Cytoplasmic Polyadenylation Element Binding Proteins CPEB1 and CPEB3 Regulate the Translation of FosB and Are Required for Maintaining Addiction-Like Behaviors Induced by Cocaine.

A recurrent and devastating feature of addiction to a drug of abuse is its persistence, which is mediated by maladaptive long-term memories of the highly pleasurable experience initially associated with the consumption of the drug. We have recently found that members of the CPEB family of proteins (Cytoplasmic Polyadenylation Element-Binding Proteins) are involved in the maintenance of spatial memory. However, their possible role in the maintenance of memories that sustain addictive behavior has yet to be explored. Little is known about any of the mechanisms for maintaining memories for addictive behavior. To address the mechanisms whereby addictive behavior is maintained over time, we utilized a conditional transgenic mouse model expressing a dominant-negative version of CPEB1 that abolishes the activity in the forebrain of two of the four CPEB isoforms (CPEB1 and CPEB3). We found that, following cocaine administration, these dominant-negative (DN) CPEB mice showed a significant decrease, when compared to wild type (WT) mice, in both locomotor sensitizations and conditioned place preference (CPP), two indices of addictive behavior. Supporting these behavioral results, we also found a difference between WT and DN-CPEB1-3 mice in the cocaine-induced synaptic depression in the core of the Nucleus Accumbens (NAc). Finally, we found that (1) CPEB is reduced in transgenic mice following cocaine injections and that (2) FosB, known for its contribution to establishing the addictive phenotype, when its expression in the striatum is increased by drug administration, is a novel target of CPEBs molecules. Thus, our study highlights how CPEB1 and CPEB3 act on target mRNAs to build the neuroadaptative implicit memory responses that lead to the development of the cocaine addictive phenotypes in mammals.

Routine Clinical Mutation Profiling of Non-Small Cell Lung Cancer Using Next-Generation Sequencing.

CONTEXT: The availability of massive, parallel-sequencing technologies makes possible efficient, simultaneous detection of driver and druggable mutations in cancer. OBJECTIVE: To develop an amplicon-based, next-generation sequencing, mutation-detection assay for lung cancer using the 454 GS Junior (Roche Applied Science, Indianapolis, Indiana) platform. DESIGN: Fusion primers incorporating target sequence, 454 adaptors, and multiplex identifiers were designed to generate 35 amplicons (median length 246 base pairs) covering 8.9 kilobases of mutational hotspots in AKT1, BRAF, EGFR, ERBB2, HRAS, KRAS, NRAS, PIK3CA, and MAP2K1 genes and all exons of the PTEN gene. RESULTS: The assay was validated on 23 formalin-fixed, paraffin-embedded lung cancer specimens. A minimum number of reads was consistently achieved with overall median read depth of 529× per amplicon. In total, 25 point mutations and 4 insertions/deletions (indels) with a frequency of 5.5% to 93.1% mutant alleles were detected. All EGFR, ERBB2, KRAS, PIK3CA, KRAS, and PTEN mutations, as detected by next-generation sequencing, were confirmed by pyrosequencing, with the exception of 3 point mutations in a tumor sample with low mutant-allele burden (below the pyrosequencing limit of detection). CONCLUSIONS: The GS Junior-based, targeted, resequencing assay for a focused set of non-small cell lung cancer driver genes allows for quick and sensitive detection of point mutations and indels for the most relevant therapeutic genes in this type of cancer.

SUMOylation Is an Inhibitory Constraint that Regulates the Prion-like Aggregation and Activity of CPEB3.

Protein synthesis is crucial for the maintenance of long-term-memory-related synaptic plasticity. The prion-like cytoplasmic polyadenylation element-binding protein 3 (CPEB3) regulates the translation of several mRNAs important for long-term synaptic plasticity in the hippocampus. Here, we provide evidence that the prion-like aggregation and activity of CPEB3 is controlled by SUMOylation. In the basal state, CPEB3 is a repressor and is soluble. Under these circumstances, CPEB3 is SUMOylated in hippocampal neurons both in vitro and in vivo. Following neuronal stimulation, CPEB3 is converted into an active form that promotes the translation of target mRNAs, and this is associated with a decrease of SUMOylation and an increase of aggregation. A chimeric CPEB3 protein fused to SUMO cannot form aggregates and cannot activate the translation of target mRNAs. These findings suggest a model whereby SUMO regulates translation of mRNAs and structural synaptic plasticity by modulating the aggregation of the prion-like protein CPEB3.

The incidence of pelvic and para-aortic lymph node metastasis in uterine papillary serous and clear cell carcinoma according to the SEER registry.

OBJECTIVE: In this study we utilized the Surveillance, Epidemiology and End-Results (SEER) registry to identify risk factors for lymphatic spread and determine the incidence of pelvic and para-aortic lymph node metastases in patients with uterine papillary serous carcinoma (UPSC) and uterine clear cell carcinoma (UCCC) who underwent complete surgical staging and lymph node dissection. METHODS: Nine hundred seventy-two eligible patients diagnosed between 1998 to 2009 with International Federation of Gynecology and Obstetrics (FIGO) 1988 stage IA-IVA UPSC (n=685) or UCCC (n=287) were identified for analysis. Binomial logistic regression was used to determine risk factors for lymph node metastasis, with the incidence of pelvic and para-aortic lymph node metastases reported for each FIGO primary tumor stage. The Cox proportional hazards regression model was used to determine factors associated with overall survival. RESULTS: FIGO primary tumor stage was the only independent risk factor for lymph node metastasis (p<0.01). The incidence of pelvis-only and para-aortic lymph node involvement according to the FIGO primary tumor stage were as follows: IA (2.3%/3.8%), IB (7.5%/5.2%), IC (22.5%/16.9%), IIA (20.8%/13.2%), IIB (25.7%/14.9%), and III/IV (25.7%/24.3%). Prognostic factors for overall survival included lymph node involvement (hazard ratio [HR], 1.42; 95% confidence interval [CI], 1.09 to 1.85; p<0.01), patient age >60 years (HR, 1.70; 95% CI, 1.21 to 2.41; p<0.01), and advanced FIGO primary tumor stage (p<0.01). Tumor grade, histologic subtype, and patient race did not predict for either lymph node metastasis or overall survival. CONCLUSION: There is a high incidence of both pelvic and para-aortic lymph node metastases for FIGO stages IC and above uterine papillary serous and clear cell carcinomas, suggesting a potential role for lymph node-directed therapy for these patients.

Biofilm formed by a hypervirulent (hypermucoviscous) variant of Klebsiella pneumoniae does not enhance serum resistance or survival in an in vivo abscess model.

A new hypervirulent (hypermucoviscous) clinical variant of Klebsiella pneumoniae (hvKP) has emerged over the last decade. Our goal is to identify new mechanisms, which increase the virulence hvKP. It has been shown that hvKP strains produce more biofilm than "classical" stains of K. pneumoniae, therefore we hypothesized that biofilm formation may contribute to the pathogenesis of systemic infection. To test this hypothesis, transposon mutants of the model pathogen hvKP1 were generated and screened for decreased production of biofilm. Three mutant constructs with disruptions in glnA [putatively encodes glutamine synthetase, hvKP1 glnA:: EZ::TN < KAN-2 > (glnA::Tn)], sucD [putatively encodes succinyl-CoA synthase α subunit, hvKP1 sucD:: EZ::TN < KAN-2 > (sucD::Tn)], and tag [putatively encodes transcriptional antiterminator of glycerol uptake operon, hvKP1 tag:: EZ::TN < KAN-2 > (tag::Tn)] were chosen for further characterization and use in biologic studies. Quantitative assays performed in rich laboratory medium and human ascites confirmed the phenotype and a hypermucoviscosity assay established that capsule production was not affected. However, compared with its wild-type parent, neither planktonic cells nor biofilms of glnA::Tn, sucD::Tn and tag::Tn displayed a change to the bactericidal activity of 90% human serum. Likewise, when assessed in a rat subcutaneous abscess model, the growth and survival of glnA::Tn, sucD::Tn and tag::Tn in abscess fluid was similar to hvKP1. In this report we identify three new genes that contribute to biofilm formation in hvKP1. However, decreased biofilm production due to disruption of these genes does not affect the sensitivity of these mutant constructs to 90% human serum when in planktonic form or within a biofilm. Further, their virulence in an in vivo abscess model was unaffected.

Entry of Yersinia pestis into the viable but nonculturable state in a low-temperature tap water microcosm.

Yersinia pestis, the causative agent of plague, has caused several pandemics throughout history and remains endemic in the rodent populations of the western United States. More recently, Y. pestis is one of several bacterial pathogens considered to be a potential agent of bioterrorism. Thus, elucidating potential mechanisms of survival and persistence in the environment would be important in the event of an intentional release of the organism. One such mechanism is entry into the viable but non-culturable (VBNC) state, as has been demonstrated for several other bacterial pathogens. In this study, we showed that Y. pestis became nonculturable by normal laboratory methods after 21 days in a low-temperature tap water microcosm. We further show evidence that, after the loss of culturability, the cells remained viable by using a variety of criteria, including cellular membrane integrity, uptake and incorporation of radiolabeled amino acids, and protection of genomic DNA from DNase I digestion. Additionally, we identified morphological and ultrastructural characteristics of Y. pestis VBNC cells, such as cell rounding and large periplasmic spaces, by electron microscopy, which are consistent with entry into the VBNC state in other bacteria. Finally, we demonstrated resuscitation of a small number of the non-culturable cells. This study provides compelling evidence that Y. pestis persists in a low-temperature tap water microcosm in a viable state yet is unable to be cultured under normal laboratory conditions, which may prove useful in risk assessment and remediation efforts, particularly in the event of an intentional release of this organism.

Enterotoxin-based mucosal adjuvants alter antigen trafficking and induce inflammatory responses in the nasal tract.

The safety of nasal vaccines containing enterotoxin-based mucosal adjuvants has not been studied in detail. Previous studies have indicated that native cholera toxin (nCT) can alter antigen trafficking when applied nasally. In this study, we determined the enterotoxin-based variables that alter antigen trafficking. To measure the influence of enterotoxin-based mucosal adjuvants on antigen trafficking in the nasal tract, native and mutant enterotoxins were coadministered with radiolabeled tetanus toxoid (TT). The nCT and heat-labile enterotoxin type 1 (LTh-1) redirected TT into the olfactory neuroepithelium (ON/E). Antigen redirection occurred mainly across the nasal epithelium without subsequent transport along olfactory neurons into the olfactory bulbs (OB). Thus, no significant accumulation of the vaccine antigen TT was observed in the OB when coadministered with nCT. In contrast, neither mutant CT nor mutant LTh-1, which lack ADP-ribosyltransferase activity, redirected TT antigen into the ON/E. Thus, ADP-ribosyltransferase activity was essential for antigen trafficking across the olfactory epithelium. Accumulation of TT in the ON/E was also due to B-subunit binding to GM1 gangliosides, as was demonstrated (i) by redirection of TT by LTh-1 in a dose-dependent manner, (ii) by ganglioside inhibition of the antigen redirection by LTh-1 and nCT, and (iii) by the use of LT-IIb, a toxin that binds to gangliosides other than GM1. Redirection of TT into the ON/E coincided with elevated production of interleukin 6 (IL-6) but not IL-1beta or tumor necrosis factor alpha in the nasal mucosa. Thus, redirection of TT is dependent on ADP-ribosyltransferase activity and GM1 binding and is associated with production of the inflammatory cytokine IL-6.

BhuR, a virulence-associated outer membrane protein of Bordetella avium, is required for the acquisition of iron from heme and hemoproteins.

Iron (Fe) is an essential element for most organisms which must be obtained from the local environment. In the case of pathogenic bacteria, this fundamental element must be acquired from the fluids and tissues of the infected host. A variety of systems have evolved in bacteria for efficient acquisition of host-bound Fe. The gram-negative bacterium Bordetella avium, upon colonization of the avian upper respiratory tract, produces a disease in birds that has striking similarity to whooping cough, a disease caused by the obligate human pathogen Bordetella pertussis. We describe a B. avium Fe utilization locus comprised of bhuR and six accessory genes (rhuIR and bhuSTUV). Genetic manipulations of B. avium confirmed that bhuR, which encodes a putative outer membrane heme receptor, mediates efficient acquisition of Fe from hemin and hemoproteins (hemoglobin, myoglobin, and catalase). BhuR contains motifs which are common to bacterial heme receptors, including a consensus FRAP domain, an NPNL domain, and two TonB boxes. An N-terminal 32-amino-acid segment, putatively required for rhuIR-dependent regulated expression of bhuR, is present in BhuR but not in other bacterial heme receptors. Two forms of BhuR were observed in the outer membrane of B. avium: a 91-kDa polypeptide consistent in size with the predicted mature protein and a smaller 82-kDa polypeptide which lacks the 104 amino acids found at the N terminus of the 91-kDa form. A mutation in hemA was engineered in B. avium to demonstrate that the bacterium transports heme into the cytoplasm in a BhuR-dependent manner. The role of BhuR in virulence was established in turkey poults by use of a competitive-infection model.