Selective expression of a dominant-negative form of peroxisome proliferator-activated receptor in keratinocytes leads to impaired epidermal healing.

Many nuclear hormone receptors are involved in the regulation of skin homeostasis. However, their role in the epithelial compartment of the skin in stress situations, such as skin healing, has not been addressed yet. The healing of a skin wound after an injury involves three major cell types: immune cells, which are recruited to the wound bed; dermal fibroblasts; and epidermal and hair follicle keratinocytes. Our previous studies have revealed important but nonredundant roles of PPARalpha and beta/delta in the reparation of the skin after a mechanical injury in the adult mouse. However, the mesenchymal or epithelial cellular compartment in which PPARalpha and beta/delta play a role could not be determined in the null mice used, which have a germ line PPAR gene invalidation. In the present work, the role of PPARalpha was studied in keratinocytes, using transgenic mice that express a PPARalpha mutant with dominant-negative (dn) activity specifically in keratinocytes. This dn PPARalpha lacks the last 13 C terminus amino acids, binds to a PPARalpha agonist, but is unable to release the nuclear receptor corepressor and to recruit the coactivator p300. When selectively expressed in keratinocytes of transgenic mice, dn PPARalphaDelta13 causes a delay in the healing of skin wounds, accompanied by an exacerbated inflammation. This phenotype, which is similar to that observed in PPARalpha null mice, strongly suggests that during skin healing, PPARalpha is required in keratinocytes rather than in other cell types.

Impact of Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry on the Clinical Management of Patients With Gram-negative Bacteremia: A Prospective Observational Study.

Background. Early identification of pathogens from blood cultures using matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry may optimize the choice of empirical antibiotic therapy in the setting of bloodstream infections. We aimed to assess the impact of this new technology on the use of antibiotic treatment in patients with gram-negative bacteremia. Methods. We conducted a prospective observational study from January to December 2010 to evaluate the sequential and separate impacts of Gram stain reporting and MALDI-TOF bacterial identification performed on blood culture pellets in patients with gram-negative bacteremia. The primary outcome was the impact of MALDI-TOF on empirical antibiotic choice. Results. Among 202 episodes of gram-negative bacteremia, Gram stain reporting had an impact in 42 cases (20.8%). MALDI-TOF identification led to a modification of empirical therapy in 71 of all 202 cases (35.1%), and in 16 of 27 cases (59.3%) of monomicrobial bacteremia caused by AmpC-producing Enterobacteriaceae. The most frequently observed impact was an early appropriate broadening of the antibiotic spectrum in 31 of 71 cases (43.7%). In total, 143 of 165 episodes (86.7%) of monomicrobial bacteremia were correctly identified at genus level by MALDI-TOF. Conclusions. In a low prevalence area for extended spectrum betalactamases (ESBL) and multiresistant gram-negative bacteria, MALDI-TOF performed on blood culture pellets had an impact on the clinical management of 35.1% of all gram-negative bacteremia cases, demonstrating a greater impact than Gram stain reporting. Thus, MALDI-TOF could become a vital second step beside Gram stain in guiding the empirical treatment of patients with bloodstream infection.

Dynamic and Regulated Association of Caveolin with Lipid Bodies: Modulation of Lipid Body Motility and Function by a Dominant Negative Mutant

Caveolins are a crucial component of caveolae but have also been localized to the Golgi complex, and, under some experimental conditions, to lipid bodies (LBs). The physiological relevance and dynamics of LB association remain unclear. We now show that endogenous caveolin-1 and caveolin-2 redistribute to LBs in lipid loaded A431 and FRT cells. Association with LBs is regulated and reversible; removal of fatty acids causes caveolin to rapidly leave the lipid body. We also show by subcellular fractionation, light and electron microscopy that during the first hours of liver regeneration, caveolins show a dramatic redistribution from the cell surface to the newly formed LBs. At later stages of the regeneration process (when LBs are still abundant), the levels of caveolins in LBs decrease dramatically. As a model system to study association of caveolins with LBs we have used brefeldin A (BFA). BFA causes rapid redistribution of endogenous caveolins to LBs and this association was reversed upon BFA washout. Finally, we have used a dominant negative LB-associated caveolin mutant (cavDGV) to study LB formation and to examine its effect on LB function. We now show that the cavDGV mutant inhibits microtubule-dependent LB motility and blocks the reversal of lipid accumulation in LBs.

Resigned but satisfied: The negative impact of public service motivation and red tape on work satisfaction

This article examines the relationship between red tape, Public Service Motivation (PSM) and a particular work outcome labelled 'resigned satisfaction'. Using data from a national survey of over 3754 public servants working at the municipal level in Switzerland, this study shows the importance of looking more closely at the concept of work satisfaction and, furthermore, of thoroughly investigating the impact of the different PSM dimensions on work outcomes. Unsurprisingly, research findings show that red tape is the most important predictor of resignation. Nevertheless, when PSM dimensions are analysed separately, results demonstrate that 'commitment to public interest/civic duty' and, to a lesser extent, 'attraction to policy-making' decrease resignation, whereas 'compassion' and 'self-sacrifice' increase it. This study thus highlights some of the negative (or undesirable) effects of PSM that have not been previously addressed in PSM literature.

Le cancer du sein sans envahissement ganglionnaire. Au-delà des consensus internationaux: une approche pragmatique [Node negative breast cancer. Beyond international consensus: a pragmatic approach].

Apart from therapeutic advances related to new treatments, our practices in the management of early breast cancer have been modified by to key organizational settings (1) mass screening, substantially altering the presentation and epidemiology of breast cancer and (2) the development of guidelines to ensure that any patient management is in agreement with the demonstrated impact in the adjuvant treatment. In daily practice, the impact of screening and guidelines recommendations has put us now in a paradoxical situation: while the majority of non-metastatic breast cancers treated in the hexagon are node negative, most of the results of clinical studies on chemotherapy and targeted therapies today arise from populations predominantly node positive. Therefore, it seemed legitimate to convene a working group around a reflection on the directions of adjuvant chemotherapy in a growing node negative population in order to better respond to the questions of the field oncologists, trying to address the discrepancies between different existing guidelines.

Regulatory roles of the GacS/GacA two-component system in plant-associated and other gram-negative bacteria.

The sensor kinase GacS and the response regulator GacA are members of a two-component system that is present in a wide variety of gram-negative bacteria and has been studied mainly in enteric bacteria and fluorescent pseudomonads. The GacS/GacA system controls the production of secondary metabolites and extracellular enzymes involved in pathogenicity to plants and animals, biocontrol of soilborne plant diseases, ecological fitness, or tolerance to stress. A current model proposes that GacS senses a still-unknown signal and activates, via a phosphorelay mechanism, the GacA transcription regulator, which in turn triggers the expression of target genes. The GacS protein belongs to the unorthodox sensor kinases, characterized by an autophosphorylation, a receiver, and an output domain. The periplasmic loop domain of GacS is poorly conserved in diverse bacteria. Thus, a common signal interacting with this domain would be unexpected. Based on a comparison with the transcriptional regulator NarL, a secondary structure can be predicted for the GacA sensor kinases. Certain genes whose expression is regulated by the GacS/GacA system are regulated in parallel by the small RNA binding protein RsmA (CsrA) at a posttranscriptional level. It is suggested that the GacS/GacA system operates a switch between primary and secondary metabolism, with a major involvement of posttranscriptional control mechanisms.

Negative antagonists promote an inactive conformation of the beta 2-adrenergic receptor.

The beta 2-adrenergic receptor undergoes isomerization between an inactive conformation (R) and an active conformation (R*). The formation of the active conformation of the receptor molecule can be promoted by adrenergic agonists or by mutations in the third cytoplasmic domain that constitutively activate the receptor. Here we show that, of several beta-adrenergic receptor-blocking drugs tested, only two, ICI 118551 and betaxolol, inhibit the basal signaling activity of the beta 2-adrenergic receptor, thus acting as negative antagonists. We document the molecular properties of the more efficacious ICI 118551; (i) it shows higher affinity for the inactive form of the receptor and (ii) it inhibits the spontaneous formation of a beta-adrenergic receptor kinase substrate by the receptor. These properties are opposite those of adrenergic agonists, indicating that, in a fashion reciprocal to that of agonists, negative antagonists promote the formation of an inactive conformation of the receptor.

False negative apraclonidine test in two patients with Horner syndrome

BACKGROUND: Because of denervation supersensitivity, a miotic pupil in a sympathetically-denervated eye dilates in response to a dilute or weak alpha-1-agonist drug. A reversal of anisocoria after topical apraclonidine is considered as a positive test result that diagnoses a unilateral Horner syndrome. HISTORY AND SIGNS: Two women aged 34 and 46 years with a cocaine-confirmed oculosympathetic defect (Horner syndrome) were tested with 1 % topical apraclonidine on separate days. THERAPY AND OUTCOME: In one patient, her miotic Horner pupil dilated marginally but not enough to reverse the baseline anisocoria. Additionally, the upper lid on the same side retracted. There was no discernable effect of apraclonidine on the normal, contralateral eye. In the second patient, there was no pupillary response to apraclonidine but there was resolution of her ptosis. CONCLUSIONS: Neither patient demonstrated a reversal of anisocoria, the current criterion for diagnosing a Horner syndrome using apraclonidine. Thus, these two patients with an established oculosympathetic defect were said to have a "negative test" for Horner syndrome. Yet both women showed subtle pupil and/or lid changes in response to apraclonidine that were consistent with sympathetic denervation supersensitivity. Reversal of anisocoria following topical apraclonidine does not occur in all patients with a unilateral oculosympathetic defect and more specific parameters for defining a positive test result might optimize apraclonidine's utility as a diagnostic test for Horner syndrome

Thermal nucleation of cavities in liquid helium at negative pressures

We have investigated the nucleation rate at which cavities are formed in 4He and 3He at negative pressures due to thermal fluctuations. To this end, we have used a density functional that reproduces the He liquid-gas interface along the coexistence line. The inclusion of thermal effects in the calculation of the barrier against nucleation results in a sizable decrease of the absolute value of the tensile strength above 1.5 K.

Nucleation in supersaturated solutions 3He in 4He at negative pressure.

Depending on the 3He concentration, thermal nucleation in 3-4He supersaturated liquid mixtures at negative pressures may be originated either by bubble or by 3rich drop formation. We have investigated this phenomenon within a density-functional approach, determining the regions in the pressure¿3He-concentration plane where bubbles or drops likely drive the nucleation process. As an illustrative example, we also give the homogeneous nucleation pressure corresponding to 50 and 100 mK temperature.

Clonal deletion and the fate of autoreactive thymocytes that survive negative selection.

Clonal deletion of autoreactive thymocytes is important for self-tolerance, but the intrathymic signals that induce clonal deletion have not been clearly identified. We now report that clonal deletion during negative selection required CD28-mediated costimulation of autoreactive thymocytes at the CD4(+)CD8(lo) intermediate stage of differentiation. Autoreactive thymocytes were prevented from undergoing clonal deletion by either a lack of CD28 costimulation or transgenic overexpression of the antiapoptotic factors Bcl-2 or Mcl-1, with surviving thymocytes differentiating into anergic CD4(-)CD8(-) double-negative thymocytes positive for the T cell antigen receptor αβ subtype (TCRαβ) that 'preferentially' migrated to the intestine, where they re-expressed CD8α and were sequestered as CD8αα(+) intraepithelial lymphocytes (IELs). Our study identifies costimulation by CD28 as the intrathymic signal required for clonal deletion and identifies CD8αα(+) IELs as the developmental fate of autoreactive thymocytes that survive negative selection.

Quorum-sensing-negative (lasR) mutants of Pseudomonas aeruginosa avoid cell lysis and death.

In Pseudomonas aeruginosa, N-acylhomoserine lactone signals regulate the expression of several hundreds of genes, via the transcriptional regulator LasR and, in part, also via the subordinate regulator RhlR. This regulatory network termed quorum sensing contributes to the virulence of P. aeruginosa as a pathogen. The fact that two supposed PAO1 wild-type strains from strain collections were found to be defective for LasR function because of independent point mutations in the lasR gene led to the hypothesis that loss of quorum sensing might confer a selective advantage on P. aeruginosa under certain environmental conditions. A convenient plate assay for LasR function was devised, based on the observation that lasR mutants did not grow on adenosine as the sole carbon source because a key degradative enzyme, nucleoside hydrolase (Nuh), is positively controlled by LasR. The wild-type PAO1 and lasR mutants showed similar growth rates when incubated in nutrient yeast broth at pH 6.8 and 37 degrees C with good aeration. However, after termination of growth during 30 to 54 h of incubation, when the pH rose to > or = 9, the lasR mutants were significantly more resistant to cell lysis and death than was the wild type. As a consequence, the lasR mutant-to-wild-type ratio increased about 10-fold in mixed cultures incubated for 54 h. In a PAO1 culture, five consecutive cycles of 48 h of incubation sufficed to enrich for about 10% of spontaneous mutants with a Nuh(-) phenotype, and five of these mutants, which were functionally complemented by lasR(+), had mutations in lasR. The observation that, in buffered nutrient yeast broth, the wild type and lasR mutants exhibited similar low tendencies to undergo cell lysis and death suggests that alkaline stress may be a critical factor providing a selective survival advantage to lasR mutants.

Exotic dynamically generated baryons with negative charm quantum number

Following a model based on the SU(8) symmetry that treats heavy pseudoscalars and heavy vector mesons on an equal footing, as required by heavy quark symmetry, we study the interaction of baryons and mesons in coupled channels within an unitary approach that generates dynamically poles in the scattering T-matrix. We concentrate in the exotic channels with negative charm quantum number for which there is the experimental claim of one state.

Validation and clinical utility of a 70-gene prognostic signature for women with node-negative breast cancer.

BACKGROUND: A 70-gene signature was previously shown to have prognostic value in patients with node-negative breast cancer. Our goal was to validate the signature in an independent group of patients. METHODS: Patients (n = 307, with 137 events after a median follow-up of 13.6 years) from five European centers were divided into high- and low-risk groups based on the gene signature classification and on clinical risk classifications. Patients were assigned to the gene signature low-risk group if their 5-year distant metastasis-free survival probability as estimated by the gene signature was greater than 90%. Patients were assigned to the clinicopathologic low-risk group if their 10-year survival probability, as estimated by Adjuvant! software, was greater than 88% (for estrogen receptor [ER]-positive patients) or 92% (for ER-negative patients). Hazard ratios (HRs) were estimated to compare time to distant metastases, disease-free survival, and overall survival in high- versus low-risk groups. RESULTS: The 70-gene signature outperformed the clinicopathologic risk assessment in predicting all endpoints. For time to distant metastases, the gene signature yielded HR = 2.32 (95% confidence interval [CI] = 1.35 to 4.00) without adjustment for clinical risk and hazard ratios ranging from 2.13 to 2.15 after adjustment for various estimates of clinical risk; clinicopathologic risk using Adjuvant! software yielded an unadjusted HR = 1.68 (95% CI = 0.92 to 3.07). For overall survival, the gene signature yielded an unadjusted HR = 2.79 (95% CI = 1.60 to 4.87) and adjusted hazard ratios ranging from 2.63 to 2.89; clinicopathologic risk yielded an unadjusted HR = 1.67 (95% CI = 0.93 to 2.98). For patients in the gene signature high-risk group, 10-year overall survival was 0.69 for patients in both the low- and high-clinical risk groups; for patients in the gene signature low-risk group, the 10-year survival rates were 0.88 and 0.89, respectively. CONCLUSIONS: The 70-gene signature adds independent prognostic information to clinicopathologic risk assessment for patients with early breast cancer.

Chemoendocrine compared with endocrine adjuvant therapies for node-negative breast cancer: predictive value of centrally reviewed expression of estrogen and progesterone receptors--International Breast Cancer Study Group.

PURPOSE: To centrally assess estrogen receptor (ER) and progesterone receptor (PgR) levels by immunohistochemistry and investigate their predictive value for benefit of chemo-endocrine compared with endocrine adjuvant therapy alone in two randomized clinical trials for node-negative breast cancer. PATIENTS AND METHODS: International Breast Cancer Study Group Trial VIII compared cyclophosphamide, methotrexate, and fluorouracil (CMF) chemotherapy for 6 cycles followed by endocrine therapy with goserelin with either modality alone in pre- and perimenopausal patients. Trial IX compared three cycles of CMF followed by tamoxifen for 5 years versus tamoxifen alone in postmenopausal patients. Central Pathology Office reviewed 883 (83%) of 1,063 patients on Trial VIII and 1,365 (82%) of 1,669 on Trial IX and determined ER and PgR by immunohistochemistry. Disease-free survival (DFS) was compared across the spectrum of expression of each receptor using the Subpopulation Treatment Effect Pattern Plot methodology. RESULTS: Both receptors displayed a bimodal distribution, with substantial proportions showing no staining (receptor absent) and most of the remainder showing a high percentage of stained cells. Chemo-endocrine therapy yielded DFS superior to endocrine therapy alone for patients with receptor-absent tumors, and in some cases also for those with low levels of receptor expression. Among patients with ER-expressing tumors, additional prediction of benefit was suggested in absent or low PgR in Trial VIII but not in Trial IX. CONCLUSION: Low levels of ER and PgR are predictive of the benefit of adding chemotherapy to endocrine therapy. Low PgR may add further prediction among pre- and perimenopausal but not postmenopausal patients whose tumors express ER.