[Genetic mutation profiles for children with congenital hypothyroidism in Fujian province].

Objective: To explore the mutation characteristics of pathogenic genes in children with congenital hypothyroidism (CH) in Fujian. Methods: The clinical data of 116 unrelated CH children diagnosed in Fujian Provincial Maternal and Child Health Hospital from January 2019 to September 2020 were retrospectively analyzed, including 50 females and 66 males, with an average age of (20±10) days at diagnosis. Targeted exome sequencing technology was used to detect the mutation frequency, type and distribution characteristics of 29 genes related to thyroxine synthesis or thyroid development. Results: Three hundred and fifty-one potential functional mutations were detected in 105 of 116 CH patients, with a detection rate of 90.5% (105/116). DUOX2 (66.4%, 77/116) was the most frequent mutated gene, followed by TG (23.3%, 27/116), DUOXA1 (23.3%, 27/116), and TPO (12.1%, 14/116), which were all involved in thyroid hormone synthesis. Among the 105 children with CH, 70 cases carried double allele mutation. Except for 3 cases of thyroid dysplasia related genes (2 cases of TSHR and 1 case of GLIS3), the rest were also related to thyroid hormone synthesis. The gene with the highest carrier rate was DUOX2 (68.8%, 59/70), followed by TG (8.6%, 6/70), TPO (4.3%, 3/70), DUOXA2 (1.4%, 1/70) and DUOXA1 (1.4%, 1/70). Conclusion: The main mutated genes in CH children in Fujian are the key genes involved in thyroid hormone synthesis, such as DUOX2, TG and TPO.

Isolation and identification of the immune-relevant ribosomal protein L10 (RPL10/QM-like gene) from the large yellow croaker Pseudosciaena crocea (Pisces: Sciaenidae).

In order to investigate the immune role of ribosomal protein L10 (RPL10/QM-like gene) in marine fish, we challenged the large yellow croaker Pseudosciaena (= Larimichthys) crocea, the most important marine fish culture species in China, by injection with a mixture of the bacteria Vibrio harveyi and V. parahaemolyticus (3:1 in volume). Microarray analysis and real-time PCR were performed 24 and 48 h post-challenge to isolate and identify the QM-like gene from the gill P. crocea (designated PcQM). The expression level of the PcQM gene did not changed significantly at 24 h post-challenge, but was significantly downregulated at 48 h post-challenge, suggesting that the gene had an immune-modulatory effect in P. crocea. Full-length PcQM cDNA and genomic sequences were obtained by rapid amplification of cDNA ends (RACE)-PCR. The sequence of the PcQM gene clustered together with those of other QM-like genes from other aquatic organisms, indicating that the QM-like gene is highly conserved in teleosts.

Genomic structure, polymorphism and expression analysis of growth hormone-releasing hormone and pituitary adenylate cyclase activating polypeptide genes in the half-smooth tongue sole (Cynoglossus semilaevis).

Growth hormone-releasing hormone (GHRH) and pituitary adenylate cyclase activating polypeptide (PACAP) regulate development and somatic growth in teleosts; they may be associated with sexual growth dimorphism in the half-smooth tongue sole (Cynoglossus semilaevis). We found that the full-length GHRH and PACAP gene sequences obtained from females and males consist of 4160, 4159, 2425, and 2446 bp, respectively, each of which includes four exons and three introns. When we analyzed normal females and males and extra-large male adults, GHRH and PACAP mRNA were found to be predominantly expressed in the brain; the expression levels were highest in normal males. The extra-large males exhibited the lowest mRNA levels of both GHRH and PACAP. Sex differences in GHRH and PACAP mRNA expression during development were also examined in a full-sib family; GHRH and PACAP mRNA were detected at all 27 times sampled from 10 to 410 days old. The GHRH expression levels in females were significantly higher than in males at most of the stages between 20 and 100 days old, while lower than those of males after 120 days old. Five microsatellite loci were identified in GHRH and PACAP genes. We used these five polymorphic markers to genotype 224 individuals, and no significant differences were found between females and males from the Bohai Sea, the Yellow Sea and hatchery samples.

Protein kinase C and intracellular calcium are involved in follicle-stimulating hormone-mediated meiotic resumption of cumulus cell-enclosed porcine oocytes in hypoxanthine-supplemented medium.

The present experiments were conducted to examine the hypothesis that follicle-stimulating hormone (FSH) can stimulate the hydrolysis of phosphoinositide, generating the intracellular second messengers to activate protein kinase C and mobilizing intracellular calcium, thus inducing oocyte meiotic resumption. Pig cumulus cell-enclosed oocytes (CEO) were cultured for 24 hr in 4 mM hypoxanthine (HX)-supplemented medium and treated with different agents in the following designs: (1) CEO were treated with neomycin (an inhibitor of phosphoinositide hydrolysis) in the presence of FSH or only treated with 7,12-dimethylbenzin(a) anthracene (DMBA, a tumor promoter which can cause phosphorylation of phospholipase C (PLC), formation of inositol triphophate, and mobilization of intracellular calcium) to mimic the direct activation of PLC; (2) CEO were challenged by FSH, together with sphingosine or staurosporine (two kinds of PKC inhibitors); or treated with phorbol myristate acetate (PMA, an activator of PKC) separately; (3) CEO were primed with BAPTA/AM (an intracellular calcium chelator) or BAPTA/AM +FSH for 60 min, and then transferred into a new culture medium supplemented with FSH but without BAPTA/AM; total culture time was 24 hr. At the end of the culture, the incidence of germinal vesicle breakdown (GVBD) was calculated. The results showed that: (1) FSH (100 U/liter) could stimulate pig CEO to override the arrest of HX and resume meiosis; DMBA (10(-8)-10(-5) M) itself also had such a kind of effect; whereas neomycin, at the level of 10-20 mM, could dramatically inhibit the stimulatory effect of FSH. (2) Staurosporine (10(-9)-10(-6) M) or sphingosine (10(-8)-10(-5) M) could also inhibit the effect of FSH in a dose-dependent manner on stimulating CEO to resume meiosis. However, PMA (10(-8)-10(-5) M) alone had a dual effect on the meiotic resumption of pig CEO. PMA, at the level of 10(-8)-10(-6) M, could stimulate CEO to resume meiosis, and at high concentration of 10(-5) M , it could even enhance the inhibitory effect of HX. (3) Priming CEO with BAPTA/AM only or BAPTA/AM +FSH for 60 min could significantly inhibit the effect of FSH in a dose-dependent manner. These results indicate that in the process of ligand-mediated meiotic resumption of pig CEO, FSH can stimulate the hydrolysis of phosphoinositide leading to the activation of PKC and mobilization of intracellular calcium; and suggest that multiple signaling pathways and signal interaction are involved in this process.

Detection of in vivo genotoxicity of haloalkanes and haloalkenes carcinogenic to rodents by the alkaline single cell gel electrophoresis (comet) assay in multiple mouse organs.

The micronucleus test is widely used to assess in vivo clastogenicity because of its convenience, but it is not appropriate for some carcinogenic chemical classes. Halogenated compounds, for example, are inconsistent micronucleus inducers. We assessed the genotoxicity of 7 haloalkanes and haloalkenes carcinogenic to rodents in 7 mouse organs-stomach, liver, kidney, bladder, lung, brain, and bone marrow-using the alkaline single cell gel electrophoresis (SCG) assay. The carcinogens we studied were 1, 2-dibromo-3-chloropropane (DBCP), 1,3-dichloropropene (mixture of cis and trans) (DCP), 1,2-dibromoethane (EDB), 1,2-dichloroethane (EDC), vinyl bromide, dichloromethane, and carbon tetrachloride; only DBCP induces micronuclei in mouse bone marrow. Except for carbon tetrachloride, halocompounds studied are mutagenic to Salmonella typhimurium. Mice were sacrificed 3 or 24 h after carcinogen administration. DCP and EDC induced DNA damage in all of the organs studied. Vinyl bromide yielded DNA damage in all of the organs except for bone marrow. DBCP induced DNA damage in the stomach, liver, kidney, lung, and bone marrow; EDB in the stomach, liver, kidney, bladder, and lung; and dichloromethane in the liver and lung. Since no deaths, morbidity, clinical signs, organ pathology, or microscopic signs of necrosis were observed, the DNA damage was not attributable to cytotoxicity. On the other hand, the positive response in the liver induced by carbon tetrachloride, which was accompanied by necrosis, was considered to be a false positive response. We suggest that the alkaline SCG assay can be used in multiple organs to detect in vivo genotoxicity that is not expressed in bone marrow cells in mice given non-necrogenic doses of halocompounds.

Organ-specific genotoxicity of the potent rodent colon carcinogen 1,2-dimethylhydrazine and three hydrazine derivatives: difference between intraperitoneal and oral administration.

We used a modification of the alkaline single cell gel electrophoresis (SCG) (Comet) assay to test the in vivo genotoxicity of four hydrazine derivatives--1,2-dimethylhydrazine (SDMH), 1,1-dimethylhydrazine (UDMH), hydrazine (HZ), and procarbazine (PCZ)--in mouse liver, lung, kidney, brain, and bone marrow, and in the mucosa of stomach, colon, and bladder. Mice were sacrificed 3 and 24 h after intra-peritoneal (i.p.) and oral (p.o.) administration. SDMH at 20 mg/kg i.p. yielded statistically significant DNA damage in all tested organs except for lung. In the gastrointestinal tract, SDMH was genotoxic in the stomach and the colon after i.p. treatment but only in the colon after 20 and 30 mg/kg p.o. treatment. UDMH at 50 mg/kg i.p. yielded DNA damage in the liver and lung at 3 h. PCZ at 200 mg/kg i.p. caused DNA damage in the liver, kidney, lung, brain, and bone marrow. UDMH and PCZ were positive in the stomach and colon p.o. but not by i.p. treatment. HZ at 100 mg/kg yielded DNA damage in the stomach, liver, and lung when given i.p. and in the brain when p.o. Thus, the administration route is important when evaluating organ-specific genotoxicity in multiple organs.

Organ-specific genotoxicity of the potent rodent bladder carcinogens o-anisidine and p-cresidine.

We used a modification of the alkaline single-cell gel electrophoresis (SCG) (Comet) assay to evaluate the in vivo genotoxicity of two potent rodent bladder carcinogens, o-anisidine and p-cresidine, in mouse liver, lung, kidney, brain, and bone marrow, and in the mucosa of stomach, colon, and bladder. Male CD-1 mice (8 weeks old) were sacrificed 3 and 24 h after oral administration of o-anisidine at 690 mg/kg or p-cresidine at 595 mg/kg. Both chemicals were dissolved in olive oil. Both chemicals yielded statistically significant DNA damage in bladder mucosa 3 and 24 h after treatment. o-Anisidine yielded DNA damage in the colon at 3 h, but not at 24 h. No significant effects were observed in any other organs. Our results suggest the importance of the urinary bladder as a sentinel organ for evaluating chemical genotoxicity in rodents.

In vivo genotoxicity of ortho-phenylphenol, biphenyl, and thiabendazole detected in multiple mouse organs by the alkaline single cell gel electrophoresis assay.

In Japan, ortho-phenylphenol (OPP), biphenyl (BP), and thiabendazole (2-(4'-thiazolyl)benzimidazole, TBZ) are commonly used as a postharvest treatment to preserve imported citrus fruits during transport and storage. We used a modification of the alkaline single cell gel electrophoresis (SCG) (Comet) assay to test the in vivo genotoxicity of those agents in mouse stomach, liver, kidney, bladder, lung, brain, and bone marrow. CD-1 male mice were sacrificed 3, 8, and 24 h after oral administration of the test compounds. OPP (2000 mg/kg) induced DNA damage in the stomach, liver, kidney, bladder, and lung, BP (2000 mg/kg) and TBZ (200 mg/kg) induced DNA damage in all the organs studied. For OPP, increased DNA damage peaked at 3-8 h and tended to decrease at 24 h. For BP, on the contrary, increased DNA migration peaked at 24 h. That delay may have been due to the fact that OPP is metabolized by cytochrome 450 and prostaglandin H synthase to phenylbenzoquinone (PBQ), a DNA binding metabolite, and BP is metabolized to PBQ via OPP and m-phenylphenol. The positive response to TBZ, an aneugen, supports the in vivo DNA-damaging action of TBZ.