Structural, Functional, and Metabolic Alterations in Human Cerebrovascular Endothelial Cells during Toxoplasma gondii Infection and Amelioration by Verapamil In Vitro.

Toxoplasma gondii (T. gondii), the causative agent of toxoplasmosis, is a frequent cause of brain infection. Despite its known ability to invade the brain, there is still a dire need to better understand the mechanisms by which this parasite interacts with and crosses the blood-brain barrier (BBB). The present study revealed structural and functional changes associated with infection and replication of T. gondii within human brain microvascular endothelial cells (BMECs) in vitro. T. gondii proliferated within the BMECs and disrupted the integrity of the cerebrovascular barrier through diminishing the cellular viability, disruption of the intercellular junctions and increasing permeability of the BMEC monolayer, as well as altering lipid homeostasis. Proton nuclear magnetic resonance (1H NMR)-based metabolomics combined with multivariate data analysis revealed profiles that can be attributed to infection and variations in the amounts of certain metabolites (e.g., amino acids, fatty acids) in the extracts of infected compared to control cells. Notably, treatment with the Ca2+ channel blocker verapamil rescued BMEC barrier integrity and restricted intracellular replication of the tachyzoites regardless of the time of treatment application (i.e., prior to infection, early- and late-infection). This study provides new insights into the structural and functional changes that accompany T. gondii infection of the BMECs, and sheds light upon the ability of verapamil to inhibit the parasite proliferation and to ameliorate the adverse effects caused by T. gondii infection.

Effects of Toxoplasma gondii infection on the function and integrity of human cerebrovascular endothelial cells and the influence of verapamil treatment in vitro.

Toxoplasma gondii can cause parasitic encephalitis, a life-threatening infection that predominately occurs in immunocompromised individuals. T. gondii has the ability to invade the brain, but the mechanisms by which this parasite crosses the blood-brain-barrier (BBB) remain incompletely understood. The present study reports the changes associated with infection and replication of T. gondii within human brain microvascular endothelial cells (BMECs) in vitro. Our results indicated that exposure to T. gondii had an adverse impact on the function and integrity of the BMECs - through induction of cell cycle arrest, disruption of the BMEC barrier integrity, reduction of cellular viability and vitality, depolarization of the mitochondrial membrane potential, increase of the DNA fragmentation, and alteration of the expression of immune response and tight junction genes. The calcium channel/P-glycoprotein transporter inhibitor verapamil was effective in inhibiting T. gondii crossing the BMECs in a dose-dependent manner. The present study showed that T. gondii can compromise several functions of BMECs and demonstrated the ability of verapamil to inhibit T. gondii crossing of the BMECs in vitro.

Mutationally activated PIK3CA(H1047R) cooperates with BRAF(V600E) to promote lung cancer progression.

Adenocarcinoma of the lung, a leading cause of cancer death, frequently displays mutational activation of the KRAS proto-oncogene but, unlike lung cancers expressing mutated EGFR, ROS1, or ALK, there is no pathway-targeted therapy for patients with KRAS-mutated lung cancer. In preclinical models, expression of oncogenic KRAS(G12D) in the lung epithelium of adult mice initiates development of lung adenocarcinoma through activation of downstream signaling pathways. In contrast, mutationally activated BRAF(V600E), a KRAS effector, fails to initiate lung carcinogenesis despite highly efficient induction of benign lung tumorigenesis. To test if phosphoinositide 3-kinase (PI3K)-α (PIK3CA), another KRAS effector, might cooperate with oncogenic BRAF(V600E) to promote lung cancer progression, we used mice carrying a conditional allele of Pik3ca that allows conversion of the wild-type catalytic subunit of PIK3CA to mutationally activated PIK3CA(H1047R). Although expression of PIK3CA(H1047R) in the lung epithelium, either alone or in combination with PTEN silencing, was without phenotype, concomitant expression of BRAF(V600E) and PIK3CA(H1047R) led to dramatically decreased tumor latency and increased tumor burden compared with BRAF(V600E) alone. Most notably, coexpression of BRAF(V600E) and PIK3CA(H1047R) elicited lung adenocarcinomas in a manner reminiscent of the effects of KRAS(G12D). These data emphasize a role for PI3K signaling, not in lung tumor initiation per se, but in both the rate of tumor growth and the propensity of benign lung tumors to progress to a malignant phenotype. Finally, biologic and biochemical analysis of BRAF(V600E)/PIK3CA(H1047R)-expressing mouse lung cancer cells revealed mechanistic clues about cooperative regulation of the cell-division cycle and apoptosis by these oncogenes.

PTEN loss and KRAS activation cooperate in murine biliary tract malignancies.

Carcinomas of the biliary tract are aggressive malignancies in humans. Loss of the tumour suppressor PTEN has previously been associated with cholangiocarcinoma development in a murine model. Activation of KRAS is reported in up to one-third of human cholangiocarcinomas and 50% of gall bladder carcinomas. In this study we aimed to test the potential interaction between PTEN and KRAS mutation in biliary tract malignancy. We used an inducible Cre-LoxP-based approach to coordinately delete PTEN and activate KRAS within the adult mouse biliary epithelium. We found that activation of KRAS alone has little effect upon biliary epithelium. Loss of PTEN alone results in the development of low-grade neoplastic lesions, following long latency and at low incidence. Combination of both mutations causes rapid development of biliary epithelial proliferative lesions, which progress through dysplasia to invasive carcinoma. We conclude that activation of the PI3'K pathway following loss of PTEN is sufficient to drive slow development of low-grade biliary lesions in mice. In contrast, mutational activation of KRAS does not result in a similar phenotype, despite a prediction that this should activate both the RAF-MEK-ERK and PI3'-kinase pathways. However, mutation of both genes results in rapid tumourigenesis, arguing that PTEN normally functions as a 'brake' on the PI3'-kinase pathway, limiting the influence of KRAS activation. Mutation of both genes creates a 'permissive' environment, allowing the full effects of both mutations to be manifested. These data reveal an in vivo synergy between these mutations and provides a new mouse model of biliary tract malignancy.

Origin and maintenance of the intestinal cancer stem cell.

Colorectal cancer is one of the most common cancers in the western world and its incidence is steadily increasing. Understanding the basic biology of both the normal intestine and of intestinal tumorigenesis is vital for developing appropriate and effective cancer therapies. However, relatively little is known about the normal intestinal stem cell or the hypothetical intestinal cancer stem cell, and there is much debate surrounding these areas. This review briefly describes our current understanding of the properties of both the intestinal stem cell and the intestinal cancer stem cell. We also discuss recent theories regarding the origin of the intestinal cancer stem cell, and the signals required for its maintenance and proliferation. Finally, we place the relevance of cancer stem cell research into context by discussing potential clinical applications of targeting the intestinal cancer stem cell.

Focal adhesion kinase is required for intestinal regeneration and tumorigenesis downstream of Wnt/c-Myc signaling.

The intestinal epithelium has a remarkable capacity to regenerate after injury and DNA damage. Here, we show that the integrin effector protein Focal Adhesion Kinase (FAK) is dispensable for normal intestinal homeostasis and DNA damage signaling, but is essential for intestinal regeneration following DNA damage. Given Wnt/c-Myc signaling is activated following intestinal regeneration, we investigated the functional importance of FAK following deletion of the Apc tumor suppressor protein within the intestinal epithelium. Following Apc loss, FAK expression increased in a c-Myc-dependent manner. Codeletion of Apc and Fak strongly reduced proliferation normally induced following Apc loss, and this was associated with reduced levels of phospho-Akt and suppression of intestinal tumorigenesis in Apc heterozygous mice. Thus, FAK is required downstream of Wnt Signaling, for Akt/mTOR activation, intestinal regeneration, and tumorigenesis. Importantly, this work suggests that FAK inhibitors may suppress tumorigenesis in patients at high risk of developing colorectal cancer.

Epithelial Pten is dispensable for intestinal homeostasis but suppresses adenoma development and progression after Apc mutation.

PTEN acts as a tumor suppressor in a range of tissue types and has been implicated in the regulation of intestinal stem cells. To study Pten function in the intestine, we used various conditional transgenic strategies to specifically delete Pten from the mouse intestinal epithelium. We show that Pten loss specifically within the adult or embryonic epithelial cell population does not affect the normal architecture or homeostasis of the epithelium. However, loss of Pten in the context of Apc deficiency accelerates tumorigenesis through increased activation of Akt, leading to rapid development of adenocarcinoma. We conclude that Pten is redundant in otherwise normal intestinal epithelium and epithelial stem cells but, in the context of activated Wnt signaling, suppresses progression to adenocarcinoma through modulation of activated Akt levels.

A limited role for p53 in modulating the immediate phenotype of Apc loss in the intestine.

BACKGROUND: p53 is an important tumour suppressor with a known role in the later stages of colorectal cancer, but its relevance to the early stages of neoplastic initiation remains somewhat unclear. Although p53-dependent regulation of Wnt signalling activity is known to occur, the importance of these regulatory mechanisms during the early stages of intestinal neoplasia has not been demonstrated. METHODS: We have conditionally deleted the Adenomatous Polyposis coli gene (Apc) from the adult murine intestine in wild type and p53 deficient environments and subsequently compared the phenotype and transcriptome profiles in both genotypes. RESULTS: Expression of p53 was shown to be elevated following the conditional deletion of Apc in the adult small intestine. Furthermore, p53 status was shown to impact on the transcription profile observed following Apc loss. A number of key Wnt pathway components and targets were altered in the p53 deficient environment. However, the aberrant phenotype observed following loss of Apc (rapid nuclear localisation of beta-catenin, increased levels of DNA damage, nuclear atypia, perturbed cell death, proliferation, differentiation and migration) was not significantly altered by the absence of p53. CONCLUSION: p53 related feedback mechanisms regulating Wnt signalling activity are present in the intestine, and become activated following loss of Apc. However, the physiological Wnt pathway regulation by p53 appears to be overwhelmed by Apc loss and consequently the activity of these regulatory mechanisms is not sufficient to modulate the immediate phenotypes seen following Apc loss. Thus we are able to provide an explanation to the apparent contradiction that, despite having a Wnt regulatory capacity, p53 loss is not associated with early lesion development.

DNA evidence for global dispersal and probable endemicity of protozoa.

BACKGROUND: It is much debated whether microbes are easily dispersed globally or whether they, like many macro-organisms, have historical biogeographies. The ubiquitous dispersal hypothesis states that microbes are so numerous and so easily dispersed worldwide that all should be globally distributed and found wherever growing conditions suit them. This has been broadly upheld for protists (microbial eukaryotes) by most morphological and some molecular analyses. However, morphology and most previously used evolutionary markers evolve too slowly to test this important hypothesis adequately. RESULTS: Here we use a fast-evolving marker (ITS1 rDNA) to map global diversity and distribution of three different clades of cercomonad Protozoa (Eocercomonas and Paracercomonas: phylum Cercozoa) by sequencing multiple environmental gene libraries constructed from 47-80 globally-dispersed samples per group. Even with this enhanced resolution, identical ITS sequences (ITS-types) were retrieved from widely separated sites and on all continents for several genotypes, implying relatively rapid global dispersal. Some identical ITS-types were even recovered from both marine and non-marine samples, habitats that generally harbour significantly different protist communities. Conversely, other ITS-types had either patchy or restricted distributions. CONCLUSION: Our results strongly suggest that geographic dispersal in macro-organisms and microbes is not fundamentally different: some taxa show restricted and/or patchy distributions while others are clearly cosmopolitan. These results are concordant with the 'moderate endemicity model' of microbial biogeography. Rare or continentally endemic microbes may be ecologically significant and potentially of conservational concern. We also demonstrate that strains with identical 18S but different ITS1 rDNA sequences can differ significantly in terms of morphological and important physiological characteristics, providing strong additional support for global protist biodiversity being significantly higher than previously thought.

Intestinal homeostasis and neoplasia studied using conditional transgenesis.

Constitutive mouse models of intestinal neoplasia, such as the Apc(min/+) (multiple intestinal neoplasia) mouse have proven valuable tools both for furthering our understanding of tumorigenesis and for the development of therapeutic strategies. However, the in vivo study of a number of genes has been precluded by their absolute requirement during embryonic development. This has led to the development of conditional strategies that allow gene regulation in vivo. This review describes the principal techniques used to achieve conditional transgenesis within the mouse intestine, with a particular focus upon the Cre-Lox and Tet-regulable systems. Further, we discuss how these techniques are being used to dissect the mechanisms governing both normal homeostasis and neoplastic development within the intestine.

Loss of Apc allows phenotypic manifestation of the transforming properties of an endogenous K-ras oncogene in vivo.

Oncogenic mutations in the K-ras gene occur in approximately 50% of human colorectal cancers. However, the precise role that K-ras oncogenes play in tumor formation is still unclear. To address this issue, we have conditionally expressed an oncogenic K-ras(V12) allele in the small intestine of adult mice either alone or in the context of Apc deficiency. We found that expression of K-ras(V12) does not affect normal intestinal homeostasis or the immediate phenotypes associated with Apc deficiency. Mechanistically we failed to find activation of the Raf/MEK/ERK pathway, which may be a consequence of the up-regulation of a number of negative feedback loops. However, K-ras(V12) expression accelerates intestinal tumorigenesis and confers invasive properties after Apc loss over the long term. In renal epithelium, expression of the oncogenic K-ras(V12) allele in the absence of Apc induces the rapid development of renal carcinoma. These tumors, unlike those of intestinal origin, display activation of the Raf/MEK/ERK and Akt signaling pathways. Taken together, these data indicate that normal intestinal and kidney epithelium are resistant to malignant transformation by an endogenous K-ras oncogene. However, activation of K-ras(V12) after Apc loss results in increased tumorigenesis with distinct kinetics. Whereas the effect of K-ras oncogenes in the intestine can been observed only after long latencies, they result in rapid carcinogenesis in the kidney epithelium. These data imply a window of opportunity for anti-K-ras therapies after tumor initiation in preventing tumor growth and invasion.

Influence of chromatin and single strand binding proteins on the activity of an archaeal MCM.

The mini-chromosome maintenance (MCM) complex is the presumptive replicative helicase in archaea and eukaryotes. In archaea, the MCM is a homo-multimer, in eukaryotes a heterohexamer composed of six related subunits, MCM 2-7. Biochemical studies using naked DNA templates have revealed that archaeal MCMs and a sub-complex of eukaryotic MCM 4, 6 and 7 have 3' to 5' helicase activity. Here, we investigate the influence of the major chromatin proteins, Alba and Sul7d, of Sulfolobus solfataricus (Sso) on the ability of the MCM complex to melt partial duplex DNA substrates. In addition, we test the effect of Sso SSB on MCM activity. We reveal that Alba represents a formidable barrier to MCM activity and further demonstrate that acetylation of Alba alleviates repression of MCM activity.

Sir2 and the acetyltransferase, Pat, regulate the archaeal chromatin protein, Alba.

The DNA binding affinity of Alba, a chromatin protein of the archaeon Sulfolobus solfataricus P2, is regulated by acetylation of lysine 16. Here we identify an acetyltransferase that specifically acetylates Alba on this residue. The effect of acetylation is to lower the affinity of Alba for DNA. Remarkably, the acetyltransferase is conserved not only in archaea but also in bacteria where it appears to play a role in metabolic regulation. Therefore, our data suggest that S. solfataricus has co-opted this bacterial regulatory system to generate a rudimentary form of chromatin regulation.

Identification of two origins of replication in the single chromosome of the archaeon Sulfolobus solfataricus.

Eukaryotic chromosomes possess multiple origins of replication, whereas bacterial chromosomes are replicated from a single origin. The archaeon Pyrococcus abyssi also appears to have a single origin, suggesting a common rule for prokaryotes. However, in the current work, we describe the identification of two active origins of replication in the single chromosome of the hyperthermophilic archaeon Sulfolobus solfataricus. Further, we identify conserved sequence motifs within the origins that are recognized by a family of three Sulfolobus proteins that are homologous to the eukaryotic initiator proteins Orc1 and Cdc6. We demonstrate that the two origins are recognized by distinct subsets of these Orc1/Cdc6 homologs. These data, in conjunction with an analysis of the levels of the three Orc1/Cdc6 proteins in different growth phases and cell cycle stages, lead us to propose a model for the roles for these proteins in modulating origin activity.