Differential expression of microRNA-411 and 376c is associated with hypertension in pregnancy

Preeclampsia is a major reason of morbidity and mortality in pregnant women and perinatal fetus. Hence, it is of prime importance that diagnostic markers are defined to predict chances of preeclampsia in pregnant women. It has been previously shown that microRNA (miRNA)-376c expression is decreased in the placenta of preeclampsia patients at term. Even though this decrease was not mimicked in the placenta at the pre-term stage, miR-376c expression was decreased in the plasma of these patients as early as the second trimester. Plasma and placenta specimens were obtained from pregnant women having unifetal gestation undergoing perinatal care between January 2014 and December 2016 (n=49). Early trimester placentas were collected from patients undergoing terminated pregnancies through dilation and curettage procedure. Our results showed that in addition to miR-376c, miR-441 levels were decreased in the placenta of preeclampsia patients, and this decrease occurred both at pre-term and at term. This decrease is also mimicked in the plasma levels at both early and late weeks of pregnancy, highlighting that miR-441 levels can serve as a diagnostic marker of risk of preeclampsia in pregnant women. Overexpression of the miR-441, as well as miR-376c, promoted cell viability, migration, and invasion in the human immortalized cytotrophoblast cell line HTR8/SVneo, indicating that their decrease in pregnant women would result in anomalous apoptosis and functional imbalance resulting in premature abortion and other complications. MiR-441 level can thus potentially serve as diagnostic marker of preeclampsia in pregnant women.

Identification and characterisation of microRNAs in young adults of Angiostrongylus cantonensis via a deep-sequencing approach

Angiostrongylus cantonensis is an important causative agent of eosinophilic meningitis and eosinophilic meningoencephalitis in humans. MicroRNAs (miRNAs) are small non-coding RNAs that participate in a wide range of biological processes. This study employed a deep-sequencing approach to study miRNAs from young adults of A. cantonensis. Based on 16,880,456 high-quality reads, 252 conserved mature miRNAs including 10 antisense miRNAs that belonging to 90 families, together with 10 antisense miRNAs were identified and characterised. Among these sequences, 53 miRNAs from 25 families displayed 50 or more reads. The conserved miRNA families were divided into four groups according to their phylogenetic distribution and a total of nine families without any members showing homology to other nematodes or adult worms were identified. Stem-loop real-time polymerase chain reaction analysis of aca-miR-1-1 and aca-miR-71-1 demonstrated that their level of expression increased dramatically from infective larvae to young adults and then decreased in adult worms, with the male worms exhibiting significantly higher levels of expression than female worms. These findings provide information related to the regulation of gene expression during the growth, development and pathogenesis of young adults of A. cantonensis.

Depressed nNOS expression during spine transition in the developing hippocampus of FMR1 KO mice

Nitric oxide (NO), synthesized as needed by NO synthase (NOS), is involved in spinogenesis and synaptogenesis. Immature spine morphology is characteristic of fragile X syndrome (FXS). The objective of this research was to investigate and compare changes of postnatal neuronal NOS (nNOS) expression in the hippocampus of male fragile X mental retardation 1 gene knockout mice (FMR1 KO mice, the animal model of FXS) and male wild-type mice (WT) at postnatal day 7 (P7), P14, P21, and P28. nNOS mRNA levels were analyzed by real-time quantitative PCR (N = 4-7) and nNOS protein was estimated by Western blot (N = 3) and immunohistochemistry (N = 1). In the PCR assessment, primers 5’-GTGGCCATCGTGTCCTACCATAC-3’ and 5’-GTTTCGAGGCAGGTGGAAGCTA-3’ were used for the detection of nNOS and primers 5’-CCGTTTCTCCTGGCTCAGTTTA-3’ and 5’-CCCCAATACCACATCATCCAT-3’ were used for the detection of β-actin. Compared to the WT group, nNOS mRNA expression was significantly decreased in FMR1 KO mice at P21 (KO: 0.2857 ± 0.0150, WT: 0.5646 ± 0.0657; P < 0.05). Consistently, nNOS immunoreactivity also revealed reduced staining intensity at P21 in the FMR1 KO group. Western blot analysis validated the immunostaining results by demonstrating a significant reduction in nNOS protein levels in the FMR1 KO group compared to the WT group at P21 (KO: 0.3015 ± 0.0897, WT: 1.7542 ± 0.5455; P < 0.05). These results suggest that nNOS was involved in the postnatal development of the hippocampus in FXS and impaired NO production may retard spine maturation in FXS.

Microarray Analysis of Differentially Expressed Genes between Cysts and Trophozoites of Acanthamoeba castellanii

Acanthamoeba infection is difficult to treat because of the resistance property of Acanthamoeba cyst against the host immune system, diverse antibiotics, and therapeutic agents. To identify encystation mediating factors of Acanthamoeba, we compared the transcription profile between cysts and trophozoites using microarray analysis. The DNA chip was composed of 12,544 genes based on expressed sequence tag (EST) from an Acanthamoeba ESTs database (DB) constructed in our laboratory, genetic information of Acanthamoeba from TBest DB, and all of Acanthamoeba related genes registered in the NCBI. Microarray analysis indicated that 701 genes showed higher expression than 2 folds in cysts than in trophozoites, and 859 genes were less expressed in cysts than in trophozoites. The results of real-time PCR analysis of randomly selected 9 genes of which expression was increased during cyst formation were coincided well with the microarray results. Eukaryotic orthologous groups (KOG) analysis showed an increment in T article (signal transduction mechanisms) and O article (posttranslational modification, protein turnover, and chaperones) whereas significant decrement of C article (energy production and conversion) during cyst formation. Especially, cystein proteinases showed high expression changes (282 folds) with significant increases in real-time PCR, suggesting a pivotal role of this proteinase in the cyst formation of Acanthamoeba. The present study provides important clues for the identification and characterization of encystation mediating factors of Acanthamoeba.

Nkx-2.3 gene in mouse epidemial maturation

In higher vertebrates, from amphibians to humans, epidemial maturation is a conserved developmental process. Using adult epidemial tissue and an established keratinocyte cell line, the mouse Nkx-2.3 homeobox gene was demonstrated, for the first time, to be expressed in mouse epidermal keratinocytes. Under the normal culture condition, the spontaneous aggregation phenomenon, a common initiation step of ES cell differentiation, and the induction of mouse adult K1 keratin, a marker of mature epidermal keratinocytes, were both observed in vitro when the Xenopus Nkx-2.3 gene was stably transfected into a mouse pluripotent P19 EC cell line. The induction of mouse K1 keratin by using its Xenopus orthologous gene in the mouse P19 cell implies that Nkx-2.3 may play a conserved role in the epidermal maturation of the mouse, as it does in that of the frog (Ma, 2004). However, the CAT assay study on frog adult keratin promoter could not find the induction of adult keratin. This implies there might not be a direct activation of its promoter.

Clonagem e expressão da glicoproteína transmembrana do retrovírus HTLV-1 em células de mamíferos / Cloning and transmembrane glycoprotein expression of the retrovirus HTLV-1 in mammals' cells

O retrovírus linfotrópico de células T humanas tipo 1 é o agente etiológico da leucemia das células T do adulto e da paraparesia espástica tropical/mielopatia associada ao HTLV-1. O genoma proviral é composto por 9.032 pares de bases, contendo genes estruturais e regulatórios. A glicoproteína transmembrana gp 21 é codificada pelo gene estrutural env. O desenvolvimento de metodologias para a expressão heteróloga de proteínas, assim como a obtencão de uma linhagem celular que expresse a gp 21 recombinante constitutivamente são os principais objetivos do trabalho. O fragmento codificante da gp 21 foi amplificado por Nested-PCR e clonado no vetor pCR2.1-TOPO. Posteriormente, foi realizada a subclonagem no vetor de expressão pcDNA3.1+. A transfeccão da linhagem celular de mamíferos HEK 293 foi realizada de maneira transitória e permanente. A producão da gp 21 recombinante foi confirmada por citometria de fluxo e a linhagem celular produtora será utilizada em ensaios de imunogenicidade.

Differentially displayed expressed sequence tags in Melipona scutellaris (Hymenoptera, Apidae, Meliponini) development

Nesse estudo nós usamos a técnica de Differential Display Reverse Transcriptase - Polymerase Chain Reaction (DDRT-PCR) para comparamos o perfil de mRNA em Melipona scutellaris durante o desenvolvimento ontogenético pós-embrionário e em operárias adultas, rainha natural e induzida pelo Hormônio Juvenil III. Fragmentos diferencialmente expressos foram detectados usando as seguintes combinações de primers: HT11G-AP05; HT11C-AP05; HT11G-OPF12; HT11G-OPA16. Dos 9 ESTs descrito nesse trabalho, 6 tiveram expressão diferencial nas fases de larva L1 e L2, sugerindo serem mecanismos chave no regulação do desenvolvimento larval em Melipona. A combinação HT11G-AP05 revelou em L1 e L2 um produto com similaridade à proteína tioredoxina redutase de Clostridium sporogenes, uma proteína importante durante os processos de oxidoredução. Esse estudo representa as primeiras evidências moleculares do perfil de expressão durante o desenvolvimento ontogenético em abelhas do gênero Melipona.

Developmental regulation of the expression of sodium currents in Xenopus primary neurons

The electrophysiological properties of neurons are determined by the expression of defined complements of ion channels. Nonetheless, the regulation mechanisms of the expression of neuronal ion channels are poorly understood, due in part to the diversity of neuron subtypes. We explored the expression of voltage-gated currents of Xenopus primary spinal neurons unequivocally identified by means of single-cell RT-PCR. We found that identified spinal neurons exhibit heterogeneity in the temporal appearance of voltage-gated currents. Nevertheless, all neurons progress to similar functional phenotypes. A physiological feature is the onset and increase of the expression of sodium currents. To understand the mechanisms underlying this process, we studied the effect of a dominant negative form of the transcriptional silencer REST/NRSF and found that it associates to an increase in the density of sodium currents. This observation is compatible with a role of this factor in the regulation of gene expression in neurons. These experiments constitute a proof of principle for the feasibility of analyzing molecular mechanisms of the regulation of ion channel genes during early neuronal development and provide direct evidence of the role of REST/NRSF in the control of neuronal sodium channel expression.

Increased expression of SNARE proteins and synaptotagmin IV in islets from pregnant rats and in vitro prolactin-treated neonatal islets

During pregnancy and the perinatal period of life, prolactin (PRL) and other lactogenic substances induce adaptation and maturation of the stimulus-secretion coupling system in pancreatic â-cells. Since the SNARE molecules, SNAP-25, syntaxin 1, VAMP-2, and synaptotagmins participate in insulin secretion, we investigated whether the improved secretory response to glucose during these periods involves alteration in the expression of these proteins. mRNA was extracted from neonatal rat islets cultured for 5 days in the presence of PRL and from pregnant rats (17th-18th days of pregnancy) and reverse transcribed. The expression of genes was analyzed by semi-quantitative RT-PCR assay. The expression of proteins was analyzed by Western blotting and confocal microscopy. Transcription and expression of all SNARE genes and proteins were increased in islets from pregnant and PRL-treated neonatal rats when compared with controls. The only exception was VAMP-2 production in islets from pregnant rats. Increased mRNA and protein expression of synaptotagmin IV, but not the isoform I, also was observed in islets from pregnant and PRL-treated rats. This effect was not inhibited by wortmannin or PD098059, inhibitors of the PI3-kinase and MAPK pathways, respectively. As revealed by confocal laser microscopy, both syntaxin 1A and synaptotagmin IV were immunolocated in islet cells, including the insulin-containing cells. These results indicate that PRL modulates the final steps of insulin secretion by increasing the expression of proteins involved in membrane fusion.

Expression of dorsal-ventral genes during early development of Rhynchosciara americana embryos

The establishment of dorsal-ventral polarity in Drosophila is a complex process which involves the action of maternal and zygotically expressed genes. Interspecific differences in the expression pattern of some of these genes have been described in other species. Here we present the expression of dorsal-ventral genes during early embryogenesis in the lower dipteran Rhynchosciara americana. The expression of four genes, the ventralizing genes snail (sna) and twist (twi) and the dorsalizing genes decapentaplegic (dpp) and zerknüllt (zen), was investigated by whole-mount in situ hybridization. Sense and antisense mRNA were transcribed in vitro using UTP-digoxigenin and hybridized at 55°C with dechorionated fixed embryos. Staining was obtained with anti-digoxigenin alkaline phosphatase-conjugated antibody revealed with NBT-BCIP solution. The results showed that, in general, the spatial-temporal expression of R. americana dorsal-ventral genes is similar to that observed in Drosophila, where twi and sna are restricted to the ventral region, while dpp and zen are expressed in the dorsal side. The differences encountered were subtle and probably represent a particular aspect of dorsal-ventral axis determination in R. americana. In this lower dipteran sna is expressed slightly later than twi and dpp expression is expanded over the lateral ectoderm during cellular blastoderm stage. These data suggest that the establishment of dorsal-ventral polarity in R. americana embryos follows a program similar to that observed in Drosophila melanogaster.

Functional proteomics of receptor-Ck in the developmental stages of human atherosclerotic arterial wall.

BACKGROUND: The study was undertaken to understand the relationship between the functional proteomics of receptor-Ck and developmental stages of human atherosclerotic aortic wall. METHODS AND RESULTS: Gene expression study of 25 aortas was undertaken and the results revealed a gradual increase in receptor-Ck gene expression paralleled by the regulatory response of its effector genes coding for sterol response element-binding protein, p27, cyclin D, interleukin-6 and CD40 from a normal to atherosclerotic arterial wall (viz. fatty streak and fibrofatty/fibrous plaque). CONCLUSIONS: Based upon this and our earlier studies, we propose that cholesterol-specific receptor-Ck-dependent gene regulation may be of crucial importance in atherogenesis.