[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.

Role of MshQ in MSHA pili biosynthesis and biofilm formation of Aeromonas hydrophila.

Biofilm formation of pathogen bacterium is currently one of the most widely studied topics; however, little is known regarding pathogen bacteria biofilms in aquaculture. Aeromonas hydrophila is a representative species of the genus Aeromonas, which has been recognized as a common pathogen, is associated with many diseases in aquatic animals, and causes significant mortality. The objectives of this study are i) to confirm that A. hydrophila can form biofilms on abiotic substrates and construct a biofilm growth curve for this bacterium; ii) to identify the genes that play crucial roles in A. hydrophila biofilm formation. The biofilm growth curve of A. hydrophila was constructed using a crystal violet assay, which showed that biofilm formation for this bacterium is a dynamic process. Next, a mutant library of pathogenic A. hydrophila B11 was constructed using the mini-Tn10 transposon mutagenesis system. A total of 861 mutants were screened, and 5 mutants were stably deficient in biofilm formation. Molecular analysis of the mutant B112 revealed that the open reading frame that encodes the protein MshQ was disrupted. Comparison of biological characteristics including growth, motility, and adhesion between the mutant B112 and the wild-type strain B11 suggested that MshQ is necessary for mannose-sensitive hemagglutinin pilus biosynthesis of A. hydrophila, and that these pili play crucial roles in A.hydrophila adherence to a solid surface during the early stages of biofilm formation.

Polymorphic microsatellite loci for Japanese Spanish mackerel (Scomberomorus niphonius).

We isolated and characterized 21 polymorphic microsatellite loci in Japanese Spanish mackerel (Scomberomorus niphonius) using a (GT)(13)-enriched genomic library. Forty individuals were collected from Qingdao, China. We found 3 to 24 alleles per locus, with a mean of 8.8. The observed and expected heterozygosities ranged from 0.263 to 0.975 and from 0.385 to 0.946, with means of 0.655 and 0.685, respectively. Deviation from Hardy-Weinberg proportions was detected at three loci. Two loci showed evidence for null alleles. These microsatellite markers will be useful for population genetic analysis of Japanese Spanish mackerel.

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.

Characterization of yellow grouper Epinephelus awoara (Serranidae) karyotype by chromosome bandings and fluorescence in situ hybridization.

The cytogenetics of yellow grouper Epinephelus awoara was studied using multiple cytogenetic markers [Giemsa staining, C-banding, Ag-NORs and fluorescence in situ hybridization (FISH)]. Giemsa staining results showed that the karyotypic formula of E. awoara was 2n = 48a, FN (fundamental number) = 48. Faint C-bandings were only detected at the centromeric regions of chromosome pair number 24, being almost indiscernible on the other chromosome pairs. After Ag-NOR staining, one pair of nucleolar organizer regions (NOR) was observed in the subcentromeric region of pair number 24. FISH results showed that 5S rDNA was located at a pair of medium-sized chromosomes, while 18S rDNA appeared at the same location in the subcentromeric region of pair number 24 where Ag-NORs were detected. The telomeric sequence (TTAGGG)(n) detected by FISH was located at both ends of each chromosome. The results suggested that E. awoara has retained general karyotypic structure stability during the evolutionary diversification process.

Polymorphic microsatellite loci for the Japanese anchovy Engraulis japonicus (Engraulidae).

We isolated and characterized 14 polymorphic microsatellite loci in the Japanese anchovy (Engraulis japonicus) using a (GT)(13)-enriched genomic library. The numbers of alleles per locus ranged from 6 to 31, with a mean of 17.8. The observed and expected heterozygosities ranged from 0.180 to 0.949 and from 0.172 to 0.966, with means of 0.731 and 0.825, respectively. All 14 loci were in Hardy-Weinberg equilibrium and no significant linkage disequilibrium between loci pairs was detected. These microsatellite markers will be useful for analyzing the population genetic structure and gene flow of E. japonicus.

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.

[Analysis on clustered regularly interspaced short palindromic repeats loci polymorphism of Yersinia pestis and its area distribution in Gansu province].

Objective: To study the clustered regularly interspaced short palindromic repeats (CRISPR) loci polymorphism of Yersinia pestis and its area distribution in Gansu province. Methods: A total of 203 strains of Yersinia pestis isolated from 1962 to 2014 were selected for the culture and extraction of DNA. Three pairs of CRISPR primers were used to amplify the strain DNA by PCR, and the PCR products were sequenced. The groups and genotypes of strains were determined according to the spacer and spacer arrangement of CRISPR loci in the strain. Cluster analysis was done by using the software BioNumerics 5.10. Results: A total of 16 spacers, including 9 species of YPa loci, 4 species of YPb loci and 3 species of YPc loci, were found in the 203 strains of Yersinia pestis. A new spacer of a1' was found. The 203 strains were divided into 5 CRISPR genotypes and classified into 5 CRISPR clusters (Cb2, Ca7, Ca7', CaΔ5' and Ca35'). Each cluster showed significant area-specific characteristics, Cb2 was mainly distributed in Huining country and Pingchuan district, Ca7 was mainly found in Aksai Kazak autonomous country, Ca7' was mainly found in Xiahe country, Ca35' was mainly found in Subei Mongolia autonomous county and Yumen city and CaΔ5' was mainly distributed in Sunan Yugur autonomous county. Conclusions: The strains from different plague foci in Gansu were distinguished by CRISPR, all kinds of clusters showed the obvious area specific characteristics. It is important to study the evolution of Yersinia pestis in Gansu and trace the molecular biology origin of human plague.

Effects of caloric restriction on peroxisome proliferator-activated receptors and positive transcription elongation factor b expression in obese rats.

OBJECTIVE: To investigate the effect of caloric restriction (CR) on expressions of peroxisome proliferators-activated receptors (PPARs) and positive transcription elongation factor b (P-TEFb) (including cyclin-dependent kinase 9 (CDK9) and cyclin T1) protein in visceral adipose tissue of obese rats. MATERIALS AND METHODS: Obese rats were induced by high-fat diet for 8 weeks. Then they were divided into three groups: Model (n=5), 50% Calorie Restricted (50% CR, n=5), Intermittent Fasting (IF) (eight cycles of 3-d fasting and 3-d refeeding, n=6) for 8 weeks. Biochemical parameters were measured. Protein and mRNA expression of Cdk9, cyclin T1 and PPARs were qualified in visceral adipose tissue. RESULTS: A significant decline in fasting plasma glucose (FPG), homeostatic model assessment of insulin resistance (HOMA-IR), body weight, and visceral fat weight was observed in 50% CR group. The IF group exhibited a significant decrease in FPG, HOMA-IR, visceral fat weight. Both 50% CR and IF down-regulated mRNA and protein expression of PPARγ and Cdk9, cyclin T1 and up-regulated mRNA and protein expression of PPARß. CONCLUSIONS: These results suggest that the effects of 50% CR and IF on HOMA-IR, body weight, visceral fat weight, P-TEFb and PPARγ expression may be related to their protective potential on obesity.

Colon-specific genotoxicity of heterocyclic amines detected by the modified alkaline single cell gel electrophoresis assay of multiple mouse organs.

The in vivo genotoxicity of five heterocyclic amines-Trp-P-2 (13 mg/kg), IQ (13 mg/kg), MeIQ (13 mg/kg), MeIQx (13 mg/kg), and PhIP (40 mg/kg)-in the mucosa of gastrointestinal and urinary tract organs (stomach, duodenum, jejunum, ileum, colon, and bladder) was studied by the alkaline single cell gel electrophoresis (SCG) (Comet) assay. Male CD-1 mice were sacrificed 1, 3, and 8 h after intraperitoneal injection. All the heterocyclic amines studied yielded statistically significant DNA damage in the colon but not the small intestine (duodenum, jejunum, and ileum) or urinary bladder. In this study, five heterocyclic amines were injected intraperitoneally to avoid the consequences of ingestion. Thus, the extensive damage to colon DNA was concluded to be due, at least in part, to a systemic effect.

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.

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 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.

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.

Molecular genetic analysis for Ax phenotype of the ABO blood group system in Chinese.

BACKGROUND AND OBJECTIVES: To elucidate the molecular genetic background of the Ax phenotype in the Chinese population. MATERIALS AND METHODS: The ABO genes of eight Ax phenotype samples, four Ax and four AxB, were amplified by polymerase chain reaction (PCR) and were cloned, along with those of 10 random A(1) Chinese subjects. We analysed the ABO gene transcript structure and the sequences of two exons and one intron at the ABO locus. RESULTS: Among the four Ax phenotype samples, we identified one Ax02, two Ax03 and one novel Ax allele with the 543G > T mutation in the A102 background. Two of five family members also carry the allele. Of the four AxB phenotypes, one was designated as cis-AB-1/B101; the other three were shown to carry one B allele and one O with the nt261G deletion. The B alleles of the latter three were identical to B101 except for single point mutation at nt700C > G, nt640A > G and nt641T > C, respectively. The novel B101-like alleles were first associated with A(weak)B phenotypes. CONCLUSIONS: Two ABO*B(A) alleles and an Ax allele clearly differ from all previously reported ABO alleles, suggesting that the molecular genetic background of Ax is heterogeneous in the Chinese population.

Molecular background of Rh D-positive, D-negative, D(el) and weak D phenotypes in Chinese.

BACKGROUND AND OBJECTIVES: The aim of this study was to elucidate the genetic background of D-negative and D(el) in the Chinese population. MATERIALS AND METHODS: We investigated nine D-positive, 76 D-negative, 26 D(el) and three weak D Chinese individuals by amplification and sequencing of the complete coding region of the RHD gene from genomic DNA. A new RHD polymerase chain reaction with sequence-specific primers (PCR-SSP) method was developed with optimized specificity for typing Chinese individuals. RESULTS: In D-positive samples the RHD sequence was in complete concordance with RHD in other populations. In 12 of 76 (15.8%) D-negative individuals we detected regions of RHD. Three new alleles were found. All 26 D(el), as well as two weak D, individuals carried an RHD 1227A allele. In the remaining weak D sample we identified a weak D type 15. CONCLUSIONS: It should now be possible to correctly predict the RhD phenotype in Chinese subjects. D(el) can also be designated as a particular weak D type.

Involvement of MEK-mitogen-activated protein kinase pathway in follicle-stimulating hormone-induced but not spontaneous meiotic resumption of mouse oocytes.

Mitogen-activated protein (MAP) kinase has been reported to be activated during oocyte meiotic maturation in a variety of mammalian species. However, the mechanism(s) responsible for MAP kinase activation and the consequence of its premature activation during gonadotropin-induced oocyte meiotic resumption have not been examined. The present experiments were conducted to investigate the possible role of MAP kinase in FSH-induced and spontaneous oocyte meiotic resumption in the mouse. MAP kinase kinase (MAPKK, MEK) inhibitor, PD98059 or U0126, produced a dose-dependent inhibitory effect on both FSH-induced oocyte meiotic resumption and MAP kinase activation in the oocytes. However, the same inhibitor did not block spontaneous meiotic resumption of either denuded or cumulus cell-enclosed mouse oocytes, despite the activity of MAP kinase being totally inhibited. Immunoblotting the oocytes and the cumulus cells with the anti-active MAP kinase antibody showed that MAP kinase activity in the oocytes was detected at 8 h of FSH treatment, prior to germinal vesicle breakdown and increased as maturation progressed in the following culture period. In the cumulus cells, MAP kinase was activated even faster, its activity was detected at 1 h of FSH stimulation and increased gradually until 8 h of FSH treatment, then decreased and diminished after 12 h of FSH action. These data demonstrated that the MEK-MAP kinase pathway is implicated in FSH-induced but not spontaneous oocyte meiotic resumption.

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.