Targeting the Hedgehog Pathway in Pediatric Medulloblastoma

Medulloblastoma (MB), a primitive neuroectomal tumor of the cerebellum, is the most common malignant pediatric brain tumor. The cause of MB is largely unknown, but aberrant activation of Hedgehog (Hh) pathway is responsible for ~30% of MB. Despite aggressive treatment with surgical resection, radiation and chemotherapy, 70%-80% of pediatric medulloblastoma cases can be controlled, but most treated patients suffer devastating side effects. Therefore, developing a new effective treatment strategy is urgently needed. Hh signaling controls transcription of target genes by regulating activities of the three Glioma-associated oncogene (Gli1-3) transcription factors. In this review, we will focus on current clinical treatment options of MB and discuss mechanisms of drug resistance. In addition, we will describe current known molecular pathways which crosstalk with the Hedgehog pathway both in the context of medulloblastoma and non-medulloblastoma cancer development. Finally, we will introduce post-translational modifications that modulate Gli1 activity and summarize the positive and negative regulations of the Hh/Gli1 pathway. Towards developing novel combination therapies for medulloblastoma treatment, current information on interacting pathways and direct regulation of Hh signaling should prove critical

Transcriptional repression by ApiAP2 factors is central to chronic toxoplasmosis

Tachyzoite to bradyzoite development in Toxoplasma is marked by major changes in gene expression resulting in a parasite that expresses a new repertoire of surface antigens hidden inside a modified parasitophorous vacuole called the tissue cyst. The factors that control this important life cycle transition are not well understood. Here we describe an important transcriptional repressor mechanism controlling bradyzoite differentiation that operates in the tachyzoite stage. The ApiAP2 factor, AP2IV-4, is a nuclear factor dynamically expressed in late S phase through mitosis/cytokinesis of the tachyzoite cell cycle. Remarkably, deletion of the AP2IV-4 locus resulted in the expression of a subset of bradyzoite-specific proteins in replicating tachyzoites that included tissue cyst wall components BPK1, MCP4, CST1 and the surface antigen SRS9. In the murine animal model, the mis-timing of bradyzoite antigens in tachyzoites lacking AP2IV-4 caused a potent inflammatory monocyte immune response that effectively eliminated this parasite and prevented tissue cyst formation in mouse brain tissue. Altogether, these results indicate that suppression of bradyzoite antigens by AP2IV-4 during acute infection is required for Toxoplasma to successfully establish a chronic infection in the immune-competent host

Bromodomains in Protozoan Parasites: Evolution, Function, and Opportunities for Drug Development

Parasitic infections remain one of the most pressing global health concerns of our day, affecting billions of people and producing unsustainable economic burdens. The rise of drug-resistant parasites has created an urgent need to study their biology in hopes of uncovering new potential drug targets. It has been established that disrupting gene expression by interfering with lysine acetylation is detrimental to survival of apicomplexan (Toxoplasma gondii and Plasmodium spp.) and kinetoplastid (Leishmania spp. and Trypanosoma spp.) parasites. As "readers" of lysine acetylation, bromodomain proteins have emerged as key gene expression regulators and a promising new class of drug target. Here we review recent studies that demonstrate the essential roles played by bromodomain-containing proteins in parasite viability, invasion, and stage switching and present work showing the efficacy of bromodomain inhibitors as novel antiparasitic agents. In addition, we performed a phylogenetic analysis of bromodomain proteins in representative pathogens, some of which possess unique features that may be specific to parasite processes and useful in future drug development

Algoriphagus machipongonensis sp. nov., co-isolated with a colonial choanoflagellate

A Gram-negative, non-motile, non-spore-forming bacterial strain, PR1[superscript T], was isolated from a mud core sample containing colonial choanoflagellates near Hog Island, Virginia, USA. Strain PR1[superscript T] grew optimally at 30 °C and with 3 % (w/v) NaCl. Strain PR1[superscript T] contained MK-7 as the major menaquinone as well as carotenoids but lacked pigments of the flexirubin-type. The predominant fatty acids were iso-C15 : 0 (29.4 %), iso-C17 : 1ω9c (18.5 %) and summed feature 3 (C16 : 1ω6c and/or C16 : 1ω7c; 11.3 %). The major polar lipids detected in strain PR1[superscript T] were phosphatidylethanolamine, an unknown phospholipid, an aminophospholipid, an aminolipid and two lipids of unknown character. The DNA G+C content was 38.7 mol%. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain PR1[superscript T] fell within the cluster comprising the genus Algoriphagus and was most closely related to Algoriphagus halophilus JC 2051[superscript T] (95.4 % sequence similarity) and Algoriphagus lutimaris S1-3[superscript T] (95.3 % sequence similarity). The 16S rRNA gene sequence similarity between strain PR1[superscript T] and the type strains of other species of the genus Algoriphagus were in the range 91–95 %. Differential phenotypic properties and phylogenetic and genetic distinctiveness of strain PR1[superscript T] demonstrated that this strain was distinct from other members of the genus Algoriphagus, including its closest relative, A. halophilus. Based on phenotypic, chemotaxonomic, phylogenetic and genomic data, strain PR1[superscript T] should be placed in the genus Algoriphagus as a representative of a novel species, for which the name Algoriphagus machipongonensis sp. nov. is proposed. The type strain is PR1[superscript T] ( = ATCC BAA-2233[superscript T]  = DSM 24695[superscript T]).Gordon and Betty Moore Foundation (Investigator Award (581))National Institutes of Health (U.S.) (NIH National Research Service Award and Fellowship grant (5F32GM086054))United States. National Aeronautics and Space Administration (NASA Astrobiology Institute (NNA08CN84A

Toxoplasma gondii AP2IX-4 Regulates Gene Expression during Bradyzoite Development

Toxoplasma gondii is a protozoan parasite of great importance to human and animal health. In the host, this obligate intracellular parasite persists as a tissue cyst that is imperceptible to the immune response and unaffected by current therapies. The tissue cysts facilitate transmission through predation and give rise to chronic cycles of toxoplasmosis in immunocompromised patients. Transcriptional changes accompany conversion of the rapidly replicating tachyzoites into the encysted bradyzoites, and yet the mechanisms underlying these alterations in gene expression are not well defined. Here we show that AP2IX-4 is a nuclear protein exclusively expressed in tachyzoites and bradyzoites undergoing division. Knockout of AP2IX-4 had no discernible effect on tachyzoite replication but resulted in a reduced frequency of tissue cyst formation following alkaline stress induction-a defect that is reversible by complementation. AP2IX-4 has a complex role in regulating bradyzoite gene expression, as the levels of many bradyzoite mRNAs dramatically increased beyond those seen under conditions of normal stress induction in AP2IX-4 knockout parasites exposed to alkaline media. The loss of AP2IX-4 also resulted in a modest virulence defect and reduced cyst burden in chronically infected mice, which was reversed by complementation. These findings illustrate that the transcriptional mechanisms responsible for tissue cyst development operate across the intermediate life cycle from the dividing tachyzoite to the dormant bradyzoite. IMPORTANCEToxoplasma gondii is a single-celled parasite that persists in its host as a transmissible tissue cyst. How the parasite converts from its replicative form to the bradyzoites housed in tissue cysts is not well understood, but the process clearly involves changes in gene expression. Here we report that parasites lacking a cell cycle-regulated transcription factor called AP2IX-4 display reduced frequencies of tissue cyst formation in culture and in a mouse model of infection. Parasites missing AP2IX-4 lose the ability to regulate bradyzoite genes during tissue cyst development. Expressed in developing bradyzoites still undergoing division, AP2IX-4 may serve as a useful marker in the study of transitional forms of the parasite

AP2IX-4, a cell cycle regulated nuclear factor, modulates gene expression during bradyzoite development in toxoplasma gondii

Indiana University-Purdue University Indianapolis (IUPUI)Toxoplasma gondii is a ubiquitous, protozoan parasite contributing significantly to global human and animal health. In the host, this obligate intracellular parasite converts into a latent tissue cyst form known as the bradyzoite, which is impervious to the immune response. The tissue cysts facilitate wide-spread transmission through the food chain and give rise to chronic toxoplasmosis in immune compromised patients. In addition, they may reactivate into replicating tachyzoites which cause tissue damage and disseminated disease. Current available drugs do not appear to have appreciable activity against latent bradyzoites. Therefore, a better understanding of the molecular mechanisms that drive interconversion between tachyzoite and bradyzoite forms is required to manage transmission and pathogenesis of Toxoplasma. Conversion to the bradyzoite is accompanied by an altered transcriptome, but the molecular players directing this process are largely uncharacterized. Studies of stage-specific promoters revealed that conventional cis-acting mechanisms operate to regulate developmental gene expression during tissue cyst formation. The major class of transcription factor likely to work through these cis-regulatory elements appears to be related to the Apetala-2 (AP2) family in plants. The Toxoplasma genome contains nearly 70 proteins harboring at least one predicted AP2 domain, but to date only three of these T. gondii AP2 proteins have been linked to bradyzoite development. We show that the putative T. gondii transcription factor, AP2IX-4, is localized to the parasite nucleus and exclusively expressed in tachyzoites and bradyzoites undergoing division. Knockout of AP2IX-4 had negligible effect on tachyzoite replication, but resulted in a reduced frequency of bradyzoite cysts in response to alkaline stress induction – a defect that is reversible by complementation. Microarray analyses revealed an enhanced activation of bradyzoite-associated genes in the AP2IX-4 knockout during alkaline conditions. In mice, the loss of AP2IX-4 resulted in a modest virulence defect and reduced brain cyst burden. Complementation of the AP2IX-4 knockout restored cyst counts to wild-type levels. These findings illustrate the complex role of AP2IX-4 in bradyzoite development and that certain transcriptional mechanisms responsible for tissue cyst development operate across parasite division

Medulloblastoma: Clinical Challenges and Emerging Molecular Discoveries

Medulloblastoma is the most common type of malignant brain tumor in children, responsible for 25% of pediatric brain cancers. Conventional treatment methods, which include surgery, radiotherapy, and chemotherapy, have improved overall survival rates for patients with medulloblastoma to over 50%. A majority of survivors, however, suffer serious long-term side effects, including developmental, neurological, and psychosocial deficits. Now entering clinical trials for sonic hedgehog-driven medulloblastomas, Smoothened inhibitors have been FDA approved for the treatment of basal cell carcinomas. However, treatment efficacy endures only for a few months before lesion relapses and drug resistance occurs. Therefore, there is an urgent need for new therapies to reduce the significant problems associated with current drug-resistant treatments. In this chapter, we will illustrate the clinical presentation and current treatment methods for medulloblastoma and detail the molecular pathways within each of the four molecular subgroups of medulloblastoma, with an eye for possible candidates for novel combination therapies

Insights Into Elevated Distortion Product Otoacoustic Emissions In Sickle Cell Disease: Comparisons of Hydroxyurea-treated and Non-treated Young Children

Distortion product otoacoustic emissions (DPOAEs) were examined in 15 normal- hearing African-American children between the ages of 6 and 14 years with homozygous sickle cell disease (SCD), who were on a regimen of hydroxyurea (HDU), a drug that reduces inflammatory processes and symptoms of SCD; a matched group of 15 African- American children with homozygous SCD not on HDU; and 15 African-American children with normal hemoglobin. DPOAEs were evoked by 13 primary tone pairs with f2 frequencies ranging from 1000 to 4500 Hz. Increased DPOAE amplitudes, believed to be a precursor of eventual hearing loss, were evident in children with SCD who were not receiving HDU. Those taking HDU had DPOAE amplitudes similar to normal controls. These findings suggest that HDU, in addition to reducing symptoms of SCD, may play a role in inhibiting or preventing cochlear pathology and hearing loss in individuals with SCD. Key Words: distortion product otoacoustic emissions; sickle cell disease; hydroxyurea Abbreviations: ABR = auditory brainstem response; DPOAE = distortion product otoacoustic emission; HDU = hydroxyurea; HbSS = homozygous sickle cell disease; ICAM = intercellular adhesion molecule; M = mean; OAE = otoacoustic emission; p = probability; PECAM = platelet-endothelial cell adhesion molecule SCD = sickle cell disease; SD = standard deviation of the mean; SOAE = spontaneous otoacoustic emission; TEOAE = transient evoked otoacoustic emission; VCAM = vascular cell adhesion molecule