Treatment with docetaxel is associated with significant prolongation of survival, and at a dose of 75 mg/m(2), the benefits of docetaxel therapy outweigh the risks.

This first randomized trial in this setting demonstrates that D75 every 3 weeks can offer clinically meaningful benefit to patients with advanced NSCLC whose disease has relapsed or progressed after platinum-based chemotherapy.

Pulmonary fibrosis is a progressive and largely untreatable group of disorders that affects up to 100,000 people on any given day in the United States. To elucidate the molecular mechanisms that lead to end-stage human pulmonary fibrosis we analyzed samples from patients with histologically proven pulmonary fibrosis (usual interstitial pneumonia) by using oligonucleotide microarrays. Gene expression patterns clearly distinguished normal from fibrotic lungs. Many of the genes that were significantly increased in fibrotic lungs encoded proteins associated with extracellular matrix formation and degradation and proteins expressed in smooth muscle. Using a combined set of scoring systems we determined that matrilysin (matrix metalloproteinase 7), a metalloprotease not previously associated with pulmonary fibrosis, was the most informative increased gene in our data set. Immunohistochemisry demonstrated increased expression of matrilysin protein in fibrotic lungs. Furthermore, matrilysin knockout mice were dramatically protected from pulmonary fibrosis in response to intratracheal bleomycin. Our results identify matrilysin as a mediator of pulmonary fibrosis and a potential therapeutic target. They also illustrate the power of global gene expression analysis of human tissue samples to identify molecular pathways involved in clinical disease.usual interstitial pneumonia ͉ microarray analysis ͉ informative genes ͉ bleomycin ͉ matrix metalloproteases

Purpose: Oral fluid (saliva) meets the demand for noninvasive, accessible, and highly efficient diagnostic medium. Recent discovery that a large panel of human RNA can be reliably detected in saliva gives rise to a novel clinical approach, salivary transcriptome diagnostics. The purpose of this study is to evaluate the diagnostic value of this new approach by using oral squamous cell carcinoma (OSCC) as the proof-of-principle disease.Experimental Design: Unstimulated saliva was collected from patients (n ‫؍‬ 32) with primary T1/T2 OSCC and normal subjects (n ‫؍‬ 32) with matched age, gender, and smoking history. RNA isolation was done from the saliva supernatant, followed by two-round linear amplification with T7 RNA polymerase. Human Genome U133A microarrays were applied for profiling human salivary transcriptome. The different gene expression patterns were analyzed by combining a t test comparison and a fold-change analysis on 10 matched cancer patients and controls. Quantitative polymerase chain reaction (qPCR) was used to validate the selected genes that showed significant difference (P < 0.01) by microarray. The predictive power of these salivary mRNA biomarkers was analyzed by receiver operating characteristic curve and classification models.Results: Microarray analysis showed there are 1,679 genes exhibited significantly different expression level in saliva between cancer patients and controls (P < 0.05).Seven cancer-related mRNA biomarkers that exhibited at least a 3.5-fold elevation in OSCC saliva (P < 0.01) were consistently validated by qPCR on saliva samples from OSCC patients (n ‫؍‬ 32) and controls (n ‫؍‬ 32). These potential salivary RNA biomarkers are transcripts of IL8, IL1B, DUSP1, HA3, OAZ1, S100P, and SAT. The combinations of these biomarkers yielded sensitivity (91%) and specificity (91%) in distinguishing OSCC from the controls.Conclusions: The utility of salivary transcriptome diagnostics is successfully demonstrated in this study for oral cancer detection. This novel clinical approach could be exploited to a robust, high-throughput, and reproducible tool for early cancer detection. Salivary transcriptome profiling can be applied to evaluate its usefulness for other major disease applications as well as for normal health surveillance.

BACKGROUND Extracellular RNAs (exRNAs) in human body fluids are emerging as effective biomarkers for detection of diseases. Saliva, as the most accessible and noninvasive body fluid, has been shown to harbor exRNA biomarkers for several human diseases. However, the entire spectrum of exRNA from saliva has not been fully characterized. METHODS Using high-throughput RNA sequencing (RNA-Seq), we conducted an in-depth bioinformatic analysis of noncoding RNAs (ncRNAs) in human cell-free saliva (CFS) from healthy individuals, with a focus on microRNAs (miRNAs), piwi-interacting RNAs (piRNAs), and circular RNAs (circRNAs). RESULTS Our data demonstrated robust reproducibility of miRNA and piRNA profiles across individuals. Furthermore, individual variability of these salivary RNA species was highly similar to those in other body fluids or cellular samples, despite the direct exposure of saliva to environmental impacts. By comparative analysis of >90 RNA-Seq data sets of different origins, we observed that piRNAs were surprisingly abundant in CFS compared with other body fluid or intracellular samples, with expression levels in CFS comparable to those found in embryonic stem cells and skin cells. Conversely, miRNA expression profiles in CFS were highly similar to those in serum and cerebrospinal fluid. Using a customized bioinformatics method, we identified >400 circRNAs in CFS. These data represent the first global characterization and experimental validation of circRNAs in any type of extracellular body fluid. CONCLUSIONS Our study provides a comprehensive landscape of ncRNA species in human saliva that will facilitate further biomarker discoveries and lay a foundation for future studies related to ncRNAs in human saliva.

Psychostimulant-induced alteration of dendritic spines on dopaminoceptive neurons in nucleus accumbens (NAcc) has been hypothesized as an adaptive neuronal response that is linked to longlasting addictive behaviors. NAcc is largely composed of two distinct subpopulations of medium-sized spiny neurons expressing high levels of either dopamine D1 or D2 receptors. In the present study, we analyzed dendritic spine density after chronic cocaine treatment in distinct D1 or D2 receptor-containing medium-sized spiny neurons in NAcc. These studies made use of transgenic mice that expressed EGFP under the control of either the D1 or D2 receptor promoter (Drd1-EGFP or Drd2-EGFP). After 28 days of cocaine treatment and 2 days of withdrawal, spine density increased in both Drd1-EGFP-and Drd2-EGFP-positive neurons. However, the increase in spine density was maintained only in Drd1-EGFP-positive neurons 30 days after drug withdrawal. Notably, increased ⌬FosB expression also was observed in Drd1-EGFP-and Drd2-EGFP-positive neurons after 2 days of drug withdrawal but only in Drd1-EGFP-positive neurons after 30 days of drug withdrawal. These results suggest that the increased spine density observed after chronic cocaine treatment is stable only in D1-receptor-containing neurons and that ⌬FosB expression is associated with the formation and͞or the maintenance of dendritic spines in D1 as well as D2 receptor-containing neurons in NAcc.T he mesolimbic dopaminergic pathway is composed of neurons in the ventral tegmental area that innervate the nucleus accumbens (NAcc), olfactory tubercle, prefrontal cortex, and amygdala (1), whereas nigrostriatal dopaminergic neurons in the substantia nigra (pars compacta) provide an ascending projection to dorsal striatum (2). Psychostimulants elevate synaptic concentrations of dopamine in NAcc: cocaine, by blocking dopamine uptake from the synaptic cleft, and amphetamine, by promoting dopamine release from nerve terminals (3-5). Repeated, intermittent administration of psychostimulants results in augmented behavioral responses (sensitization) to the acute stimulatory effects of these drugs (6-8). Most lines of evidence suggest that adaptive changes in the ventral tegmental areaNAcc dopaminergic system are central to alterations in the experience-dependent plasticity that underlies drug-induced behavior.In addition to dopamine, glutamate is required for the development of behavioral sensitization in response to psychostimulants (9, 10). Medium-sized spiny neurons (MSNs) in ventral striatum receive excitatory glutamatergic projections from prefrontal cortex that synapse onto the heads of dendritic spines. MSNs also are the major target for dopaminergic axons that synapse onto spine necks (1,11,12). Therefore, dendritic spines in MSNs represent the cellular compartment where dopaminergic and glutamatergic transmission are initially integrated. Dopamine acts on two major receptor subfamilies, the D1 subfamily (D1 and D5 subtypes) and the D2 subfamily (D2, D3, and D4 subtypes) (13). In dorsal striatum, ...

WAVE1--the Wiskott-Aldrich syndrome protein (WASP)--family verprolin homologous protein 1--is a key regulator of actin-dependent morphological processes in mammals, through its ability to activate the actin-related protein (Arp2/3) complex. Here we show that WAVE1 is phosphorylated at multiple sites by cyclin-dependent kinase 5 (Cdk5) both in vitro and in intact mouse neurons. Phosphorylation of WAVE1 by Cdk5 inhibits its ability to regulate Arp2/3 complex-dependent actin polymerization. Loss of WAVE1 function in vivo or in cultured neurons results in a decrease in mature dendritic spines. Expression of a dephosphorylation-mimic mutant of WAVE1 reverses this loss of WAVE1 function in spine morphology, but expression of a phosphorylation-mimic mutant does not. Cyclic AMP (cAMP) signalling reduces phosphorylation of the Cdk5 sites in WAVE1, and increases spine density in a WAVE1-dependent manner. Our data suggest that phosphorylation/dephosphorylation of WAVE1 in neurons has an important role in the formation of the filamentous actin cytoskeleton, and thus in the regulation of dendritic spine morphology.

Using purified proteins from calf and a synthetic substrate, we have reconstituted the enzymatic reactions required for mammalian Okazaki fragment processing in vitro. The required reactions are removal of initiator RNA, synthesis fromanupstre fragment to generate a nick, and then ligation. (4)(5)(6) has prompted several proposed roles for the latter enzyme in DNA replication (7)(8)(9)(10). While the viral model has clarified priming and elongation steps in the replication fork, the exact steps involved in completion of lagging-strand synthesis are not yet clear. Simian virus 40 reconstitution reactions (11-13) indicated roles for the mammalian 5'-to-3' exonuclease, a nuclear DNA polymerase, DNA ligase, and possibly RNase H type I (RNase HI) in joining of nascent segments of the lagging strand. DNA ligase I, rather than III, was shown to be required (12, 13). The specific function of RNase HI and the cleavage mechanism involved remained to be determined.In Escherichia coli, initiator RNA of Okazaki fragments is thought to be removed by the 5'-to-3' exonuclease of DNA polymerase I, although a role for RNase H cannot be totally excluded (14). Double strand-specific 5'-to-3' exonucleases, which appear to be functional homologs of each other, have been isolated from human cells, mice, and calf (11-13, 15, 16). The calf nuclease displayed an endonuclease function that cleaves the unannealed 5' tail of a primer on a template, in the presence of a directly adjacent upstream primer (17). This very specific function is also exhibited by E. coli DNA polymerase 1 (18), which strongly suggests that the bacterial and mammalian enzymes are also functional homologs.Mammalian RNase H enzymes have been identified and characterized in detail. RNase H enzymes can be placed into one of two classes (I and II) based on subunit structure, peptide molecular mass, and the ability to use manganese as a cofactor (19,20). They have been postulated to be involved in DNA replication and transcription (21), but no specific roles have been established for either class.We had shown (22) that the calf 5'-to-3' exonuclease could work with calf DNA polymerases a, 8, or e and DNA ligase I to join two DNA primers annealed to a template with a four-base gap. In addition, we have demonstrated that 5'-to-3' exonuclease activity is stimulated by synthesis from an upstream primer and that the stimulation is the result of the formation of a nick between the two primers, which is a preferred substrate for the exonuclease (17). These results suggest that a nick translation process occurs prior to the joining reaction catalyzed by DNA ligase I. Goulian et al. (15) previously showed that a combination of RNase HI and 5'-to-3' exonuclease can remove initiator RNA from primertemplates in vitro. Here we define the specific reactions by which these two nucleases can remove initiator RNA in a model Okazaki fragment system before polymerization can make the substrate for ligation.MATERIALS AND METHODS Protein Purification. DNA polymerase E, DNA ligase I, a...