Evaluation of the transcriptional immune biomarkers in peripheral blood from warao indigenous associate with the infection by mycobacterium tuberculosis

Abstract INTRODUCTION: Biomarkers are critical tools for finding new approaches for controlling the spread of tuberculosis (TB), including for predicting the development of TB therapeutics, vaccines, and diagnostic tools. METHODS: Expression of immune biomarkers was analyzed in peripheral blood cells stimulated and non-stimulated with M. tuberculosis antigens ESAT-6, CFP10 and TB7.7. in Warao indigenous individuals. These biomarkers may be able to differentiate TB states, such as active tuberculosis (ATB) cases and latent tuberculosis infection (LTBI) from non-infected controls (NIC). A real-time reverse transcription polymerase chain reaction (RT-qPCR) assay was performed on 100 blood samples under non-stimulation or direct ex vivo conditions (NS=50) and stimulation conditions (S=50). RESULTS: The findings are shown as the median and interquartile range (IQR) of relative gene expression levels of IFN-γ, CD14, MMP9, CCR5, CCL11, CXCL9/MIG, and uPAR/PLAUR immune biomarkers. MMP9 levels were significantly higher in the LTBI-NS and LTBI-S groups compared with the NIC-NS and NIC-S groups. However, CCR5 levels were significantly lower in the LTBI-S group compared with both NIC-NS and NIC-S groups. CCL11 levels were significantly lower in the LTBI-S group compared with the NIC-NS group. CONCLUSIONS: Preliminary findings showed that MMP9 immune biomarkers separated LTBI indigenous individuals from NIC indigenous individuals, while CCR5, CCL11, CD14, and IFN-γ did not differentiate TB states from NIC. MMP9 may be useful as a potential biomarker for LTBI and new infected case detection among Warao indigenous individuals at high risk of developing the disease. It may also be used to halt the epidemic, which will require further validation in larger studies.

Clinical significance of FAM19A4 gene promotor methylation in cervical cancer / 实用医学杂志

Objective To investigate the expression of FAM19A4 gene promotor methylation in different cervical lesions and its diagnostic value. Methods 31 cervical cancers cases ,22 HSILs and 23 normal cervical tissues of formalin-fixed and parrffin-embedded specimen diagnosed by pathology were selected. Taqman probe-based quantitative PCR(qPCR)was used to detect the differences of FAM19A4 methylation in different cervical lesions, and then corresponding analyses were made. Results Statistical differences of FAM19A4 methylationrates were observed in cervical caner ,HSIL and normalcervix,respectively96.8%(30/31),72.7%(16/22)and 8.7%(2/23) (P < 0.05 ).FAM19A4 methylation rates increased with severity of cervical lesion (P < 0.05).The methylation rates of FAM19A4 were not statistically different in different clinicopathological characteristics of cervical cancer (P>0.05). Conclusions FAM19A4 gene promoter methylation is probably a specific biomarker of cervical cancer,andmay play a role in the development and progress of cervical cancer,but may not participate in the invasion and metastasis.

Relationship between gene expression of lysine decarboxylase and accumulation of matrine and oxymatrine in Sophora alopecuroides / 中国药学杂志

OBJECTIVE: To study the effect of lysine decarboxylase (LDC) gene on the accumulation of matrine (MA) and oxymatrine (OMA) in cotyledon of Sophora alopecuroides L germinating seeds. METHODS: The S. alopecuroides germinating seeds were stressed by different mass fractions of PEG 6000, and the contents of MA and OMA were determined by high performance liquid chromatography (HPLC) and the expression level of LDC was analyzed by real-time fluorescence quantitative PCR (qPCR) after 72 h treatment. RESULTS: The contents of MA and OMA decreased in the cotyledon under light stress (PEG mass fraction 20%). The analysis of qPCR revealed that the LDC expression level was decreased first, and then increased with the stress rising. The changes of the contents of MA and OMA were parallel with the expression level of LDC especially under light and severe stress. CONCLUSION: There is certain association between the accumulation of MA and OMA and the gene expression quantity of LDC. The results is of significance for illustrating the role of LDC in the biosynthetic pathways of MA and OMA.

Differential analysis of the bacterial community composition in mouse feces and intestinal contents / 中国实验动物学报

Objective To explore the objectivity and scientificity of fecal sampling , and to provide reference for investigating the relationship between intestinal microbes and diseases . Methods Terminal restriction fragment length polymorphism, degeneration gradient gel electrophoresis and real time fluorescent quantitative PCR techniques were applied to differentially analyze the bacterial community composition and abundance of intestinal contents and feces taken from dif -ferent sites of BALB/c mouse intestine .Results The predominant T-RFLP fragments ( T-RFs) in feces in the rectum and colon were 244 bp, 255 bp and 449 bp, however , those in feces of the small intestine including duodenum , jejunum and il-eum were 60 bp, 73 bp, 261 bp, 268 bp and 272 bp, and with a larger variation of the bacterial community composition in various parts of the small intestine .The bacterial abundance in the contents of duodenum and jejunum were 6.9 log ( cop-ies)/g and 8.3 log (copies)/g, fewer than in the other parts of the intestine , while the bacterial abundance in the feces was as high as 11.8 log (copies)/g, being about 2 times higher than that in the duodenum and jejunum (P0.05).Conclusions The inter-mouse variations of bacterial communi-ty composition in the large intestine contents are small .The bacterial composition and abundance are similar suggest that studies on the relationship between large intestine especially colorectal microbiota and diseases may be conducted via fecal sampling.

Methylobacterium-plant interaction genes regulated by plant exudate and quorum sensing molecules

Bacteria from the genus Methylobacterium interact symbiotically (endophytically and epiphytically) with different plant species. These interactions can promote plant growth or induce systemic resistance, increasing plant fitness. The plant colonization is guided by molecular communication between bacteria-bacteria and bacteria-plants, where the bacteria recognize specific exuded compounds by other bacteria (e.g. homoserine molecules) and/or by the plant roots (e.g. flavonoids, ethanol and methanol), respectively. In this context, the aim of this study was to evaluate the effect of quorum sensing molecules (N-acyl-homoserine lactones) and plant exudates (including ethanol) in the expression of a series of bacterial genes involved in Methylobacterium-plant interaction. The selected genes are related to bacterial metabolism (mxaF), adaptation to stressful environment (crtI, phoU and sss), to interactions with plant metabolism compounds (acdS) and pathogenicity (patatin and phoU). Under in vitro conditions, our results showed the differential expression of some important genes related to metabolism, stress and pathogenesis, thereby AHL molecules up-regulate all tested genes, except phoU, while plant exudates induce only mxaF gene expression. In the presence of plant exudates there is a lower bacterial density (due the endophytic and epiphytic colonization), which produce less AHL, leading to down regulation of genes when compared to the control. Therefore, bacterial density, more than plant exudate, influences the expression of genes related to plant-bacteria interaction.

Molecular Identification and Real-time Quantitative PCR (qPCR) for Rapid Detection of Thelohanellus kitauei, a Myxozoan Parasite Causing Intestinal Giant Cystic Disease in the Israel Carp

Intestinal giant-cystic disease (IGCD) of the Israel carp (Cyprinus carpio nudus) has been recognized as one of the most serious diseases afflicting inland farmed fish in the Republic of Korea, and Thelohanellus kitauei has been identified as the causative agent of the disease. Until now, studies concerning IGCD caused by T. kitauei in the Israel carp have been limited to morphological and histopathological examinations. However, these types of diagnostic examinations are relatively time-consuming, and the infection frequently cannot be detected in its early stages. In this study, we cloned the full-length 18S rRNA gene of T. kitauei isolated from diseased Israel carps, and carried out molecular identification by comparing the sequence with those of other myxosporeans. Moreover, conventional PCR and real-time quantitative PCR (qPCR) using oligonucleotide primers for the amplification of 18S rRNA gene fragment were established for further use as methods for rapid diagnosis of IGCD. Our results demonstrated that both the conventional PCR and real-time quantitative PCR systems applied herein are effective for rapid detection of T. kitauei spores in fish tissues and environmental water.