Real-time reverse transcription PCR (RT-qPCR) has emerged as an accurate and widely used technique for expression profiling of selected genes. However, obtaining reliable measurements depends on the selection of appropriate reference genes for gene expression normalization. The aim of this work was to assess the expression stability of 15 candidate genes to determine which set of reference genes is best suited for transcript normalization in citrus in different tissues and organs and leaves challenged with five pathogens (Alternaria alternata, Phytophthora parasitica, Xylella fastidiosa and Candidatus Liberibacter asiaticus). We tested traditional genes used for transcript normalization in citrus and orthologs of Arabidopsis thaliana genes described as superior reference genes based on transcriptome data. geNorm and NormFinder algorithms were used to find the best reference genes to normalize all samples and conditions tested. Additionally, each biotic stress was individually analyzed by geNorm. In general, FBOX (encoding a member of the F-box family) and GAPC2 (GAPDH) was the most stable candidate gene set assessed under the different conditions and subsets tested, while CYP (cyclophilin), TUB (tubulin) and CtP (cathepsin) were the least stably expressed genes found. Validation of the best suitable reference genes for normalizing the expression level of the WRKY70 transcription factor in leaves infected with Candidatus Liberibacter asiaticus showed that arbitrary use of reference genes without previous testing could lead to misinterpretation of data. Our results revealed FBOX, SAND (a SAND family protein), GAPC2 and UPL7 (ubiquitin protein ligase 7) to be superior reference genes, and we recommend their use in studies of gene expression in citrus species and relatives. This work constitutes the first systematic analysis for the selection of superior reference genes for transcript normalization in different citrus organs and under biotic stress.
Schistosomiasis is a water-borne parasitic illness caused by neoophoran trematodes of the genus Schistosoma. Using classical histological techniques and whole-mount preparations, the present work describes the embryonic development of Schistosoma mansoni eggs in the murine host and compares it with eggs maintained under in vitro conditions. Two pre-embryonic stages occur inside the female worm: the prezygotic stage is characterized by the release of mature oocytes from the female ovary until its fertilization. The zygotic stage encompasses the migration of the zygote through the ootype, where the eggshell is formed, to the uterus. Fully formed eggs are laid still undeveloped, without having suffered any cleavage. In the outside environment, eight embryonic stages can be defined: stage 1 refers to early cleavages and the beginning of yolk fusion. Stage 2 represents late cleavage, with the formation of a stereoblastula and the onset of outer envelope differentiation. Stage 3 is defined by the elongation of the embryonic primordium and the onset of inner envelope formation. At stage 4, the first organ primordia arise. During stages 5 to 7, tissue and organ differentiation occurs (neural mass, epidermis, terebratorium, musculature, and miracidial glands). Stage 7 is characterized by the nuclear condensation of neurons of the central neural mass. Stage 8 refers to the fully formed larva, presenting muscular contraction, cilia, and flame-cell beating. This staging system was compared to a previous classification and could underlie further studies on egg histoproteomics (morphological localizome). The differentiation of embryonic structures and their probable roles in granulomatogenesis are discussed herein.
The increased reliability and efficiency of the quantitative polymerase chain reaction (qPCR) makes it a promising tool for performing large-scale screening for infectious disease among high-risk individuals. To date, no study has evaluated the specificity and sensitivity of different qPCR assays for leprosy diagnosis using a range of clinical samples that could bias molecular results such as difficult-to-diagnose cases. In this study, qPCR assays amplifying different M. leprae gene targets, sodA, 16S rRNA, RLEP and Ag 85B were compared for leprosy differential diagnosis. qPCR assays were performed on frozen skin biopsy samples from a total of 62 patients: 21 untreated multibacillary (MB), 26 untreated paucibacillary (PB) leprosy patients, as well as 10 patients suffering from other dermatological diseases and 5 healthy donors. To develop standardized protocols and to overcome the bias resulted from using chromosome count cutoffs arbitrarily defined for different assays, decision tree classifiers were used to estimate optimum cutoffs and to evaluate the assays. As a result, we found a decreasing sensitivity for Ag 85B (66.1%), 16S rRNA (62.9%), and sodA (59.7%) optimized assay classifiers, but with similar maximum specificity for leprosy diagnosis. Conversely, the RLEP assay showed to be the most sensitive (87.1%). Moreover, RLEP assay was positive for 3 samples of patients originally not diagnosed as having leprosy, but these patients developed leprosy 5–10 years after the collection of the biopsy. In addition, 4 other samples of patients clinically classified as non-leprosy presented detectable chromosome counts in their samples by the RLEP assay suggesting that those patients either had leprosy that was misdiagnosed or a subclinical state of leprosy. Overall, these results are encouraging and suggest that RLEP assay could be useful as a sensitive diagnostic test to detect M. leprae infection before major clinical manifestations.
BackgroundCitrus huanglongbing (HLB) disease is caused by endogenous, phloem-restricted, Gram negative, uncultured bacteria named Candidatus Liberibacter africanus (CaLaf), Ca. L. asiaticus (CaLas), and Ca. L. americanus (CaLam), depending on the continent where the bacteria were first detected. The Asian citrus psyllid vector, Diaphorina citri, transmits CaLas and CaLam and both Liberibacter species are present in Brazil. Several studies of the transcriptional response of citrus plants manifesting HLB symptoms have been reported, but only for CaLas infection. This study evaluated the transcriptional reprogramming of a susceptible genotype of sweet orange challenged with CaLam, using a customized 385K microarray containing approximately 32,000 unigene transcripts. We analyzed global changes in gene expression of CaLam-infected leaves of sweet orange during the symptomatic stage of infection and compared the results with previously published microarray studies that used CaLas-infected plants. Twenty candidate genes were selected to validate the expression profiles in symptomatic and asymptomatic PCR-positive leaves infected with CaLas or CaLam.ResultsThe microarray analysis identified 633 differentially expressed genes during the symptomatic stage of CaLam infection. Among them, 418 (66%) were upregulated and 215 (34%) were down regulated. Five hundred and fourteen genes (81%) were orthologs of genes from Arabidopsis thaliana. Gene set enrichment analysis (GSEA) revealed that several of the transcripts encoded transporters associated with the endomembrane system, especially zinc transport. Among the most biologically relevant gene transcripts in GSEA were those related to signaling, metabolism and/or stimulus to hormones, genes responding to stress and pathogenesis, biosynthesis of secondary metabolites, oxidative stress and transcription factors belonging to different families. Real time PCR of 20 candidate genes validated the expression pattern of some genes in symptomatic and asymptomatic leaves infected with CaLam or CaLas.ConclusionsMany gene transcripts and biological processes are significantly altered upon CaLam infection. Some of them had been identified in response to CaLas infection, while others had not been previously reported. These data will be useful for selecting target genes for genetic engineering to control HLB.
Mycobacterium leprae and Mycobacterium tuberculosis antimicrobial resistance has been followed with great concern during the last years, while the need for new drugs able to control leprosy and tuberculosis, mainly due to extensively drug-resistant tuberculosis (XDR-TB), is pressing. Our group recently showed that M. leprae is able to induce lipid body biogenesis and cholesterol accumulation in macrophages and Schwann cells, facilitating its viability and replication. Considering these previous results, we investigated the efficacies of two statins on the intracellular viability of mycobacteria within the macrophage, as well as the effect of atorvastatin on M. leprae infections in BALB/c mice. We observed that intracellular mycobacteria viability decreased markedly after incubation with both statins, but atorvastatin showed the best inhibitory effect when combined with rifampin. Using Shepard's model, we observed with atorvastatin an efficacy in controlling M. leprae and inflammatory infiltrate in the BALB/c footpad, in a serum cholesterol level-dependent way. We conclude that statins contribute to macrophage-bactericidal activity against Mycobacterium bovis, M. leprae, and M. tuberculosis. It is likely that the association of statins with the actual multidrug therapy effectively reduces mycobacterial viability and tissue lesion in leprosy and tuberculosis patients, although epidemiological studies are still needed for confirmation.
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