Enrichment of Cryptosporidium parvum from in vitro culture as measured by total RNA and subsequent sequence analysis.

Cryptosporidium parvum is an apicomplexan parasite that infects a wide range of hosts including humans. Due to the parasite's quasi-intracellular, intermembrane location on the host cell, it is difficult to purify parasites from in vitro and in vivo infections for molecular studies. We have developed a method to greatly enrich in vitro C. parvum merozoites from host cells. The efficiency of the protocol was assessed with C. parvum (KSU-1 isolate) parasites of different developmental stages isolated following a synchronized infection of HCT-8 host cells. Total RNA was extracted from the samples and used to evaluate the quantity of host cell contamination in enriched parasite fractions. The quality of the RNA was verified using an Agilent BioAnalyzer. cDNA libraries of RNA isolated from 24 and 48 h C. parvum in vitro preparations isolated via this protocol were sequenced at the Broad Institute via an NIH Microbial Sequencing (GSCID) Contract. Cryptosporidium sequences comprised 30% of the cDNA reads, demonstrating significant enrichment.

Accumulation-associated protein enhances Staphylococcus epidermidis biofilm formation under dynamic conditions and is required for infection in a rat catheter model.

Biofilm formation is the primary virulence factor of Staphylococcus epidermidis. S. epidermidis biofilms preferentially form on abiotic surfaces and may contain multiple matrix components, including proteins such as accumulation-associated protein (Aap). Following proteolytic cleavage of the A domain, which has been shown to enhance binding to host cells, B domain homotypic interactions support cell accumulation and biofilm formation. To further define the contribution of Aap to biofilm formation and infection, we constructed an aap allelic replacement mutant and an icaADBC aap double mutant. When subjected to fluid shear, strains deficient in Aap production produced significantly less biofilm than Aap-positive strains. To examine the in vivo relevance of our findings, we modified our previously described rat jugular catheter model and validated the importance of immunosuppression and the presence of a foreign body to the establishment of infection. The use of our allelic replacement mutants in the model revealed a significant decrease in bacterial recovery from the catheter and the blood in the absence of Aap, regardless of the production of polysaccharide intercellular adhesin (PIA), a well-characterized, robust matrix molecule. Complementation of the aap mutant with full-length Aap (containing the A domain), but not the B domain alone, increased initial attachment to microtiter plates, as did in trans expression of the A domain in adhesion-deficient Staphylococcus carnosus. These results demonstrate Aap contributes to S. epidermidis infection, which may in part be due to A domain-mediated attachment to abiotic surfaces.

Rat jugular catheter model of biofilm-mediated infection.

Staphylococcus epidermidis is now recognized as the primary cause of nosocomial catheter-mediated infections. Bacteria may be introduced exogenously via contamination of the catheter hub or insertion site and endogenously from sepsis. The in vivo model described in this chapter examines the infection resulting from hematogenous seeding of jugular vein catheters.

A dysfunctional tricarboxylic acid cycle enhances fitness of Staphylococcus epidermidis during ß-lactam stress.

UNLABELLED: A recent controversial hypothesis suggested that the bactericidal action of antibiotics is due to the generation of endogenous reactive oxygen species (ROS), a process requiring the citric acid cycle (tricarboxylic acid [TCA] cycle). To test this hypothesis, we assessed the ability of oxacillin to induce ROS production and cell death in Staphylococcus epidermidis strain 1457 and an isogenic citric acid cycle mutant. Our results confirm a contributory role for TCA-dependent ROS in enhancing susceptibility of S. epidermidis toward ß-lactam antibiotics and also revealed a propensity for clinical isolates to accumulate TCA cycle dysfunctions presumably as a way to tolerate these antibiotics. The increased protection from ß-lactam antibiotics could result from pleiotropic effects of a dysfunctional TCA cycle, including increased resistance to oxidative stress, reduced susceptibility to autolysis, and a more positively charged cell surface. IMPORTANCE: Staphylococcus epidermidis, a normal inhabitant of the human skin microflora, is the most common cause of indwelling medical device infections. In the present study, we analyzed 126 clinical S. epidermidis isolates and discovered that tricarboxylic acid (TCA) cycle dysfunctions are relatively common in the clinical environment. We determined that a dysfunctional TCA cycle enables S. epidermidis to resist oxidative stress and alter its cell surface properties, making it less susceptible to ß-lactam antibiotics.

Aminopyrimidinone cdc7 kinase inhibitors.

We have investigated the SAR of a series of pyrimidinone-containing Cdc7 kinase inhibitors. A wide range of amine substitutions give potent compounds with activities (K(i)) less than 1nM. Kinase selectivity is reasonable and cytotoxicity corresponds to inhibition of MCM2 phosphorylation.

CR2+ marginal zone B cell production of pathogenic natural antibodies is C3 independent.

Intestinal ischemia-reperfusion (IR)-induced damage requires complement receptor 2 (CR2) for generation of the appropriate natural Ab repertoire. Pathogenic Abs recognize neoantigens on the ischemic tissue, activate complement, and induce intestinal damage. Because C3 cleavage products act as ligands for CR2, we hypothesized that CR2(hi) marginal zone B cells (MZBs) require C3 for generation of the pathogenic Abs. To explore the ability of splenic CR2(+) B cells to generate the damaging Ab repertoire, we adoptively transferred either MZBs or follicular B cells (FOBs) from C57BL/6 or Cr2(-/-) mice into Rag-1(-/-) mice. Adoptive transfer of wild type CR2(hi) MZBs but not CR2(lo) FOBs induced significant damage, C3 deposition, and inflammation in response to IR. In contrast, similarly treated Rag-1(-/-) mice reconstituted with either Cr2(-/-) MZB/B1 B cells (B1Bs) or FOBs lacked significant intestinal damage and displayed limited complement activation. To determine whether C3 cleavage products are critical in CR2-dependent Ab production, we evaluated the ability of the natural Ab repertoire of C3(-/-) mice to induce damage in response to IR. Infusion of C3(-/-) serum into Cr2(-/-) mice restored IR-induced tissue damage. Furthermore, Rag-1(-/-) mice sustained significant damage after infusion of Abs from C3(-/-) but not Cr2(-/-) mice. Finally, adoptive transfer of MZBs from C3(-/-) mice into Rag-1(-/-) mice resulted in significant tissue damage and inflammation. These data indicate that CR2 expression on MZBs is sufficient to induce the appropriate Abs required for IR-induced tissue damage and that C3 is not critical for generation of the pathogenic Abs.

Domain V peptides inhibit beta2-glycoprotein I-mediated mesenteric ischemia/reperfusion-induced tissue damage and inflammation.

Reperfusion of ischemic tissue induces significant tissue damage in multiple conditions, including myocardial infarctions, stroke, and transplantation. Although not as common, the mortality rate of mesenteric ischemia/reperfusion (IR) remains >70%. Although complement and naturally occurring Abs are known to mediate significant damage during IR, the target Ags are intracellular molecules. We investigated the role of the serum protein, ß2-glycoprotein I as an initiating Ag for Ab recognition and ß2-glycoprotein I (ß2-GPI) peptides as a therapeutic for mesenteric IR. The time course of ß2-GPI binding to the tissue indicated binding and complement activation within 15 min postreperfusion. Treatment of wild-type mice with peptides corresponding to the lipid binding domain V of ß2-GPI blocked intestinal injury and inflammation, including cellular influx and cytokine and eicosanoid production. The optimal therapeutic peptide (peptide 296) contained the lysine-rich region of domain V. In addition, damage and most inflammation were also blocked by peptide 305, which overlaps with peptide 296 but does not contain the lysine-rich, phospholipid-binding region. Importantly, peptide 296 retained efficacy after replacement of cysteine residues with serine. In addition, infusion of wild-type serum containing reduced levels of anti-ß2-GPI Abs into Rag-1(-/-) mice prevented IR-induced intestinal damage and inflammation. Taken together, these data suggest that the serum protein ß2-GPI initiates the IR-induced intestinal damage and inflammatory response and as such is a critical therapeutic target for IR-induced damage and inflammation.

Cryspovirus: a new genus of protozoan viruses in the family Partitiviridae.

The family Partitiviridae includes plant and fungal viruses with bisegmented dsRNA genomes and isometric virions in which the two genome segments are packaged separately and used as templates for semiconservative transcription by the viral polymerase. A new genus, Cryspovirus, has been approved for this family. Its name is based on that of the host genus, Cryptosporidium, which encompasses several species of apicomplexan parasites that infect a wide range of mammals, birds, and reptiles, and are a major cause of human diarrheal illness worldwide. The type species of the new genus is Cryptosporidium parvum virus 1. Distinguishing characteristics include infection of a protozoan host, a smaller capsid protein than found in other members of the family Partitiviridae, and sequence-based phylogenetic divergence.

Synthesis and SAR of novel tricyclic quinoxalinone inhibitors of poly(ADP-ribose)polymerase-1 (PARP-1).

Based on screening hit 1, a series of tricyclic quinoxalinones have been designed and evaluated for inhibition of PARP-1. Substitutions at the 7- and 8-positions of the quinoxalinone ring led to a number of compounds with good enzymatic and cellular potency. The tricyclic quinoxalinone class is sensitive to modifications of both the amine substituent and the tricyclic core. The synthesis and structure-activity relationship studies are presented.

Macrophage cell lines use CD81 in cell growth regulation.

CD81 is an integral membrane protein belonging to the tetraspanin superfamily. It has two extracellular domains that interact with cell surface proteins and two intracellular tails that contribute to cellular processes. Although there are considerable data about how CD81 affects T- and B-cell function, not much is known about how it impacts macrophages. To address this, we established four cell lines from mouse bone marrow in the presence of macrophage colony-stimulating factor and transfection with SV40 large T antigen. Two were CD81(-/-) (ASD1 and ASD2) and two were CD81(+/-) (2ASD1.10 and 2BSD1.10). Cells were Mac-2-, PU.1-, and c-fms-positive and all the cell lines were phagocytic indicating that they were macrophage-like. In mixtures of the two cell types in tissue culture, CD81(-/-) cells out competed CD81(+/-) cells with CD81-bearing cells being undetectable after 50 cell culture passages. Although cell divisions during log-phase growth were not significantly different between CD81(+/-) macrophage cells and CD81(-/-) macrophage cells, we found that CD81(-/-) macrophage cells reached a higher density at confluency than CD81(+/-) macrophage cells. CD81 transcript levels increased as cultures became confluent, but transcript levels of other tetraspanin-related molecules remained relatively constant. Transfection of CD81 into ASD1 (CD81(-/-)) cells reduced the density of confluent cultures of transformants compared to cells transfected with vector alone. These data suggest that CD81 potentially plays a role in macrophage cell line growth regulation.

Akt inhibitor a-443654 interferes with mitotic progression by regulating aurora a kinase expression.

Both Akt and Aurora A kinase have been shown to be important targets for intervention for cancer therapy. We report here that Compound A (A-443654), a specific Akt inhibitor, interferes with mitotic progression and bipolar spindle formation. Compound A induces G(2)/M accumulation, defects in centrosome separation, and formation of either monopolar arrays or disorganized spindles. On the basis of gene expression array studies, we identified Aurora A as one of the genes regulated transcriptionally by Akt inhibitors including Compound A. Inhibition of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway, either by PI3K inhibitor LY294002 or by Compound A, dramatically inhibits the promoter activity of Aurora A, whereas the mammalian target of rapamycin inhibitor has little effect, suggesting that Akt might be responsible for up-regulating Aurora A for mitotic progression. Further analysis of the Aurora A promoter region indicates that the Ets element but not the Sp1 element is required for Compound A-sensitive transcriptional control of Aurora A. Overexpression of Aurora A in cells treated with Compound A attenuates the mitotic arrest and the defects in bipolar spindle formation induced by Akt inhibition. Our studies suggest that that Akt may promote mitotic progression through the transcriptional regulation of Aurora A.

Pharmacological and functional comparison of the polo-like kinase family: insight into inhibitor and substrate specificity.

PLK1 (polo-like kinase 1) is a key mitotic kinase and a therapeutic target in the treatment of proliferative diseases. Here we investigate the relative substrate specificity and pharmacological relatedness of PLK1, -2, -3, and -4 that together comprise a conserved family of Ser/Thr kinases (PLK family). We report consensus substrate sequences for PLK2, -3, and -4 and an expanded consensus sequence for PLK1, which we use to design an optimal peptide substrate, PLKtide. We report inhibitory activity for the entire PLK family across a diverse set of small-molecule ATP-competitive inhibitors including several clinical compounds. With respect to both substrate and ATP-site specificity, highest similarity is observed between PLK2 and PLK3, PLK1 is next most similar, and PLK4 is least similar. Further, we have identified and report time-dependent inhibition by two potent and selective PLK inhibitors.

Syntheses of potent, selective, and orally bioavailable indazole-pyridine series of protein kinase B/Akt inhibitors with reduced hypotension.

Compound 7 was identified as a potent (IC50 = 14 nM), selective, and orally bioavailable (F = 70% in mouse) inhibitor of protein kinase B/Akt. While promising efficacy was observed in vivo, this compound showed effects on depolarization of Purkinje fibers in an in vitro assay and CV hypotension in vivo. Guided by an X-ray structure of 7 bound to protein kinase A, which has 80% homology with Akt in the kinase domain, our efforts have focused on structure-activity relationship (SAR) studies of the phenyl moiety, in an attempt to address the cardiovascular liability and further improve the Akt potency. A novel and efficient synthetic route toward diversely substituted phenyl derivatives of 7 was developed utilizing a copper-mediated aziridine ring-opening reaction as the key step. To improve the selectivity of these Akt inhibitors over other protein kinases, a nitrogen atom was incorporated into selected phenyl analogues of 7 at the C-6 position of the methyl indazole scaffold. These modifications resulted in the discovery of inhibitor 37c with greater potency (IC50 = 0.6 nM vs Akt), selectivity, and improved cardiovascular safety profile. The SARs, pharmacokinetic profile, and CV safety of selected Akt inhibitors will be discussed.

microRNA expression in the prefrontal cortex of individuals with schizophrenia and schizoaffective disorder.

BACKGROUND: microRNAs (miRNAs) are small, noncoding RNA molecules that are now thought to regulate the expression of many mRNAs. They have been implicated in the etiology of a variety of complex diseases, including Tourette's syndrome, Fragile x syndrome, and several types of cancer. RESULTS: We hypothesized that schizophrenia might be associated with altered miRNA profiles. To investigate this possibility we compared the expression of 264 human miRNAs from postmortem prefrontal cortex tissue of individuals with schizophrenia (n = 13) or schizoaffective disorder (n = 2) to tissue of 21 psychiatrically unaffected individuals using a custom miRNA microarray. Allowing a 5% false discovery rate, we found that 16 miRNAs were differentially expressed in prefrontal cortex of patient subjects, with 15 expressed at lower levels (fold change 0.63 to 0.89) and 1 at a higher level (fold change 1.77) than in the psychiatrically unaffected comparison subjects. The expression levels of 12 selected miRNAs were also determined by quantitative RT-PCR in our lab. For the eight miRNAs distinguished by being expressed at lower microarray levels in schizophrenia samples versus comparison samples, seven were also expressed at lower levels with quantitative RT-PCR. CONCLUSION: This study is the first to find altered miRNA profiles in postmortem prefrontal cortex from schizophrenia patients.

Design and synthesis of pyridine-pyrazolopyridine-based inhibitors of protein kinase B/Akt.

Thr-211 is one of three different amino acid residues in the kinase domain of protein kinase B/Akt as compared to protein kinase A (PKA), a closely related analog in the same AGC family. In an attempt to improve the potency and selectivity of our indazole-pyridine series of Akt inhibitors over PKA, efforts have focused on the incorporation of a chemical functionality to interact with the hydroxy group of Thr-211. Several substituents including an oxygen anion, amino, and nitro groups have been introduced at the C-6 position of the indazole scaffold, leading to a significant drop in Akt potency. Incorporation of a nitrogen atom into the phenyl ring at the same position (i.e., 9f) maintained the Akt activity and, in some cases, improved the selectivity over PKA. The structure-activity relationships of the new pyridine-pyrazolopyridine series of Akt inhibitors and their structural features when bound to PKA are also discussed.

Direct regulation of an oncogenic micro-RNA cluster by E2F transcription factors.

Micro-RNAs (miRNAs) are a class of non-coding RNAs that post-transcriptionally regulate gene expression via the RNA interference pathway. In addition to roles in normal development, miRNAs have recently been implicated in a range of human diseases, including cancer. We recently demonstrated that a polycistronic cluster of miRNAs, miR-17-92, is oncogenic in a mouse model for Burkitt's lymphoma. This is due, in part, to a reduced apoptotic program. In an effort to understand the regulation of miR-17-92, we have studied the promoter structure of this miRNA cluster. The primary transcript initiates from a consensus initiator sequence downstream of a nonconsensus TATA box. The core promoter region contains two functional E2F transcription factor binding sites. Chromatin immunoprecipitation demonstrates that E2F3 is the primary E2F family member that occupies the promoter. These data place miR-17-92 in a regulatory loop between E2F3 and the miR-17 target E2F1. We propose a model whereby miR-17-92 promotes cell proliferation by shifting the E2F transcriptional balance away from the pro-apoptotic E2F1 and toward the proliferative E2F3 transcriptional network.

MicroRNAs modulate the angiogenic properties of HUVECs.

MicroRNAs (miRNAs) have recently come into focus as key posttranscriptional modulators of gene expression. In this work, we addressed whether in vitro angiogenesis is an miRNA-regulated process. We performed large-scale analysis of miRNA expression in human umbilical vein endothelial cells (HUVECs) and found that 15 highly expressed miRNAs have the receptors of angiogenic factors as putative targets. In particular, we demonstrated that miR-221 and miR-222 affect c-Kit expression and, as a consequence, the angiogenic properties of its ligand stem cell factor. Interaction between miR-222 and c-Kit is likely to be part of a complex circuit that controls the ability of endothelial cells to form new capillaries.

Synthesis and SAR of indazole-pyridine based protein kinase B/Akt inhibitors.

A series of heteroaryl-pyridine containing inhibitors of Akt are reported. The synthesis and structure-activity relationships are discussed, leading to the discovery of a indazole-pyridine analogue (K(i)=0.16 nM). These compounds bind in the ATP binding site, are potent, ATP competitive, and reversible inhibitors of Akt activity. No selectivity amongst the Akt isoforms is observed for this analogue, but there is good selectivity against an panel of other kinases. It is least selective for other members of the AGC family of kinases but is nonetheless 40-fold selective for Akt over PKA. The compound shows cellular activity and significantly slows tumor growth in vivo.

Identification of a novel 3,5-disubstituted pyridine as a potent, selective, and orally active inhibitor of Akt1 kinase.

Based on lead compounds 2 and 3 a series of 3,5-disubstituted pyridines have been designed and evaluated for inhibition of AKT/PKB. Modifications at the 3 position of the pyridine ring led to a number of potent compounds with improved physical properties, resulting in the identification of 11g as a promising, orally active Akt inhibitor. The synthesis, structure-activity relationship studies, and pharmacokinetic data are presented in this paper.

Isoquinoline-pyridine-based protein kinase B/Akt antagonists: SAR and in vivo antitumor activity.

The structure-activity relationships of a series of isoquinoline-pyridine-based protein kinase B/Akt antagonists have been investigated in an effort to improve the major short-comings of the lead compound 3, including poor pharmacokinetic profiles in several species (e.g., mouse i.v. t(1/2) = 0.3 h, p.o. F = 0%). Chlorination at C-1 position of the isoquinoline improved its pharmacokinetic property in mice (i.v. t(1/2) = 5.0 h, p.o. F = 51%) but resulted in >500-fold drop in potency. In a mouse MiaPaCa-2 xenograft model, an amino analog 10y significantly slowed the tumor growth, however was accompanied by toxicity.