Cross-genera amplification and identification of Colpodella sp. with Cryptosporidium primers in fecal samples of zoo felids from northeast China

Abstract The protozoans include many intracellular human pathogens. Accurate detection of these pathogens is necessary to treat the diseases. In clinical epidemiology, molecular identification of protozoan is considered a more reliable and rapid method for identification than microscopy. Among these protozoans, Cryptosporidium considered being one of the important water-borne zoonotic pathogens and a major cause of a diarrheal disease named cryptosporidiosis in humans, domestic animals, and wild animals. This study was aimed to identify Cryptosporidium in zoo felids (N= 56) belonging to different zoo of China, but accidentlly Colpodella was encountered in the zoo felids sample and phylogenetic data confirmed this unexpected amplification from fecal samples using two-step nested-PCR. Phylogenetic analysis revealed the fact about the specific primers used previously by many researchers and cross-genera amplification. We came to know that genetically sequenced amplicon gives more accurate identification of species. This study suggests more investigation on Colpodella which has been neglected previously but gains the attention of researchers after identified from humans and animals and has been known to correlate with neurological symptoms in patients.

Resumo Os protozoários incluem muitos patógenos humanos intracelulares. A detecção acurada desses patógenos é necessária para tratar as doenças. Na epidemiologia clínica, a identificação molecular de protozoários é considerada o método de identificação mais confiável e rápido do que a microscopia. Entre esses protozoários, o Cryptosporidium é considerado um dos importantes patógenos zoonóticos transmitidos pela água e uma das principais causas de uma doença diarreica denominada criptosporidiose em humanos, animais domésticos e selvagens. Este estudo teve como objetivo identificar Cryptosporidium em zoofelídeos (N = 56) pertencentes a diferentes zoológicos da China, mas acidentalmente Colpodella foi encontrada na amostra de zoofelídeos e os dados filogenéticos confirmaram essa amplificação inesperada de amostras fecais usando nested-PCR em duas etapas. A análise filogenética revelou o fato sobre os primers específicos usados anteriormente por muitos pesquisadores e a amplificação entre gêneros. Ficamos sabendo que o amplicon sequenciado geneticamente fornece uma identificação mais acurada das espécies. Este estudo sugere mais investigação sobre Colpodella, que foi negligenciada anteriormente, mas ganha a atenção dos pesquisadores depois de identificada em humanos e animais e é conhecida por se correlacionar com sintomas neurológicos em pacientes.

Cross-genera amplification and identification of Colpodella sp. with Cryptosporidium primers in fecal samples of zoo felids from northeast China / Amplificação cruzada de gêneros e identificação de Colpodella sp. com iniciadores de Cryptosporidium em amostras fecais de zoofilia do nordeste da China

Abstract The protozoans include many intracellular human pathogens. Accurate detection of these pathogens is necessary to treat the diseases. In clinical epidemiology, molecular identification of protozoan is considered a more reliable and rapid method for identification than microscopy. Among these protozoans, Cryptosporidium considered being one of the important water-borne zoonotic pathogens and a major cause of a diarrheal disease named cryptosporidiosis in humans, domestic animals, and wild animals. This study was aimed to identify Cryptosporidium in zoo felids (N= 56) belonging to different zoo of China, but accidentlly Colpodella was encountered in the zoo felids sample and phylogenetic data confirmed this unexpected amplification from fecal samples using two-step nested-PCR. Phylogenetic analysis revealed the fact about the specific primers used previously by many researchers and cross-genera amplification. We came to know that genetically sequenced amplicon gives more accurate identification of species. This study suggests more investigation on Colpodella which has been neglected previously but gains the attention of researchers after identified from humans and animals and has been known to correlate with neurological symptoms in patients.

Resumo Os protozoários incluem muitos patógenos humanos intracelulares. A detecção acurada desses patógenos é necessária para tratar as doenças. Na epidemiologia clínica, a identificação molecular de protozoários é considerada o método de identificação mais confiável e rápido do que a microscopia. Entre esses protozoários, o Cryptosporidium é considerado um dos importantes patógenos zoonóticos transmitidos pela água e uma das principais causas de uma doença diarreica denominada criptosporidiose em humanos, animais domésticos e selvagens. Este estudo teve como objetivo identificar Cryptosporidium em zoofelídeos (N = 56) pertencentes a diferentes zoológicos da China, mas acidentalmente Colpodella foi encontrada na amostra de zoofelídeos e os dados filogenéticos confirmaram essa amplificação inesperada de amostras fecais usando nested-PCR em duas etapas. A análise filogenética revelou o fato sobre os primers específicos usados ​​anteriormente por muitos pesquisadores e a amplificação entre gêneros. Ficamos sabendo que o amplicon sequenciado geneticamente fornece uma identificação mais acurada das espécies. Este estudo sugere mais investigação sobre Colpodella, que foi negligenciada anteriormente, mas ganha a atenção dos pesquisadores depois de identificada em humanos e animais e é conhecida por se correlacionar com sintomas neurológicos em pacientes.

Cross-genera amplification and identification of Colpodella sp. with Cryptosporidium primers in fecal samples of zoo felids from northeast China.

The protozoans include many intracellular human pathogens. Accurate detection of these pathogens is necessary to treat the diseases. In clinical epidemiology, molecular identification of protozoan is considered a more reliable and rapid method for identification than microscopy. Among these protozoans, Cryptosporidium considered being one of the important water-borne zoonotic pathogens and a major cause of a diarrheal disease named cryptosporidiosis in humans, domestic animals, and wild animals. This study was aimed to identify Cryptosporidium in zoo felids (N= 56) belonging to different zoo of China, but accidentlly Colpodella was encountered in the zoo felids sample and phylogenetic data confirmed this unexpected amplification from fecal samples using two-step nested-PCR. Phylogenetic analysis revealed the fact about the specific primers used previously by many researchers and cross-genera amplification. We came to know that genetically sequenced amplicon gives more accurate identification of species. This study suggests more investigation on Colpodella which has been neglected previously but gains the attention of researchers after identified from humans and animals and has been known to correlate with neurological symptoms in patients.

CDC6 is up-regulated and a poor prognostic signature in glioblastoma multiforme.

PURPOSE: Glioblastoma multiforme (GBM) represents the most common and the most malignant type of brain tumor. Cell division cycle 6 (CDC6), a gene associated with DNA replication initiation, has been proven to be associated with the prognosis of multiple tumors. In this study, we aim to explore the association between CDC6 expression and GBM carcinogenesis and prognosis. METHODS: CDC6 expression in normal cells and GBM cells was explored by analyzing TCGA dataset, as well as by RT-PCR and western blot methods. Survival analysis was performed by the Kaplan-Meier method. Multivariate Cox-regression analysis was adopted to estimate the independence of CDC6 as a GBM prognostic factor. RESULTS AND CONCLUSIONS: Elevated CDC6 levels in GBM tumor tissues compared with those in normal brain tissues were illustrated by analyzing the gene expression profiles from TCGA dataset, and confirmed by RT-PCR and western blot assays in GBM tumor and normal human astrocyte cell lines. Kaplan-Meier analysis indicated the negative influence of high CDC6 expression on GBM overall survival (OS) probability and days to progression (D2P) after initial treatment, but not on days to recurrence (D2R) after initial treatment. Multivariate Cox regression analysis showed CDC6 as an independent signature marker gene for GBM prognosis. In addition, the combination of CDC6 mRNA expression and CpG island methylator phenotype (CIMP) could sensitively predict 3-year OS and D2P. In conclusion, our study uncovered the role of CDC6 in GBM carcinogenesis and prognosis for the first time, which could shed new light on GBM diagnosis and treatment.

Gene polymorphisms are associated with eggshell ultrastructure organization in hens

Eggshell ultrastructure organization, including effective layer thickness, mammillary layer thickness, and average size of mammillary cones, is important for breeding and significantly influences eggshell mechanical properties. Several matrix proteins were known to be important in eggshell formation. However, the proteins and variations that determine eggshell ultrastructure organization are not known. Results: In this study, 17 single-nucleotide polymorphisms of three major genes in a hen population using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Five single-nucleotide polymorphisms with a very low minor allele frequency ( 1%) were excluded from further analysis. The remaining 12 single-nucleotide polymorphisms in Hardy-Weinberg equilibrium were used for analysis of associations with eggshell ultrastructure organization. Associations were found for (i) ovocleidin-116 with effective layer thickness (EFF), mammillary layer thickness (MAM), and average size of mammillary cones (SMAM); (ii) ovalbumin with eggshell thickness (ESH), effective layer thickness, and density of the mammillary cone (DMAM); and (iii) calmodulin1 with density of the mammillary cone. Conclusions: The single-nucleotide polymorphisms identified in the present study may be used as potential markers to improve eggshell quality.(AU)

Gene polymorphisms are associated with eggshell ultrastructure organization in hens

Eggshell ultrastructure organization, including effective layer thickness, mammillary layer thickness, and average size of mammillary cones, is important for breeding and significantly influences eggshell mechanical properties. Several matrix proteins were known to be important in eggshell formation. However, the proteins and variations that determine eggshell ultrastructure organization are not known. Results: In this study, 17 single-nucleotide polymorphisms of three major genes in a hen population using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Five single-nucleotide polymorphisms with a very low minor allele frequency ( 1%) were excluded from further analysis. The remaining 12 single-nucleotide polymorphisms in Hardy-Weinberg equilibrium were used for analysis of associations with eggshell ultrastructure organization. Associations were found for (i) ovocleidin-116 with effective layer thickness (EFF), mammillary layer thickness (MAM), and average size of mammillary cones (SMAM); (ii) ovalbumin with eggshell thickness (ESH), effective layer thickness, and density of the mammillary cone (DMAM); and (iii) calmodulin1 with density of the mammillary cone. Conclusions: The single-nucleotide polymorphisms identified in the present study may be used as potential markers to improve eggshell quality.

Expression and clinical significance of the obesity-related gene TNFAIP9 in obese children.

To investigate the expression of tumor necrosis factor-alpha inducible protein 9 (TNFAIP9) gene in obese children and its clinical significance, 36 simple obese children and 17 non-obese children were recruited as research subjects. The adipose tissue was obtained by abdominal operation. The expression of TNFAIP9 was detected using real-time fluorescence quantitative polymerase chain reaction and western blot. The relationship between the expression of TNFAIP9 and blood lipid, blood glucose, and obesity indexes was analyzed. The levels of TNFAIP9 mRNA and protein in obese children were significantly lower than those in the control group (P < 0.05). The waist circumference (wc), body mass, body mass index (BMI), fat, total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C), insulin resistance index (HOMA-IR), and endothelin (ET) in obese children were significantly higher than those in the control group. The level of high-density lipoprotein cholesterol (HDL-C) was significantly lower than that in the control group (P < 0.05). The level of TNFAIP9 protein was negatively correlated with the wc, body mass, BMI, fat, TC, TG, LDL-C, HOMA-IR, and ET (P < 0.05) and was positively correlated with the level of HDL-C (P < 0.05). In conclusion, the expression of TNFAIP9 significantly decreased in the adipose tissue of obese children, and its levels are closely related to blood lipid level, insulin resistance, and obesity.

Avian sarcoma and leukosis virus gag gene in the Anser anser domesticus genome.

Endogenous retroviruses are regarded as ideal genetic markers for evolutionary analyses. Birds were some of the initial vertebrates found to contain endogenous retroviruses. However, few studies have investigated the presence and distribution of endogenous retroviruses in goose. In this study, we detected the avian sarcoma and leukosis virus gag gene in the genomic DNA of 8 Chinese native breeds using polymerase chain reaction method. The results indicated that a 1.2-kb avian sarcoma and leukosis virus gag sequence was integrated into all 8 goose breeds. The mean genetic pairwise distance was 0.918% among the investigated geese. To the best of our knowledge, this is the first report demonstrating the presence of the endogenous retroviruses in the domestic goose genome. The genetic structure should be further examined in the domestic goose.