Distinct role of nuclear receptor corepressor 1 regulated de novo fatty acids synthesis in liver regeneration and hepatocarcinogenesis in mice.

It is urgent that the means to improve liver regeneration (LR) be found, while mitigating the concurrent risk of hepatocarcinogenesis (HCG). Nuclear receptor corepressor 1 (NCoR1) is a co-repressor of nuclear receptors, which regulates the expression level of metabolic genes; however, little is known about its potential contribution for LR and HCG. Here, we found that liver-specific NCoR1 knockout in mice (NCoR1Δhep ) dramatically enhances LR after partial hepatectomy and, surprisingly, blocks the process of diethylnitrosamine (DEN)-induced HCG. Both RNA-sequencing and metabolic assay results revealed improved expression of Fasn and Acc2 in NCoR1Δhep mice, suggesting the critical role of de novo fatty acid synthesis (FAS) in LR. Continual enhanced de novo FAS in NCoR1Δhep mice resulted in overwhelmed adenosine triphosphate ATP and nicotinamide adenine dinucleotide phosphate (NADPH) consumption and increased mitochondrial reactive oxygen species production, which subsequently attenuated HCG through inducing apoptosis of hepatocytes at an early stage after DEN administration. CONCLUSION: NCoR1 functions as a negative modulator for hepatic de novo FAS and mitochondria energy adaptation, playing distinct roles in regeneration or carcinogenesis. (Hepatology 2018;67:1071-1087).

Choline Kinase α Mediates Interactions Between the Epidermal Growth Factor Receptor and Mechanistic Target of Rapamycin Complex 2 in Hepatocellular Carcinoma Cells to Promote Drug Resistance and Xenograft Tumor Progression.

BACKGROUND & AIMS: Choline kinase α (CHKA) catalyzes conversion of choline to phosphocholine and can contribute to carcinogenesis. Little is known about the role of CHKA in the pathogenesis of hepatocellular carcinoma (HCC). METHODS: We performed whole-exome and transcriptome sequence analyses of 9 paired HCC and non-tumor-adjacent tissues. We performed tissue chip analyses of 120 primary HCC and non-tumor-adjacent tissues from patients who received surgery in Shanghai, China from January 2006 through December 2009; 48 sets of specimens (HCC and non-tumor-adjacent tissues) were also analyzed. CHKA gene copy number was quantified and findings were validated by quantitative reverse transcription polymerase chain reaction analysis. CHKA messenger RNA and protein levels were determined by polymerase chain reaction, immunohistochemical, and immunoblot analyses. CHKA was examined in 2 hepatocyte cell lines and 7 HCC-derived cell lines, and knocked down with small interfering RNAs in 3 HCC cell lines. Cells were analyzed in proliferation, wound healing, migration, and invasion assays. Cells were injected into tail veins of mice and tumor growth and metastasis were quantified. Immunoprecipitation and immunofluorescence assays were conducted to determine interactions between CHKA and the epidermal growth factor receptor (EGFR) and the mechanistic target of rapamycin complex 2. RESULTS: Levels of CHKA messenger RNA were frequently increased in HCC tissues compared with nontumor tissues; increased expression was associated with amplification at the CHKA loci. Tumors that expressed high levels of CHKA had more aggressive phenotypes, and patients with these tumors had shorter survival times after surgery compared to patients whose tumors expressed low levels of CHKA. HCC cell lines that stably overexpressed CHKA had higher levels of migration and invasion than control HCC cells, and formed larger xenograft tumors with more metastases in mice compared to HCC cells that did not overexpress CHKA. CHKA was required for physical interaction between EGFR and mechanistic target of rapamycin complex 2. This complex was required for HCC cells to form metastatic xenograft tumors in mice and to become resistant to EGFR inhibitors. CONCLUSIONS: We found levels of CHKA to be increased in human HCCs compared to nontumor tissues, and increased expression to be associated with tumor aggressiveness and reduced survival times of patients. Overexpression of CHKA in HCC cell lines increased their invasiveness, resistance to EGFR inhibitors, and ability to form metastatic tumors in mice by promoting interaction of EGFR with mechanistic target of rapamycin complex 2.

[Results of Soil-transmitted Nematode Monitoring in Henan Province in 2011-2015].

To understand the endemic situation of soil-transmitted nematodiasis (Ascaris lumbricoides, Trichuris trichiura and Ancylostoma sp.) in Huaiyang County, Henan Province. Over 1 000 fecal samples from inhabitants in Huaiyang County of Henan Province were collected each year during 2011-2015, in which the soil-transmitted nematodes eggs and other intestinal helminth eggs were examined by Kato-Katz technique. The helminth-positive samples were examined by filter paper culture method to identify the species of hookworm. The cellophane swab method was used to detect Enterobius vermicularis eggs in children aged 3～12 years. Soil samples were collected from vegetable field, lavatory, courtyard and kitchen of 10 families randomly selected in each year to examine Ascaris eggs by a modified saturated sodium nitrate floatation method. In 2011-2015, 5 229 people were examined and 54 person infected with intestinal helminths were found. Five intestinal helminthes, A. lumbricoides, T. trichiura, Ancylostoma duodenale, Enterobius vermicularis and Trichostrongylus orientalis were found with 13, 2, 9, 29 and 1 infection person respectively. All showed mild infection and no multiple infections were found. There was no significant difference between the year 2015 which had the highest soil-transmitted nematode infection rate 0.6%（7/1 134） and the year 2013 which had the lowest infection rate 0.3%（3/1 037）（P>0.05）. The infection rate of intestinal helminths was highest in group of <10 years（2.8%, 25/905）, followed by the groups of >70 years（1.6%, 4/256） and 30～40 years（1.2%, 8/671）（P>0.05）. The average infection rate of E. vermicularis was 1.8%（18/993） The infection rate of E. vermicularis was relatively higher in kindergarten kids（1.6%, 6/366） and students（1.3%, 13/1 005） than that in farmers（0.3%, 10/3 782）（P<0.01）. No Ascaris eggs were found in the 200 randomly collected soil samples. The intestinal helminth infection status maintaines at low level in Henan Province during 2011-2015.

[The Levels of IL-4, IL-9, and IgE in Patients Infected with Intestinal Helminths and their Clinical Values].

OBJECTIVE: To investigate the serum levels of interleukin-4 (IL-4), interleukin-9 (IL-9), and immunoglobulin E (IgE) in the patients infected with intestinal helminths, and study their relationship to the clinical symptoms or species of the helminths. METHODS: This study was carried out in the Department of Paediatrics, Henan Provincial People's Hospital from January 2010 to July 2014. The blood samples were collected from 55 infected patients. Among the 55 cases, 18 cases (32.7%) were with ascaris infection, 8 cases (14.5%) of hookworm infection, 7 cases (12.7%) of whipworm infection, and 22 cases (40%) of pinworm infection. ELISA were used to measure the levels of IL-4, IL-9, and IgE in peripheral blood samples from the patients and 15 healthy volunteers. The relationship between the concentration of the cytokines and clinical symptoms or species of the parasites was analyzed. RESULTS: The serum levels of IL-4, IL-9, and IgE in infection group were (157.42 ± 41) pg/ml, (59.9 ± 21.7) pg/ml, and (316.6 ± 129) IU/ml, respectively, which were higher than that of the healthy control[ IL-4 (39.01 ± .5) pg/ml, IL-9 (21.3 ± 12.5) pg/m, IgE (127.7 ± 57.6) IU/ml] (P > 0.01). After treatment by albendazole in the infection group, the level of IL-4, IL-9, and IgE decreased to (98.1 ± 41.7) pg/ml, (38.7 ± 14.1) pg/ml, and (253.1 ± 94.0) IU/ml, respectively, but still higher than that of the control (P < 0.05). IL-9 level in patients with upper gastrointestinal hemorrhage was (76.1 ± 23.5) pg/ml, which was higher than that of those with abdominal discomfort or disruption to bowel habits [(54.3 ± 22.1) g/ml] (P < 0.05), but lower than that of those with allergic dermatitis [(108.5 ± 33.4) pg/ml] (P < 0.05). No significant difference was found in the levels of IL-4 and IgE among the above three groups. The level of IL-9 in patients infected with pinworms was (120.3 ± 41.0) pg/ml, which was higher than that of ascaris infection group [(90.1 ± 29.7) pg/ml], hookworm infection group [(77.3 ± 18.3) pg/ml], and whipworm infection group [(62.5 ± 24.3) pg/ml] (P < 0.01). There was no significant difference in the serum level of IL-9 between ascaris infection group and hookworm infection group (P > 0.05), whereas the IL-9 level in ascaris infection group and hookworm infection group was higher than that of whipworm infection group [(62.5 ± 24.3) pg/ ml] (P < 0.01). There were no significant difference in the serum level of IL-4 and IgE among the patients infected with the species of different helminthes (P > 0.05). CONCLUSION: The levels of IL-4, IgE, and IL-9 are considerably related with intestinal helminth infection, while IL-9 level varied with different helminth species and clinical symptoms.

Serodiagnosis of experimental sparganum infections of mice and human sparganosis by ELISA using ES antigens of Spirometra mansoni spargana.

We conducted a study of serodiagnosis of experimental sparganum infections of mice and human sparganosis by enzyme-linked immunosorbent assay (ELISA) using excretory-secretory (ES) antigens of Spirometra mansoni spargana and compared the sensitivity and specificity of crude and ES antigens for detecting the specific anti-sparganum IgG antibodies. By crude antigen ELISA and ES antigen ELISA, anti-sparganum IgG was detected in all of 30 serum samples of the infected mice; no cross-reactions were observed in serum samples of the mice infected with Trichinella spiralis, Schistosoma japanicum, Toxoplasma gondii, and normal mice. Anti-sparganum IgG was detected by ES antigen ELISA in sera of mice infected with one, two, four, six, and eight spargana at 3 weeks post-infection (wpi), with a detection rate of 100%, and lasted to 18 wpi when the experiment was ended. The difference in anti-sparganum antibody levels among five groups of the infected mice was statistically significant (F=245.296, p<0.05); the antibody levels were correlated with infecting doses of spargana (r=0.323, p<0.05). The sensitivity of both ELISA in detecting the serum samples of patients with sparganosis was 100% (20/20), but 96.72% (59/61) of specificity of ES antigen ELISA in detecting serum samples of patients with cysticercosis, echinococcosis, paragonimiosis, clonorchiosis, and schistosomiasis, and healthy persons was significantly greater than 72.13% (44/61) of crude antigen ELISA (χ (2) = 14.027, p<0.05). Our finding indicates that ELISA using ES antigens of S. mansoni spargana may be applied to the specific early serodiagnosis of sparganosis.

[Epidemiological investigation on sparganosis mansoni and animal experiments].

The investigation was made in Luohe City of Henan Province from April to November, 2008. Cyclops were collected and plerocercoids of Spirometra mansoni were examined by microscope. Skin, muscle and internal organs of frogs and tadpoles were checked to detect spargana by naked eye and/or anatomical microscope. Feces of cats and dogs were collected to examine eggs after washing and precipitation. Spargana from tadpoles were collected to infect cats by oral inoculation. Results showed that the infection rate of plerocercoids in cyclops was 3.5% (3/85) and that of spagarna in tadpoles and frogs was 35.9% (120/334) and 16.8% (75/446), respectively. Among 3 cats and 31 dogs investigated, 1 and 6 (19.4%) were found infected respectively. Eggs of Spirometra mansoni were found in feces of cats 12 days after infection. 17 adult worms were found in the intestine of the cat on the 25th day. The habit of eating live tadpoles was found in local residents. The investigation reveals a high prevalence of Spirometra mansoni in the intermediate and final hosts. Eating live tadpoles seems a main reason of getting sparganosis mansoni.

A case of group infections with Paraginimus species in Henan, Central China.

In July of 2012, mass infections with Paragonimus species were detected in the Henan province sickening 11 of 51 people. In May 2011, these individuals had participated in an excursion during which freshwater crabs were caught and served after being toasted. Before the group infections with Paraginimus species was confirmed, 5 of the 11 patients had been misdiagnosed as tuberculosis (TB) and treated with an anti-TB drug regimen for six months. The most common and typical manifestations were eosinophilia (11/11, 100%) and pulmonary manifestations including, among others, stethalgia and cough (7/11 63.6%). Sero-examination revealed that all 11 patients were seropositive for Paragonimus species. Surprisingly, in our case, one patient presented with hemoptysis and eggs in respiratory secretions, and this is the first time P. skrjabini eggs are detected in the sputum of a patient from the Henan province. Paragonimus metacercariae were collected from 6 of 11 (54.5%) crabs caught at the infection site and were identified as Paraginiumus skrjabini by morphological and molecular examinations. Epidemiological and laboratory evidence confirmed that this is a case of group infection with P. skrjabini. As one of the most neglected tropical diseases (NTD), paragonimiasis should be differentiated diagnosed from TB to avoid the delay of treatment. To our knowledge, this is the second report of a case of group infections with Paraginimus species in Henan, Central China. The first case was reported in 1995. As a kind of food-borne parasitic disease, paragonimiasis should be included in the public health education agenda.

Targeted sequencing reveals the mutational landscape responsible for sorafenib therapy in advanced hepatocellular carcinoma.

Rationale: The existence of primary and acquired drug resistance is the main obstacle for the effect of multi-kinase inhibitor sorafenib and regorafenib in advanced hepatocellular carcinoma (HCC). However, plenty of patients did not significantly benefit from sorafenib treatment and little is known about the mechanism of drug resistance. Methods: Laser capture microdissection was used to acquire matched normal liver and tumor tissues on formalin-fixed paraffin-embedded specimens collected before sorafenib therapy from the first surgery of 119 HCC patients. Ultra-deep sequencing (~1000×) targeting whole exons of 440 genes in microdissected specimens and siRNA screen in 7 cell lines were performed to find mutations associated with differential responses to sorafenib. Patient-derived xenograft models were employed to determine the role of TP53 in response to sorafenib. Lentiviruses harboring wild-type and c.G52C-mutant OCT4 were applied to explore the function of OCT4 in resistance to sorafenib. ChIP-PCR assay for analysis of OCT4 transcriptional activity was performed to explore the affinity with the KITLG promoter. Statistical analyses were used to associate levels of p53 and OCT4 with tumor features and patient outcomes. Results: Total 1,050 somatic mutations and 26 significant driver genes were identified. SiRNA screening in 7 HCC cell lines was further performed to identify mutations associated with differential responses to sorafenib. A recurrent nonsynonymous mutation c.G52C in OCT4 (OCT4mut) was strongly associated with good response to sorafenib, whereas the stop-gain mutation in TP53 showed the opposite outcome both in vitro and in vivo. OCT4wt-induced stem cell factor (encoded by KITLG gene, SCF) expression and cross-activation of c-KIT/FLT3-BRAF signals were identified indispensably for sorafenib resistance, which could be reversed by the combination of c-KIT tyrosine kinase inhibitors or neutralizing antibody against SCF. Mechanistically, an OCT4 binding site in upstream of KITLG promoter was identified with a higher affinity to wildtype of OCT4 rather than G52C-mutant form, which is indispensable for OCT4-induced expression of KITLG and sorafenib resistance. Conclusion: Our study reported a novel somatic mutation in OCT4 (c.G52C) responsible for the sorafenib effect, and also shed new light on the treatment of HCC through the combination of specific tyrosine kinase inhibitors according to individual genetic patterns.

Circular RNAs negatively regulate cancer stem cells by physically binding FMRP against CCAR1 complex in hepatocellular carcinoma.

Circular RNA (circRNA) possesses great pre-clinical diagnostic and therapeutic potentials in multiple cancers. It has been reported playing roles in multiple malignant behaviors including proliferation, migration, metastasis and chemoresistance. However, the underlying correlation between circRNAs and cancer stem cells (CSCs) has not been reported yet. Methods: circZKSCAN1 level was detected in HCC tissue microarrays to clarify its prognostic values. Gain and loss function experiments were applied to investigate the role of circZKSCAN1 in HCC stemness. Bioinformatic analysis was used to predict the possible downstream RNA binding protein and further RNA immunoprecipitation sequencing was carried out to identify the RBP-regulated genes. Results: The absence of circZKSCAN1 endowed several malignant properties including cancer stemness and tightly correlated with worse overall and recurrence-free survival rate in HCC. Bioinformatics analysis and RNA immunoprecipitation-sequencing (RIP-seq) results revealed that circZKSCAN1 exerted its inhibitive role by competitively binding FMRP, therefore, block the binding between FMRP and ß-catenin-binding protein-cell cycle and apoptosis regulator 1 (CCAR1) mRNA, and subsequently restrain the transcriptional activity of Wnt signaling. In addition, RNA-splicing protein Quaking 5 was found downregulated in HCC tissues and responsible for the reduction of circZKSCAN1. Conclusion: Collectively, this study revealed the mechanisms underlying the regulatory role of circZKSCAN1 in HCC CSCs and identified the newly discovered Qki5-circZKSCAN1-FMRP-CCAR1-Wnt signaling axis as a potentially important therapeutic target for HCC treatment.

A neglected risk for sparganosis: eating live tadpoles in central China.

A 29-year-old farmer from central China was sent into the Emergency Department of the Affiliated Hospital of Zhengzhou University. He had a 15-day history of persistent high fever, abdominal distention and pain. The patient was clinically diagnosed as appendicitis and peritonitis, and treated with antibiotics in a local hospital, did not improve. On exploratory laparotomy, the appendicular perforation and peritonitis were seen; appendicectomy were performed, and antibiotics were given. However, high fever and abdominal pain still persisted; intestinal adhesion and obstruction, ascites appeared. He was given the "critically ill notice". He had eosinophilia (12.95%) and the history of eating live frog tadpoles for treating his cutaneous pruritus 3 days before onset of the disease. Serum anti-sparganum antibodies assayed by ELISA were positive. This patient has hospitalized for one and half months and spend more than US$ 12 000. This patient was primarily diagnosed as visceral sparganosis, and cured with praziquantel.Sparganosis is one neglected but important parasitic zoonosis of poverty. Human infections were mainly acquired by eating raw or uncooked meat of frogs and snakes infected with plerocercoids, using frog or snake flesh as poultices, or drinking raw water contaminated with infected copepods. However, sparganosis caused by ingestion of live tadpoles are emerging in central China. Our surveys showed that 11.93% of tadpoles in Henan province are infected with plerocercoids. Eating live tadpoles is a high risk for sparganum infection. The comprehensive public health education should be carried out for people in endemic areas and the bad habit of eating live tadpoles must be discouraged.

[Study on the methods for detection of Toxoplasma antigen].

A sandwich ELISA was established with monoclonal antibody as detecting antibody and rabbit anti-Toxoplasma polyclonal antibody as capturing antibody. ABC (avidin-biotin-peroxidase complex)-ELISA and immuno-PCR were also used to detect different concentrations of Toxoplasma antigen. The lowest concentration of antigen to be detected by sandwich ELISA, ABC-ELISA and immuno-PCR was 0. 6 microg/ml, 0. 075 microg/ml and 0.1 ng/ml respectively. The immuno-PCR shows a much higher sensitivity than the other two methods.

[Investigation on prevalence of Spirometra mansoni sparganum infection in frogs from Henan Province].

OBJECTIVE: To understand the prevalence of Spirometra mansoni sparganum infection in frogs from Henan Province and analyze the risk of people suffering from sparganum infection. METHODS: The spargana were examined in the muscle of frogs collected from 12 sites of 5 regions in Henan Province, and the amount of spargana and their location were recorded. RESULTS: The natural infection rate of Spirometra mansoni spargana in frogs was 26.63% (306/1 149). There were totally 1 897 spargana found in those frogs, and the average was 6.2 per frog. The infection rate of pond frogs was higher than that of toad, the difference was statically significant (chi2 = 30.42, P < 0.01). The natural infection rate of pond frogs in the nearby patients' residence was higher than that of pond frogs collected from the other sites, and the natural infection rate of frogs was lower in the west region of Henan than that in the other areas. The muscles of hind legs of the frogs was the most common parasitic location compared with other parts of the frogs, and the ratio was 73.74% (1 365/1 851). CONCLUSION: The frogs living in the nature are widely infected with Spirometra mansoni spargana in Henan Province, and the comprehensive measures should be taken for the prevention and control of sparganosis mansoni.