Mycobacterial phosphodiesterase Rv0805 is a virulence determinant and its cyclic nucleotide hydrolytic activity is required for propionate detoxification.

Cyclic AMP (cAMP) signaling is essential to Mycobacterium tuberculosis (Mtb) pathogenesis. However, the roles of phosphodiesterases (PDEs) Rv0805, and the recently identified Rv1339, in cAMP homeostasis and Mtb biology are unclear. We found that Rv0805 modulates Mtb growth within mice, macrophages and on host-associated carbon sources.

Live Vaccination Generates Both Disease Tolerance and Host Resistance During Chronic Pulmonary Infection With Highly Virulent Francisella tularensis SchuS4.

Disease tolerance can preserve host homeostasis and limit the negative impact of infections. We report that vaccinated mice survived pulmonary challenge with the extremely virulent SchuS4 strain of Francisella tularensis for at least 100 days, despite the persistence of large numbers (~104) of organisms. Transfer of 100 of these resident bacteria to naive animals caused 100% lethality, demonstrating that virulence was maintained.

Asthma increases susceptibility to heterologous but not homologous secondary influenza.

Unlabelled: Asthma was the most common comorbidity observed among patients hospitalized with influenza A virus during the 2009 pandemic. However, little remains known about how the asthmatic phenotype influences protective immune responses against respiratory viral pathogens. Using the ovalbumin-induced allergic lung inflammation model, we found that asthmatic mice, unlike nonasthmatic mice, were highly susceptible to secondary heterologous virus challenge.

Natural history of Yersinia pestis pneumonia in aerosol-challenged BALB/c mice.

After a relatively short untreated interval, pneumonic plague has a mortality approaching 100%. We employed a murine model of aerosol challenge with Yersinia pestis to investigate the early course of pneumonic plague in the lung, blood, and spleen. We fit a mathematical model to all data simultaneously.

Cyclin-dependent kinase 8 mediates chemotherapy-induced tumor-promoting paracrine activities.

Conventional chemotherapy not only kills tumor cells but also changes gene expression in treatment-damaged tissues, inducing production of multiple tumor-supporting secreted factors. This secretory phenotype was found here to be mediated in part by a damage-inducible cell-cycle inhibitor p21 (CDKN1A). We developed small-molecule compounds that inhibit damage-induced transcription downstream of p21.

Tumor-specific silencing of COPZ2 gene encoding coatomer protein complex subunit ζ 2 renders tumor cells dependent on its paralogous gene COPZ1.

Anticancer drugs are effective against tumors that depend on the molecular target of the drug. Known targets of cytotoxic anticancer drugs are involved in cell proliferation; drugs acting on such targets are ineffective against nonproliferating tumor cells, survival of which leads to eventual therapy failure. Function-based genomic screening identified the coatomer protein complex ζ1 (COPZ1) gene as essential for different tumor cell types but not for normal cells.

Antifungal testing and high-throughput screening of compound library against Geomyces destructans, the etiologic agent of geomycosis (WNS) in bats.

Bats in the northeastern U.S. are affected by geomycosis caused by the fungus Geomyces destructans (Gd).

The 3 prime paradigm of the miR-200 family and other microRNAs.

The number of predicted human microRNAs in Sanger miRBase currently stands at over a thousand, with each of these in turn predicted to target numerous mRNAs. However, those microRNAs for which mRNA targets have been evaluated, verified and reported in the literature are still in the minority and the bulk of microRNA/mRNA interactions are yet to be confirmed. Confirmation of microRNA interaction with predicted mRNA targets represents a considerable undertaking, made more complex by potential synergistic effects of multiple microRNAs and the three possible outcomes (translational repression, degradation or a mixture of both).

Identification of single nucleotide polymorphisms in the p21 (CDKN1A) gene and correlations with longevity in the Italian population.

Longevity in humans is determined by multiple environmental and genetic factors. We have investigated possible associations between longevity and Single Nucleotide Polymorphisms (SNPs) in the p21 (CDKN1A) gene, a stress-inducible senescence-associated cell cycle inhibitor, expression of which upregulates genes implicated in several age-related diseases. By sequencing the promoter and exons of p21 in genomic DNA of ten individuals over 90 years old, we have identified 30 SNPs, many of which had not been previously characterized.

Stable expression of miR-200c alone is sufficient to regulate TCF8 (ZEB1) and restore E-cadherin expression.

Regulation of the transcription factor TCF8 (ZEB1) by the microRNA miR-200c and was first identified using a bioinformatic and relative quantitative PCR based approach.(1) Using stable ectopic expression of miR-200c we then demonstrated loss of TCF8 (ZEB1) mRNA and restoration of its primary regulatory target, E-cadherin.(2) Recently, other members of the miR-200 'family' and an additional unrelated microRNA, miR-205, have been reported to be essential for the regulation of TCF8 (ZEB1) and restoration of E-cadherin.

Overexpression of the microRNA hsa-miR-200c leads to reduced expression of transcription factor 8 and increased expression of E-cadherin.

MicroRNAs are approximately 22-nucleotide sequences thought to interact with multiple mRNAs resulting in either translational repression or degradation. We previously reported that several microRNAs had variable expression in mammalian cell lines, and we examined one, miR-200c, in more detail. A combination of bioinformatics and quantitative reverse transcription-PCR was used to identify potential targets and revealed that the zinc finger transcription factor transcription factor 8 (TCF8; also termed ZEB1, deltaEF1, Nil-2-alpha) had inversely proportional expression levels to miR-200c.

Validity of messenger RNA expression analyses of human saliva.

Purpose: The origins of expression microarray and reverse transcription-PCR (RT-PCR) signals in human saliva were evaluated.

Experimental Design: The "RNA" extracts from human saliva samples were treated with vehicle, DNase, or RNase. Two-step amplification and hybridization to Affymetrix 133A cDNA microarrays were then done.

Potential mRNA degradation targets of hsa-miR-200c, identified using informatics and qRT-PCR.

Using an anchored oligo(dT) based RT-PCR approach we quantified endogenous expression of ten microRNAs in six cell lines. This identified a miRNA, miR-200c, with variable expression, ranging from undetectable in MDA-MB-231 and HT1080 to highly expressed in MCF7. The variable expression provided a model system to investigate endogenous interactions between miRNAs and their computationally predicted targets.

Attenuation of the pulmonary inflammatory response following butylated hydroxytoluene treatment of cytosolic phospholipase A2 null mice.

Administration of butylated hydroxytoluene (BHT) to mice causes lung damage characterized by the death of alveolar type I pneumocytes and the proliferation and subsequent differentiation of type II cells to replace them. Herein, we demonstrate this injury elicits an inflammatory response marked by chemokine secretion, alveolar macrophage recruitment, and elevated expression of enzymes in the eicosanoid pathway. Cytosolic phospholipase A(2) (cPLA(2)) catalyzes release of arachidonic acid from membrane phospholipids to initiate the synthesis of prostaglandins and other inflammatory mediators.

Targeted over-expression of mPGES-1 and elevated PGE2 production is not sufficient for lung tumorigenesis in mice.

There is a significant body of evidence suggesting that enzymes involved in arachidonic acid metabolism and their eicosanoid products play a role in various cancers, having both pro- and antitumorigenic effects. The goal of this study was to further define the role microsomal prostaglandin E synthases (mPGES-1) play in lung tumorigenesis. Transgenic mice were created with targeted over-expression of human mPGES-1 in the alveolar and airway epithelial cells using an SP-C promoter driven construct.

Gene-environment interaction signatures by quantitative mRNA profiling in exfoliated buccal mucosal cells.

Exfoliated cytologic specimens from mouth (buccal) epithelium may contain viable cells, permitting assay of gene expression for direct and noninvasive measurement of gene-environment interactions, such as for inhalation (e.g., tobacco smoke) exposures.

Decreased lung tumorigenesis in mice genetically deficient in cytosolic phospholipase A2.

Epidemiological investigations suggest that chronic lung inflammation increases lung cancer risk. Pharmacologic and genetic evidence in mouse models indicates that lipid mediators released during pulmonary inflammation enhance lung tumor formation. Cytosolic phospholipase A2 (cPLA2) catalyzes arachidonic acid (AA) release from membrane phospholipids.

Phase I and II carcinogen metabolism gene expression in human lung tissue and tumors.

Purpose: The regulation of carcinogen metabolism machinery may involve proximate tobacco smoke exposure, hormonal and other endogenous coregulatory factors, and an individual's underlying genetic responsiveness. The mRNA and protein expression patterns of known carcinogen metabolism genes encoding the aromatic hydrocarbon receptor Ahr; the cytochromes P450 CYP1A1 and CYP1B1; glutathione S-transferases GSTM1, GSTM3, GSTP1, and GSTT1; and NADPH quinone oxidoreductase NQO1 were examined.

Experimental Design: Paired tumor and nontumor lung tissue from 45 subjects was subject to a recently devised RNA-specific qualitative reverse transcription-PCR strategy, as well as Western immunoblotting.

Peroxisome proliferator-activated receptor-gamma is a target of nonsteroidal anti-inflammatory drugs mediating cyclooxygenase-independent inhibition of lung cancer cell growth.

Nonsteroidal anti-inflammatory drugs (NSAIDs) inhibit the growth of different cancer cell types, suggesting a broad role for their cyclooxygenase (COX) targets and eicosanoid products in tumor cell growth. Sulindac sulfide, a COX inhibitor, inhibited the growth of non-small-cell lung cancers (NSCLC) both in soft agar and as xenografts in nude mice. Importantly, the concentration of sulindac sulfide required to inhibit NSCLC cell growth greatly exceeded the concentration required to inhibit prostaglandin (PG) E(2) synthesis in NSCLC cells, suggesting that NSAID inhibition of cell growth is mediated by additional targets distinct from COX.

mRNA-specific reverse transcription-polymerase chain reaction from human tissue extracts.

Reverse transcription-polymerase chain reaction (RT-PCR) has become the method of choice for detection of mRNA transcripts, including those of low abundance obtained from small precious samples of human tissue. A major confounding problem for standard reverse-transcription-priming strategies is the presence of contaminating genomic DNA (gDNA) carried over from the original "RNA" extract into the RT and PCR steps. The contaminating gDNA contains a processed pseudogene sequence-which lacks introns but contains a poly(A) tail-for commonly studied internal reference genes beta-actin and GAPDH, and target genes GSTM1, GSTP1, and others.

Gender-dependent expression of alpha and beta estrogen receptors in human nontumor and tumor lung tissue.

Estrogen receptor (ER) expression in human lung has been understudied, particularly in light of its potential biological importance in the female lung cancer epidemic. Reverse transcription-polymerase chain reaction was used to probe mRNA expression of wild-type ERalpha and ERbeta and their splice variants in human bronchogenic tumor and adjacent nontumor specimens. In tumor tissue from 13 women and 13 men, ERalpha was expressed in 85% of women versus 15% in men [P=0.

CYP1B1 expression in human lung.

Cytochrome P450 1B1 is a recently recognized phase I bioactivating enzyme with high affinity for both inhaled tobacco carcinogens and 17beta-estradiol. We evaluated the human lung expression of this multifunctional member of the P450 superfamily across 16 individuals. Expression of CYP1B1 was evaluated by qualitative reverse transcription-polymerase chain reaction and Western immunoblots performed on human tumor and nontumor lung tissue.

Highly attenuated HIV type 2 recombinant poxviruses, but not HIV-2 recombinant Salmonella vaccines, induce long-lasting protection in rhesus macaques.

Immunization schemes employing priming with vector-based vaccine candidates followed by subunit booster administrations have been explored and shown to have merit in the human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus systems. In this study, we have assessed the priming capacity of highly attenuated poxvirus vector (NYVAC and ALVAC)-based HIV-2 recombinants, as well as Salmonella typhimurium HIV-2 recombinants in rhesus macaques. ALVAC- and NYVAC-based vaccine candidates expressing the HIV-2 gag, pol, and env genes or NYVAC-based recombinants expressing either gp160 or gp120 were used to immunize rhesus macaques in combination protocols with alum-adjuvanted HIV-2 rgp160.