Identification of positive and negative regulators in the stepwise developmental progression towards infectivity in Trypanosoma brucei.

Trypanosoma brucei is a protozoan parasite that causes important human and livestock diseases in sub-Saharan Africa. By overexpressing a single RNA-binding protein, RBP6, in non-infectious procyclics trypanosomes, we previously recapitulated in vitro the events occurring in the tsetse fly vector, namely the development of epimastigotes and infectious, quiescent metacyclic parasites. To identify genes involved in this developmental progression, we individually targeted 86 transcripts by RNAi in the RBP6 overexpression cell line and assessed the loss-of-function phenotypes on repositioning the kinetoplast, an organelle that contains the mitochondrial genome, the expression of BARP or brucei alanine rich protein, a marker for epimastigotes, and metacyclic variant surface glycoprotein.

Pregnancy outcomes in patients with pulmonary arterial hypertension: A retrospective study.

The mortality of pregnant women with pulmonary arterial hypertension (PAH) remains high. The aim of this study was to evaluate and analyze perinatal and postpartum outcomes in patients with PAH.A total of 79 pregnant patients with PAH who underwent abortion or parturition were reviewed retrospectively.

The vault RNA of plays a role in the production of -spliced mRNA.

The vault ribonucleoprotein (RNP), comprising vault RNA (vtRNA) and telomerase-associated protein 1 (TEP1), is found in many eukaryotes. However, previous studies of vtRNAs, for example in mammalian cells, have failed to reach a definitive conclusion about their function. vtRNAs are related to Y RNAs, which are complexed with Ro protein and influence Ro's function in noncoding RNA (ncRNA) quality control and processing.

Differential expression analysis of transcriptome data of RBP6 induction in procyclics leading to infectious metacyclics and bloodstream forms .

We used an system based on the inducible expression of the RNA binding protein 6 (RBP6) to monitor transcriptome changes during the differentiation of from non-infectious procyclics to infectious metacyclics and from metacyclics to bloodstream forms. This data file describes the bioinformatics analysis of 20 distinct RNA-Seq samples, with four biological replicates each, highlighting differential transcript abundance. Additional functional annotation analysis using Gene Ontology is also presented.

A single-point mutation in the RNA-binding protein 6 generates Trypanosoma brucei metacyclics that are able to progress to bloodstream forms in vitro.

We previously established an in vitro differentiation system based on the inducible expression of the RNA binding protein 6 (RBP6), which initiated differentiation of Trypanosoma brucei non-infectious procyclics to infectious metacyclics (MFs). However, further differentiation to bloodstream forms (BFs) required infection of mice. Here we report the serendipitous isolation of a single point mutation in RBP6 (Q109K), whose expression not only generated MFs, but purified MFs continued the developmental cycle in vitro to BFs expressing variant surface glycoprotein-2 (VSG-2), formerly known as VSG 221.

The proteome and transcriptome of the infectious metacyclic form of Trypanosoma brucei define quiescent cells primed for mammalian invasion.

The infectious metacyclic forms of Trypanosoma brucei result from a complex development in the tsetse fly vector. When they infect mammals, they cause African sleeping sickness in humans. Due to scarcity of biological material and difficulties of the tsetse fly as an experimental system, very limited information is available concerning the gene expression profile of metacyclic forms.

Role of the Trypanosoma brucei HEN1 family methyltransferase in small interfering RNA modification.

Parasitic protozoa of the flagellate order Kinetoplastida represent one of the deepest branches of the eukaryotic tree. Among this group of organisms, the mechanism of RNA interference (RNAi) has been investigated in Trypanosoma brucei and to a lesser degree in Leishmania (Viannia) spp. The pathway is triggered by long double-stranded RNA (dsRNA) and in T.

The structure and repertoire of small interfering RNAs in Leishmania (Viannia) braziliensis reveal diversification in the trypanosomatid RNAi pathway.

Among trypanosomatid protozoa the mechanism of RNA interference (RNAi) has been investigated in Trypanosoma brucei and to a lesser extent in Leishmania braziliensis. Although these two parasitic organisms belong to the same family, they are evolutionarily distantly related raising questions about the conservation of the RNAi pathway. Here we carried out an in-depth analysis of small interfering RNAs (siRNAs) associated with L.

[Diagnosis of placenta previa accreta by two dimensional ultrasonography and color doppler in patients with cesarean section].

Objective: To determine the accuracy of two dismensional sonography and color doppler in diagnosing placenta previa accreta in patients with previous cesarean section.

Methods: Forty-one patients with previous cesarean sections were confirmed to have partial or total placenta previa in the current pregnancy and were given ultrasound examinations after the 28th week of gestation. Specific ultrasound features of the placenta and its interphase with the uterus and the bladder for placenta accreta were checked by two-dimensional ultrasonography and color Doppler.

Small interfering RNA-producing loci in the ancient parasitic eukaryote Trypanosoma brucei.

Background: At the core of the RNA interference (RNAi) pathway in Trypanosoma brucei is a single Argonaute protein, TbAGO1, with an established role in controlling retroposon and repeat transcripts. Recent evidence from higher eukaryotes suggests that a variety of genomic sequences with the potential to produce double-stranded RNA are sources for small interfering RNAs (siRNAs).

Results: To test whether such endogenous siRNAs are present in T.

Comparative genomics reveals two novel RNAi factors in Trypanosoma brucei and provides insight into the core machinery.

The introduction ten years ago of RNA interference (RNAi) as a tool for molecular exploration in Trypanosoma brucei has led to a surge in our understanding of the pathogenesis and biology of this human parasite. In particular, a genome-wide RNAi screen has recently been combined with next-generation Illumina sequencing to expose catalogues of genes associated with loss of fitness in distinct developmental stages. At present, this technology is restricted to RNAi-positive protozoan parasites, which excludes T.

Retention and loss of RNA interference pathways in trypanosomatid protozoans.

RNA interference (RNAi) pathways are widespread in metaozoans but the genes required show variable occurrence or activity in eukaryotic microbes, including many pathogens. While some Leishmania lack RNAi activity and Argonaute or Dicer genes, we show that Leishmania braziliensis and other species within the Leishmania subgenus Viannia elaborate active RNAi machinery. Strong attenuation of expression from a variety of reporter and endogenous genes was seen.

The transcriptome of the human pathogen Trypanosoma brucei at single-nucleotide resolution.

The genome of Trypanosoma brucei, the causative agent of African trypanosomiasis, was published five years ago, yet identification of all genes and their transcripts remains to be accomplished. Annotation is challenged by the organization of genes transcribed by RNA polymerase II (Pol II) into long unidirectional gene clusters with no knowledge of how transcription is initiated. Here we report a single-nucleotide resolution genomic map of the T.

Small nucleolar RNA interference in Trypanosoma brucei: mechanism and utilization for elucidating the function of snoRNAs.

Expression of dsRNA complementary to small nucleolar RNAs (snoRNAs) in Trypanosoma brucei results in snoRNA silencing, termed snoRNAi. Here, we demonstrate that snoRNAi requires the nuclear TbDCL2 protein, but not TbDCL1, which is involved in RNA interference (RNAi) in the cytoplasm. snoRNAi depends on Argonaute1 (Slicer), and on TbDCL2, suggesting that snoRNA dicing and slicing takes place in the nucleus, and further suggesting that AGO1 is active in nuclear silencing.

RNA interference in Trypanosoma brucei: role of the n-terminal RGG domain and the polyribosome association of argonaute.

Argonaute proteins (AGOs) are central to RNA interference (RNAi) and related silencing pathways. At the core of the RNAi pathway in the ancient parasitic eukaryote Trypanosoma brucei is a single Argonaute protein, TbAGO1, with an established role in the destruction of potentially harmful retroposon transcripts. One notable feature of TbAGO1 is that a fraction sediments with polyribosomes, and this association is facilitated by an arginine/glycine-rich domain (RGG domain) at the N terminus of the protein.

Distinct and overlapping roles for two Dicer-like proteins in the RNA interference pathways of the ancient eukaryote Trypanosoma brucei.

Trypanosoma brucei is one of the most ancient eukaryotes where RNA interference (RNAi) is operational and is the only single-cell pathogen where RNAi has been extensively studied and used as a tool for functional analyses. Here, we report that the T. brucei RNAi pathway, although relying on a single Argonaute protein (AGO1), is initiated by the activities of two distinct Dicer-like enzymes.

Genomic rearrangements and transcriptional analysis of the spliced leader-associated retrotransposon in RNA interference-deficient Trypanosoma brucei.

The Trypanosoma brucei genome is colonized by the site-specific non-LTR retrotransposon SLACS, or spliced leader-associated conserved sequence, which integrates exclusively into the spliced leader (SL) RNA genes. Although there is evidence that the RNA interference (RNAi) machinery regulates SLACS transcript levels, we do not know whether RNAi deficiency affects the genomic stability of SLACS, nor do we understand the mechanism of SLACS transcription. Here, we report that prolonged culturing of RNAi-deficient T.

Depletion of newly synthesized Argonaute1 impairs the RNAi response in Trypanosoma brucei.

In Trypanosoma brucei, Argonaute1 (TbAGO1) is an essential component of the RNA interference (RNAi) pathway. While characterizing a TbAGO1 conditional knockout cell line, we discovered that, upon blockage of TbAGO1 transcription, the RNAi response to transfected double-stranded RNA was severely inhibited, although there was no change in the TbAGO1 protein level. This observation suggested that steady-state TbAGO1 was not sufficient to fully support the RNAi response to transfected dsRNA and implicated newly synthesized Argonaute in this phenomenon.

An unusual Dicer-like1 protein fuels the RNA interference pathway in Trypanosoma brucei.

RNA interference (RNAi) is an evolutionarily conserved gene-silencing pathway that is triggered by double-stranded RNA (dsRNA). Central to this pathway are two ribonucleases: Dicer, a multidomain RNase III family enzyme that initiates RNAi by generating small interfering RNAs (siRNAs), and Argonaute or Slicer, an RNase H signature enzyme that affects cleavage of mRNA. Previous studies in the early diverging protozoan Trypanosoma brucei have established a key role for Argonaute 1 in RNAi.

Functional replacement of Trypanosoma brucei Argonaute by the human slicer Argonaute2.

RNA interference (RNAi) is widespread throughout the eukaryotic lineage, from protozoa to man. Central to all RNAi phenomena is a member of the Argonaute protein family, and, in the case of dsRNA-triggered mRNA cleavage, the Ago protein functions as the RNAi endonuclease or slicer. However, at present there is no definite experimental evidence that slicer Argonautes can be interchanged between distantly related organisms.

Selection and characterization of RNA interference-deficient trypanosomes impaired in target mRNA degradation.

Genetic analysis of the RNA interference (RNAi) pathway in Trypanosoma brucei has so far revealed one essential component, namely, TbAGO1, encoding a member of the Argonaute protein family. To gain further insight into the RNAi mechanism and its biological significance, we selected RNAi-deficient trypanosomes by using repeated cycles of electroporation with alpha-tubulin double-stranded RNA, a treatment that blocks cytokinesis in wild-type cells. Two independent clones, termed RiD-1 (for RNAi-deficient clone 1) and RiD-2, were characterized.

Function of the Trypanosome Argonaute 1 protein in RNA interference requires the N-terminal RGG domain and arginine 735 in the Piwi domain.

Argonaute proteins are central components of RNA interference (RNAi) and related phenomena in a wide variety of eukaryotes, including the early diverging protozoan Trypanosoma brucei. The single T. brucei Argonaute protein (TbAGO1) is in a complex with small interfering RNAs (siRNAs), and a fraction of this ribonucleoprotein particle is associated with polyribosomes.

Argonaute protein in the early divergent eukaryote Trypanosoma brucei: control of small interfering RNA accumulation and retroposon transcript abundance.

Members of the Argonaute protein family have been linked through a combination of genetic and biochemical studies to RNA interference (RNAi) and related phenomena. Here, we describe the characterization of the first Argonaute protein (AGO1) in Trypanosoma brucei, the earliest divergent eukaryote where RNAi has been described so far. AGO1 is predominantly cytoplasmic and is found in a ribonucleoprotein particle with small interfering RNAs (siRNAs), and this particle is present in a soluble form, as well as associated with polyribosomes.