Ductal carcinoma in situ of the breast: morphological and molecular features implicated in progression.

The spread of mammographic screening programmes around the world, including in developing countries, has substantially contributed to the diagnosis of small non-palpable lesions, which has increased the detection rate of DCIS (ductal carcinoma in situ). DCIS is heterogeneous in several ways, such as its clinical presentation, morphology and genomic profile. Excellent outcomes have been reported; however, many questions remain unanswered. For example, which patients groups are overtreated and could instead benefit from minimal intervention and which patient groups require a more traditional multidisciplinary approach. The development of a comprehensive integrated analysis that includes the radiological, morphological and genetic aspects of DCIS is necessary to answer these questions. This review focuses on discussing the significant findings about the morphological and molecular features of DCIS and its progression that have helped to uncover the biological and genetic heterogeneity of this disease. The knowledge gained in recent years might allow the development of tailored clinical management for women with DCIS in the future.

Toxoplasma gondii lysine acetyltransferase GCN5-A functions in the cellular response to alkaline stress and expression of cyst genes.

Parasitic protozoa such as the apicomplexan Toxoplasma gondii progress through their life cycle in response to stimuli in the environment or host organism. Very little is known about how proliferating tachyzoites reprogram their expressed genome in response to stresses that prompt development into latent bradyzoite cysts. We have previously linked histone acetylation with the expression of stage-specific genes, but the factors involved remain to be determined. We sought to determine if GCN5, which operates as a transcriptional co-activator by virtue of its histone acetyltransferase (HAT) activity, contributed to stress-induced changes in gene expression in Toxoplasma. In contrast to other lower eukaryotes, Toxoplasma has duplicated its GCN5 lysine acetyltransferase (KAT). Disruption of the gene encoding for TgGCN5-A in type I RH strain did not produce a severe phenotype under normal culture conditions, but here we show that the TgGCN5-A null mutant is deficient in responding to alkaline pH, a common stress used to induce bradyzoite differentiation in vitro. We performed a genome-wide analysis of the Toxoplasma transcriptional response to alkaline pH stress, finding that parasites deleted for TgGCN5-A fail to up-regulate 74% of the stress response genes that are induced 2-fold or more in wild-type. Using chromatin immunoprecipitation, we verify an enrichment of TgGCN5-A at the upstream regions of genes activated by alkaline pH exposure. The TgGCN5-A knockout is also incapable of up-regulating key marker genes expressed during development of the latent cyst form, and is impaired in its ability to recover from alkaline stress. Complementation of the TgGCN5-A knockout restores the expression of these stress-induced genes and reverses the stress recovery defect. These results establish TgGCN5-A as a major contributor to the alkaline stress response in RH strain Toxoplasma.

A decade of epigenetic research in Toxoplasma gondii.

In the past 10 years, the field of parasitology has witnessed an explosion of studies investigating gene regulation. In this review, we will describe recent advances largely stemming from the study of Toxoplasma gondii, a significant opportunistic pathogen and useful model for other apicomplexan protozoa. Surprising findings have emerged, including the discovery of a wealth of epigenetic machinery in these primitive eukaryotes, unusual histone variants, and a battery of plant-like transcription factors. We will elaborate on how these unusual features impact parasite physiology and potential therapeutics as we summarize some of the key discoveries from the last decade. We will close by proposing a few questions to address in the next 10 years.

ORF-C4 from the early branching eukaryote Giardia lamblia displays characteristics of alpha-crystallin small heat-shock proteins.

Giardia lamblia is a medically important protozoan parasite with a basal position in the eukaryotic lineage and is an interesting model to explain the evolution of biochemical events in eukaryotic cells. G. lamblia trophozoites undergo significant changes in order to survive outside the intestine of their host by differentiating into infective cysts. In the present study, we characterize the previously identified Orf-C4 (G. lamblia open reading frame C4) gene, which is considered to be specific to G. lamblia. It encodes a 22 kDa protein that assembles into high-molecular-mass complexes during the entire life cycle of the parasite. ORF-C4 localizes to the cytoplasm of trophozoites and cysts, and forms large spherical aggregates when overexpressed. ORF-C4 overexpression and down-regulation do not affect trophozoite viability; however, differentiation into cysts is slightly delayed when the expression of ORF-C4 is down-regulated. In addition, ORF-C4 protein expression is modified under specific stress-inducing conditions. Neither orthologous proteins nor conserved domains are found in databases by conventional sequence analysis of the predicted protein. However, ORF-C4 contains a region which is similar structurally to the alpha-crystallin domain of sHsps (small heat-shock proteins). In the present study, we show the potential role of ORF-C4 as a small chaperone which is involved in the response to stress (including encystation) in G. lamblia.

Antigenic variation in Giardia lamblia is regulated by RNA interference.

Giardia lamblia (also called Giardia intestinalis) is one of the most common intestinal parasites of humans. To evade the host's immune response, Giardia undergoes antigenic variation-a process that allows the parasite to develop chronic and recurrent infections. From a repertoire of approximately 190 variant-specific surface protein (VSP)-coding genes, Giardia expresses only one VSP on the surface of each parasite at a particular time, but spontaneously switches to a different VSP by unknown mechanisms. Here we show that regulation of VSP expression involves a system comprising RNA-dependent RNA polymerase, Dicer and Argonaute, known components of the RNA interference machinery. Clones expressing a single surface antigen efficiently transcribe several VSP genes but only accumulate transcripts encoding the VSP to be expressed. Detection of antisense RNAs corresponding to the silenced VSP genes and small RNAs from the silenced but not for the expressed vsp implicate the RNA interference pathway in antigenic variation. Remarkably, silencing of Dicer and RNA-dependent RNA polymerase leads to a change from single to multiple VSP expression in individual parasites.

Characterization of SNAREs determines the absence of a typical Golgi apparatus in the ancient eukaryote Giardia lamblia.

Giardia is a eukaryotic protozoal parasite with unusual characteristics, such as the absence of a morphologically evident Golgi apparatus. Although both constitutive and regulated pathways for protein secretion are evident in Giardia, little is known about the mechanisms involved in vesicular docking and fusion. In higher eukaryotes, soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) of the vesicle-associated membrane protein and syntaxin families play essential roles in these processes. In this work we identified and characterized genes for 17 SNAREs in Giardia to define the minimal set of subcellular organelles present during growth and encystation, in particular the presence or not of a Golgi apparatus. Expression and localization of all Giardia SNAREs demonstrate their presence in distinct subcellular compartments, which may represent the extent of the endomembrane system in eukaryotes. Remarkably, Giardia SNAREs, homologous to Golgi SNAREs from other organisms, do not allow the detection of a typical Golgi apparatus in either proliferating or differentiating trophozoites. However, some features of the Golgi, such as the packaging and sorting function, seem to be performed by the endoplasmic reticulum and/or the nuclear envelope. Moreover, depletion of individual genes demonstrated that several SNAREs are essential for viability, whereas others are dispensable. Thus, Giardia requires a smaller number of SNAREs compared with other eukaryotes to accomplish all of the vesicle trafficking events that are critical for the growth and differentiation of this important human pathogen.

Active and passive mechanisms drive secretory granule biogenesis during differentiation of the intestinal parasite Giardia lamblia.

The parasitic protozoan Giardia lamblia undergoes important changes to survive outside the intestine of its host by differentiating into infective cysts. During encystation, three cyst wall proteins (CWPs) are specifically expressed and concentrated within encystation-specific secretory vesicles (ESVs). ESVs are electron-dense secretory granules that transport CWPs before exocytosis and extracellular polymerization into a rigid cyst wall. Because secretory granules form at the trans-Golgi in higher eukaryotes and because Giardia lacks an identifiable Golgi apparatus, the aim of this work was to investigate the molecular basis of secretory granule formation in Giardia by examining the role of CWPs in this process. Although CWP1, CWP2, and CWP3 are structurally similar in their 26-kDa leucine-rich overlapping region, CWP2 is distinguished by the presence of a 13-kDa C-terminal basic extension. In non-encysting trophozoites, expression of different CWP chimeras showed that the CWP2 basic extension is necessary for biogenesis of ESVs, which occurs in a compartment derived from the endoplasmic reticulum. Nevertheless, the CWP2 basic extension per se is insufficient to trigger ESV formation, indicating that other domains in CWPs are also required. We found that CWP2 is a key regulator of ESV formation by acting as an aggregation factor for CWP1 and CWP3 through interactions mediated by its conserved region. CWP2 also acts as a ligand for sorting via its C-terminal basic extension. These findings show that granule biogenesis requires complex interactions among granule components and membrane receptors.