and TPX2 silencing could inhibit EC-derived cell proliferation and invasion, and to induce apoptosis. We also found that miR-29a-5p might target and repress TPX2, thereby inhibiting EC-derived cell proliferation and invasion and enhancing apoptosis. We conclude that miR-29a-5p could inhibit the proliferation and invasion of EC-derived cells and enhance the apoptosis of EC-derived cells via TPX2 down-regulation. A high TPX2 expression in primary EC tissues was found to be associated with a poor prognosis. As such, these biomarkers may serve as promising prognostic indicators. ABBREVIATIONS: EC: Endometrial cancer; 3'-UTR: 3'-untranslated regions; TPX2: target protein for Xenopus kinesin-like protein 2; TCGA: the Cancer Genome Atlas; UCEC: uterine corpus endometrial carcinoma; CCK-8: cell counting kit-8; OD: optical density; FCM: flow cytometry; EMT: epithelial-mesenchymal transition.

Distinct Clinical Characteristics of Pediatric Guillain-Barré Syndrome: A Comparative Study between Children and Adults in Northeast China.

OBJECTIVE: Clinical characteristics of pediatric Guillain-Barré syndrome (GBS) have been extensively studied whereas scarcely been compared with those of adult GBS. Herein we compared the clinical features of GBS between pediatric and adult patients. METHODS: We retrospectively collected the clinical data of 750 patients with GBS (541 adults and 209 children), and compared the clinical characteristics between children and adults. RESULTS: Pain was a more frequent complaint in children (17.2% vs 9.6%, p < 0.01), who were also found with shorter interval from disease onset to nadir (6.3d vs 7.3d, p < 0.01) and higher incidence of bulbar dysfunction (22.0% vs 14.8%, p < 0.05). The disease severity in children was comparable with adults. In addition, a higher incidence of pediatric GBS was found in summer, especially in July and August (both p < 0.01). However, the incidence of antecedent infections of different seasons in adult and pediatric patients was comparable (p > 0.05). The clinical features of acute motor axonal neuropathy (AMAN) and acute inflammatory demyelinating polyneuropathy (AIDP) in children were overall comparable with adult ones (p > 0.05). Similar to adults, bulbar dysfunction (odds ratio [OR]: 4.621, 95% confidence interval [CI]: 1.240-17.218, p < 0.05) and lower nadir Medical Research Council (MRC) sum score (OR: 0.897, 95% CI: 0.855-0.941, p < 0.01) were also risk factors for mechanical ventilation in children. However, distinct from adult ones, autonomic dysfunction was significantly higher in mechanically ventilated childhood GBS (39.1% vs 8.8%, p < 0.01), which also served as a predictor for mechanical ventilation in pediatric GBS (OR: 70.415, 95% CI: 9.265-535.158, p < 0.01). As to the efficacy of intravenous immunoglobulin, insignificant difference was identified between children and adults. CONCLUSION: The clinical features of pediatric GBS differ from those of adults. Autonomic dysfunction is an independent risk factor for mechanical ventilation in pediatric patients.

Prevalence and molecular characterization of Giardia duodenalis isolates from dairy cattle in northeast China.

Giardia duodenalis is an important zoonotic intestinal parasite responsible for diarrhea in humans and other animals worldwide. The present study was conducted to assess the prevalence of bovine giardiosis and to perform molecular characterization of Giardia duodenalis in the northeast of China. A total of 655 fecal specimens were collected from dairy cattle in 15 farms located in three different provinces. G. duodenalis assemblages and subtypes were determined by sequence analysis of the triosephosphate isomerase (TPI) gene. As a whole, the G. duodenalis infection rate in dairy cattle was 7.9% (52/655), as determined by Lugol's iodine staining. Two assemblages were identified, namely, the potentially zoonotic assemblage A (n = 1), the livestock-specific assemblage E (n = 50), and a mixed infection case of assemblages A and E. Seven distinct subtypes of E assemblages were identified and E-XI, E-I and E-III are the major subtypes. Only subtype A-I was identified in assemblage A. Findings relevant to assemblage A are of public health importance. The results indicated the livestock-specific assemblage E is the major genotype and zoonotic assemblage A or B occurs very seldomly which is significantly different with previous report in the same area. So that determination of genotypes in individual epidemiological setting can make important contributions to public health.

Higher frequency of peripheral blood follicular regulatory T cells in patients with new onset ankylosing spondylitis.

Follicular helper T (TFH) cells and B cells are linked to the pathogenesis of ankylosing spondylitis (AS). Follicular regulatory T (TFR) cells suppress TFH cell and germinal center B cell numbers in vivo. The role of TFR cells in AS is unknown. The frequency of peripheral blood inducible FOXP3+CXCR5+CD4+TFR cells and CXCR5+CD4+TFH cells were taken from 20 onset AS patients and 10 healthy controls, and were examined by flow cytometry, their disease activity were measured by the Bath Ankylosing Spondylitis Disease Activity Index. The concentrations of serum interleukin (IL)-21, immunoglobulin G, immunoglobulin A, immunoglobulin M and C-reactive protein were examined, and the values of erythrocyte sedimentation rate were measured. The frequency of peripheral blood FOXP3+CXCR5+CD4+TFR cells, CXCR5+CD4+TFH cells, the ratio of FOXP3+CXCR5+CD4+TFR/CXCR5+CD4+TFH cells and the concentration of serum IL-21 in the AS patients were significantly higher than those in the healthy controls (P < 0.0001, P = 0.0027, P < 0.0001, P = 0.0039, respectively). The frequency of FOXP3+CXCR5+CD4+TFR cells and the ratio of FOXP3+CXCR5+CD4+TFR/CXCR5+CD4+TFH cells still significantly rose in those patients after standard treatment (P = 0.0006, P < 0.0001), the concentration of serum IL-21 decreased after treatment (P = 0.0049), accompanied by significantly minimized disease activities. Furthermore, the TFR cells were negatively correlated with serum immunoglobulin A in those patients before treatment (r = -0.582, P = 0.0071), and the frequency of TFR cells was negatively correlated with that of TFH cells and the concentration of serum IL-21 after treatment (r = -0.550, P = 0.046; r = -0.581, P = 0.0371). TFR cells might participate in the pathogenesis of AS, and might be responsible for controlling the autoantibodies, the frequency and function of TFH cells to inhibit the development of AS.

[Serological detection of Cryptosporidium spp. infection in outpatients in Changchun].

CP23 gene of Cryptosporidium parvum was expressed in Escherichia coli, and the recombinant protein was purified. Its immunoreactivity was analyzed by Western blotting. Serum samples were collected from outpatients of different ages from August to November, 2010 in Changchun. Indirect ELISA was established to detect the anti-CP23 IgG in sera. Western blotting analysis indicated that the recombinant CP23 protein was recognized by sera from Cryptosporidium panum-infected calves and positive human sera, but not recognized by sera of mice infected with Schistosoma japonicum, sera from falciparum malaria patients and negative human sera. The overall anti-CP23 IgG positive rate was 3.2% (65/2 046). The seropositive rate was 2.7% (28/1 036) in men and 3.7% (37/1 010) in women (P > 0.05). The seropositive rates were significantly different among age groups (P < 0.05), and the age group of 71-80 had the highest positive rate (8.6%, 13/152).

Seroprevalence of Toxoplasma gondii infection in female sterility patients in China.

Toxoplasmosis is an important parasitic disease worldwide and is related to certain psychiatric disorders and sterility. In the present study, serum samples from 882 female sterility patients and 107 pregnant-puerperant women were assayed for anti- T. gondii IgG antibodies using ELISA. The overall T. gondii seroprevalence was 14.8%. In the female sterility patients, 15.9% (140/882) were seropositive and, in the pregnant-puerperant women, 5.6% (6/107) were positive for anti- T. gondii IgG antibodies. There was a significant difference between the 2 groups ( P < 0.05). The samples were further divided into 5 groups based on age, but no significant difference was found among the 5 groups (P > 0.05). Results of the present study argue for more attention to prevention of T. gondii infection in the female population and, in particular, women of childbearing age.